Allele and Genotype Frequencies of the ABO Blood

Group System in a Palestinian Population

DECLARATION

The work provided in this thesis unless otherwise referenced is the

researchers own work and has not been submitted elsewhere for any other

degree or qualification

Students name لمياء صبحي صقراسم الطالب

Lamiaa Signature التوقيع

Date 2013 التاريخ

ii

Allele and Genotype Frequencies of the ABO Blood

Group System in a Palestinian Population

Prepared by

Lamiaa Sobhi Saqer

Supervisor

Prof Fadel A Sharif

A Thesis Submitted in Partial Fulfillment of the Requirements for the

Degree of Master in Biological Sciences Medical Technology

1434هـ -2013م

The Islamic University ndash Gaza

Deanery of Higher Education

Faculty of Science

Master of Biology Sciences

Medical Technology

iii

i

Abstract

The ABO blood group antigens are of clinical importance in blood transfusion organ

transplantation autoimmune hemolytic anemia and fetomaternal blood group

incompatibility The ABO locus are located on chromosome 9 Till now more than 200

ABO alleles have been identified by molecular investigations The objective of this

study was to determine the major ABO alleles and genotypes frequencies in a

Palestinian population residing in Gaza Strip A four separatendashreaction multiplex allele

specific polymerase chain reaction (AS-PCR) was used to determine the ABO

genotypes Our study population consisted of 201 unrelated subjects (50 males and 151

females) whose DNA extracted from peripheral blood was subjected to genotyping

The genotypes of 201 samples were found to be A1A1 (n=3) A1O 1(n=24) A1O2 (n=25)

A1A2 (n=4) A2A2 (n=2) A2O1 (n=13) A2O2 (n=2) B1B1 (n=5) B1O1 (n=26) B1O2 (n=14)

A1B (n=11) A2B (n=4) O1O1 (n=31) O1O2 (n=26) and O2O2 (n=11) from which the

deduced phenotypes were A (n=73) B (n=45) AB (n=15) and O (n=68)Moreover

there was no significant difference between observed and expected genotypes and the

genotyping results were consistent with Hardy-Weinberg law The frequencies of A1

A2 B1 O1 and O2 alleles were 0 174 0067 0162 0376 and 0221 respectively The

rare cis-ABO1 allele was not encountered in the study population The genotype results

were compared with serologically determined phenotypes and there were no deviation

To our knowledge this is the first study in Gaza strip investigating the ABO genotypes

ABO genotyping has practical applications in blood transfusion tissueorgan

transplantation blood typing discrepancies and forensicpaternity testing investigations

Key words

ABO alleles ABO genotypes AS-PCR allele frequencies ABO phenotype

ii

Allele and Genotype Frequencies of the ABO Blood

Group System in a Palestinian Population

Prepared by

Lamiaa Sobhi Saqer

Supervisor

Prof Fadel A Sharif

A Thesis Submitted in Partial Fulfillment of the Requirements for the

Degree of Master in Biological Sciences Medical Technology

1434هـ -2013م

The Islamic University ndash Gaza

Deanery of Higher Education

Faculty of Science

Master of Biology Sciences

Medical Technology

iii

i

Abstract

The ABO blood group antigens are of clinical importance in blood transfusion organ

transplantation autoimmune hemolytic anemia and fetomaternal blood group

incompatibility The ABO locus are located on chromosome 9 Till now more than 200

ABO alleles have been identified by molecular investigations The objective of this

study was to determine the major ABO alleles and genotypes frequencies in a

Palestinian population residing in Gaza Strip A four separatendashreaction multiplex allele

specific polymerase chain reaction (AS-PCR) was used to determine the ABO

genotypes Our study population consisted of 201 unrelated subjects (50 males and 151

females) whose DNA extracted from peripheral blood was subjected to genotyping

The genotypes of 201 samples were found to be A1A1 (n=3) A1O 1(n=24) A1O2 (n=25)

A1A2 (n=4) A2A2 (n=2) A2O1 (n=13) A2O2 (n=2) B1B1 (n=5) B1O1 (n=26) B1O2 (n=14)

A1B (n=11) A2B (n=4) O1O1 (n=31) O1O2 (n=26) and O2O2 (n=11) from which the

deduced phenotypes were A (n=73) B (n=45) AB (n=15) and O (n=68)Moreover

there was no significant difference between observed and expected genotypes and the

genotyping results were consistent with Hardy-Weinberg law The frequencies of A1

A2 B1 O1 and O2 alleles were 0 174 0067 0162 0376 and 0221 respectively The

rare cis-ABO1 allele was not encountered in the study population The genotype results

were compared with serologically determined phenotypes and there were no deviation

To our knowledge this is the first study in Gaza strip investigating the ABO genotypes

ABO genotyping has practical applications in blood transfusion tissueorgan

transplantation blood typing discrepancies and forensicpaternity testing investigations

Key words

ABO alleles ABO genotypes AS-PCR allele frequencies ABO phenotype

iii

i

Abstract

The ABO blood group antigens are of clinical importance in blood transfusion organ

transplantation autoimmune hemolytic anemia and fetomaternal blood group

incompatibility The ABO locus are located on chromosome 9 Till now more than 200

ABO alleles have been identified by molecular investigations The objective of this

study was to determine the major ABO alleles and genotypes frequencies in a

Palestinian population residing in Gaza Strip A four separatendashreaction multiplex allele

specific polymerase chain reaction (AS-PCR) was used to determine the ABO

genotypes Our study population consisted of 201 unrelated subjects (50 males and 151

females) whose DNA extracted from peripheral blood was subjected to genotyping

The genotypes of 201 samples were found to be A1A1 (n=3) A1O 1(n=24) A1O2 (n=25)

A1A2 (n=4) A2A2 (n=2) A2O1 (n=13) A2O2 (n=2) B1B1 (n=5) B1O1 (n=26) B1O2 (n=14)

A1B (n=11) A2B (n=4) O1O1 (n=31) O1O2 (n=26) and O2O2 (n=11) from which the

deduced phenotypes were A (n=73) B (n=45) AB (n=15) and O (n=68)Moreover

there was no significant difference between observed and expected genotypes and the

genotyping results were consistent with Hardy-Weinberg law The frequencies of A1

A2 B1 O1 and O2 alleles were 0 174 0067 0162 0376 and 0221 respectively The

rare cis-ABO1 allele was not encountered in the study population The genotype results

were compared with serologically determined phenotypes and there were no deviation

To our knowledge this is the first study in Gaza strip investigating the ABO genotypes

ABO genotyping has practical applications in blood transfusion tissueorgan

transplantation blood typing discrepancies and forensicpaternity testing investigations

Key words

ABO alleles ABO genotypes AS-PCR allele frequencies ABO phenotype

i

Abstract

The ABO blood group antigens are of clinical importance in blood transfusion organ

transplantation autoimmune hemolytic anemia and fetomaternal blood group

incompatibility The ABO locus are located on chromosome 9 Till now more than 200

ABO alleles have been identified by molecular investigations The objective of this

study was to determine the major ABO alleles and genotypes frequencies in a

Palestinian population residing in Gaza Strip A four separatendashreaction multiplex allele

specific polymerase chain reaction (AS-PCR) was used to determine the ABO

genotypes Our study population consisted of 201 unrelated subjects (50 males and 151

females) whose DNA extracted from peripheral blood was subjected to genotyping

The genotypes of 201 samples were found to be A1A1 (n=3) A1O 1(n=24) A1O2 (n=25)

A1A2 (n=4) A2A2 (n=2) A2O1 (n=13) A2O2 (n=2) B1B1 (n=5) B1O1 (n=26) B1O2 (n=14)

A1B (n=11) A2B (n=4) O1O1 (n=31) O1O2 (n=26) and O2O2 (n=11) from which the

deduced phenotypes were A (n=73) B (n=45) AB (n=15) and O (n=68)Moreover

there was no significant difference between observed and expected genotypes and the

genotyping results were consistent with Hardy-Weinberg law The frequencies of A1

A2 B1 O1 and O2 alleles were 0 174 0067 0162 0376 and 0221 respectively The

rare cis-ABO1 allele was not encountered in the study population The genotype results

were compared with serologically determined phenotypes and there were no deviation

To our knowledge this is the first study in Gaza strip investigating the ABO genotypes

ABO genotyping has practical applications in blood transfusion tissueorgan

transplantation blood typing discrepancies and forensicpaternity testing investigations

Key words

ABO alleles ABO genotypes AS-PCR allele frequencies ABO phenotype

ii

ABO

ABO

ABO

Antisera

DNA AS-PCR

A1A1A1O1

A1O2 A1A2 A2A2A2O1A2O2B1B1B1O1B1O2

A1BA2BO1O1O1O2O2O2A

BAB

O A1 A2

B O1O2cis-ABO1

iii

TABLE OF CONTENTS CONTENTS Page

ABSTRACT (English)------------------------------------------------------------------------- i

ABSTRACT (Arabic)-------------------------------------------------------------------------- ii

TABLE OF CONTENTS--------------------------------------------------------------------- iii

LIST OF TABLES----------------------------------------------------------------------------- v

LIST OF FIGURES---------------------------------------------------------------------------- vi

ABBREVIATIONS----------------------------------------------------------------------------- vii

DEDICATION---------------------------------------------------------------------------------- x

ACKNOWLEDGEMENTS------------------------------------------------------------------ xi

CHAPTER 1

INTRODUCTION------------------------------------------------------------------------------

1

11 Background-------------------------------------------------------------------------------- 2

12 Objectives of the Study------------------------------------------------------------------ 4

121 General Objective-------------------------------------------------------------------- 4

122 Specific objectives------------------------------------------------------------------- 4

CHAPTER 2

LITIRATURE REVIEW---------------------------------------------------------------------

6

21 Background-------------------------------------------------------------------------------- 6

22 Biosynthesis of ABH antigens---------------------------------------------------------- 6

221 H antigen------------------------------------------------------------------------------ 8

222 A and B antigens------------------------------------------------------------------- 9

23 ABO Glycosyltransferases------------------------------------------------------------- 11

24 ABO Subgroups-------------------------------------------------------------------------- 11

241 A and B Subgroups ----------------------------------------------------------------- 11

2411 A Subgroups --------------------------------------------------------------------- 12

2412 B Subgroups---------------------------------------------------------------------- 13

242 O Subgroups-------------------------------------------------------------------------- 13

243 Weak subgroups-------------------------------------------------------------------- 14

2431 Weak A alleles-------------------------------------------------------------------- 15

2432 Weak B alleles-------------------------------------------------------------------- 15

25 ABO antibodies-------------------------------------------------------------------------- 17

26 Studies on ABO Genotypes------------------------------------------------------------ 18

27 ABO Genotyping and susceptibility to diseases------------------------------------- 22

CHAPTER 3

MATERIALS and METHODS--------------------------------------------------------------

24

31 Materials---------------------------------------------------------------------------------- 24

311 PCR primers-------------------------------------------------------------------------- 24

312 Kits------------------------------------------------------------------------------------- 25

313 Reagents and Chemicals------------------------------------------------------------ 25

314 Apparatus and Equipments--------------------------------------------------------- 25

32 Methods------------------------------------------------------------------------------------ 26

321 Study population--------------------------------------------------------------------- 26

iv

TABLE OF CONTENTS CONTENTS Page

322 Sample collection------------------------------------------------------------------- 26

323 Ethical Considerations------------------------------------------------------------- 26

324 Data Analysis-------------------------------------------------------------------------- 26

33Blood ABO-typing---------------------------------------------------------------------- 26

331 Forward blood group----------------------------------------------------------------- 26

34 ABO Genotyping------------------------------------------------------------------------ 27

341 DNA Extraction---------------------------------------------------------------------- 27

342 Detection of extracted DNA-------------------------------------------------------- 28

35 PCR reactions---------------------------------------------------------------------------- 28

351 Temperature cycling program------------------------------------------------------ 29

352 Expected PCR results---------------------------------------------------------------- 29

CHAPTER 4

RESULTS----------------------------------------------------------------------------------------

31

41 Study Population------------------------------------------------------------------------- 31

42 Phenotypic Frequency of ABO Blood Groups--------------------------------------- 31

43 The Allele Frequencies of ABO Antigens----------------------------------------- 32

44 PCR Results------------------------------------------------------------------------------- 33

441 Quality of the isolated DNA-------------------------------------------------------- 33

442 Blood group genotyping by allele specific PCR-------------------------------- 33

443 Genotype Frequencies--------------------------------------------------------------- 36

CHAPTER 5

DISCUSSION-----------------------------------------------------------------------------------

39

CHAPTER 6

CONCLUSION and RECOMMENDATIONS-------------------------------------------

43

CHAPTER 7

REFERENCES----------------------------------------------------------------------------------

45

v

List of Tables Table Page

Table 21 Some of Human blood group systems recognized by the ISBT----- 6

Table 22 Serological reaction patterns------------------------------------------------- 15

Table 23 Characteristics of some more frequent B weak phenotypes----------------- 16

Table 31 PCR primers sequences used for ABO genotype---------------------- 24

Table 32 Sample Anti A Anti B reaction for forward blood grouping------- 27

Table 32 Composition of PCR master mix----------------------------------------- 29

Table 41 Phenotypic frequencies of various blood groups in the studied

population-------------------------------------------------------------------

31

Table 42 Observed and expected genotypes for the 201 samples -------------- 32

Table 43 PCR products according to the genotype-------------------------------- 34

Table 44 The frequency of recognized genotypes using multiplex AS-PCR

method for population residing in Gaza Strip--------------------------

37

Table 51 Frequency of ABO alleles in Gaza Strip in comparison to some

other countries--------------------------------------------------------------

41

vi

List of Figures Figure Page

Figure 11 Schematic representation of the genomic organization of the ABO

locus-------------------------------------------------------------------------- 2

Figure 21 Biosynthesis of ABO Antigens------------------------------------------- 8

Figure 22 H antigen structure--------------------------------------------------------- 9

Figure 23 A B and O(H) blood group structures and their synthesis----------- 10

Figure 24 A and B antigen structure------------------------------------------------- 10

Figure 41 Distribution of the subjects according to gender----------------------- 13

Figure 42 Gel electrophoresis for DNA extracted from human blood

samples----------------------------------------------------------------------

33

Figure 43 The electrophoresis pattern of recognized genotypes using the

multiplex AS-PCR method------------------------------------------------

36

vii

ABBREVIATIONS

A Adenine

aa Amino acid

Ala Alanine

Arg Arginine

Asn Asparagine

Asp Aspartic acid

AS-PCR Allele Specific ndash Polymerase Chain Reaction

BGMUT Blood Group antigen gene Mutation database

bp Base pair

degC Celsius

C Cytosine

C-2 position Carbon -2 position

CAZy Carbohydrate Active enZyme

cDNA Complementary DNA

C-terminus Carbon terminus

dATP deoxyadenosine Triphosphate

dCTP deoxyacytidine Triphosphate

dGTP deoxyguanosine Triphosphate

dNTPs Deoxynucleotide Triphosphates

dTTP deoxytymidine triphosphate

Del deletion

E coli Escherichia coli

EDTA Ethylenediaminetetraacetic acid

Fuc Fucose

FUT fucosyltransferases

G Guanine

Gal Galactose

GalNAc N-acetylgalactosamine

GalNAcα3 N-acetylgalactosylamine

Galα3 galactosyl

GDP guanosine diphosphate

iii

TABLE OF CONTENTS CONTENTS Page

ABSTRACT (English)------------------------------------------------------------------------- i

ABSTRACT (Arabic)-------------------------------------------------------------------------- ii

TABLE OF CONTENTS--------------------------------------------------------------------- iii

LIST OF TABLES----------------------------------------------------------------------------- v

LIST OF FIGURES---------------------------------------------------------------------------- vi

ABBREVIATIONS----------------------------------------------------------------------------- vii

DEDICATION---------------------------------------------------------------------------------- x

ACKNOWLEDGEMENTS------------------------------------------------------------------ xi

CHAPTER 1

INTRODUCTION------------------------------------------------------------------------------

1

11 Background-------------------------------------------------------------------------------- 2

12 Objectives of the Study------------------------------------------------------------------ 4

121 General Objective-------------------------------------------------------------------- 4

122 Specific objectives------------------------------------------------------------------- 4

CHAPTER 2

LITIRATURE REVIEW---------------------------------------------------------------------

6

21 Background-------------------------------------------------------------------------------- 6

22 Biosynthesis of ABH antigens---------------------------------------------------------- 6

221 H antigen------------------------------------------------------------------------------ 8

222 A and B antigens------------------------------------------------------------------- 9

23 ABO Glycosyltransferases------------------------------------------------------------- 11

24 ABO Subgroups-------------------------------------------------------------------------- 11

241 A and B Subgroups ----------------------------------------------------------------- 11

2411 A Subgroups --------------------------------------------------------------------- 12

2412 B Subgroups---------------------------------------------------------------------- 13

242 O Subgroups-------------------------------------------------------------------------- 13

243 Weak subgroups-------------------------------------------------------------------- 14

2431 Weak A alleles-------------------------------------------------------------------- 15

2432 Weak B alleles-------------------------------------------------------------------- 15

25 ABO antibodies-------------------------------------------------------------------------- 17

26 Studies on ABO Genotypes------------------------------------------------------------ 18

27 ABO Genotyping and susceptibility to diseases------------------------------------- 22

CHAPTER 3

MATERIALS and METHODS--------------------------------------------------------------

24

31 Materials---------------------------------------------------------------------------------- 24

311 PCR primers-------------------------------------------------------------------------- 24

312 Kits------------------------------------------------------------------------------------- 25

313 Reagents and Chemicals------------------------------------------------------------ 25

314 Apparatus and Equipments--------------------------------------------------------- 25

32 Methods------------------------------------------------------------------------------------ 26

321 Study population--------------------------------------------------------------------- 26

iv

TABLE OF CONTENTS CONTENTS Page

322 Sample collection------------------------------------------------------------------- 26

323 Ethical Considerations------------------------------------------------------------- 26

324 Data Analysis-------------------------------------------------------------------------- 26

33Blood ABO-typing---------------------------------------------------------------------- 26

331 Forward blood group----------------------------------------------------------------- 26

34 ABO Genotyping------------------------------------------------------------------------ 27

341 DNA Extraction---------------------------------------------------------------------- 27

342 Detection of extracted DNA-------------------------------------------------------- 28

35 PCR reactions---------------------------------------------------------------------------- 28

351 Temperature cycling program------------------------------------------------------ 29

352 Expected PCR results---------------------------------------------------------------- 29

CHAPTER 4

RESULTS----------------------------------------------------------------------------------------

31

41 Study Population------------------------------------------------------------------------- 31

42 Phenotypic Frequency of ABO Blood Groups--------------------------------------- 31

43 The Allele Frequencies of ABO Antigens----------------------------------------- 32

44 PCR Results------------------------------------------------------------------------------- 33

441 Quality of the isolated DNA-------------------------------------------------------- 33

442 Blood group genotyping by allele specific PCR-------------------------------- 33

443 Genotype Frequencies--------------------------------------------------------------- 36

CHAPTER 5

DISCUSSION-----------------------------------------------------------------------------------

39

CHAPTER 6

CONCLUSION and RECOMMENDATIONS-------------------------------------------

43

CHAPTER 7

REFERENCES----------------------------------------------------------------------------------

45

v

List of Tables Table Page

Table 21 Some of Human blood group systems recognized by the ISBT----- 6

Table 22 Serological reaction patterns------------------------------------------------- 15

Table 23 Characteristics of some more frequent B weak phenotypes----------------- 16

Table 31 PCR primers sequences used for ABO genotype---------------------- 24

Table 32 Sample Anti A Anti B reaction for forward blood grouping------- 27

Table 32 Composition of PCR master mix----------------------------------------- 29

Table 41 Phenotypic frequencies of various blood groups in the studied

population-------------------------------------------------------------------

31

Table 42 Observed and expected genotypes for the 201 samples -------------- 32

Table 43 PCR products according to the genotype-------------------------------- 34

Table 44 The frequency of recognized genotypes using multiplex AS-PCR

method for population residing in Gaza Strip--------------------------

37

Table 51 Frequency of ABO alleles in Gaza Strip in comparison to some

other countries--------------------------------------------------------------

41

vi

List of Figures Figure Page

Figure 11 Schematic representation of the genomic organization of the ABO

locus-------------------------------------------------------------------------- 2

Figure 21 Biosynthesis of ABO Antigens------------------------------------------- 8

Figure 22 H antigen structure--------------------------------------------------------- 9

Figure 23 A B and O(H) blood group structures and their synthesis----------- 10

Figure 24 A and B antigen structure------------------------------------------------- 10

Figure 41 Distribution of the subjects according to gender----------------------- 13

Figure 42 Gel electrophoresis for DNA extracted from human blood

samples----------------------------------------------------------------------

33

Figure 43 The electrophoresis pattern of recognized genotypes using the

multiplex AS-PCR method------------------------------------------------

36

vii

ABBREVIATIONS

A Adenine

aa Amino acid

Ala Alanine

Arg Arginine

Asn Asparagine

Asp Aspartic acid

AS-PCR Allele Specific ndash Polymerase Chain Reaction

BGMUT Blood Group antigen gene Mutation database

bp Base pair

degC Celsius

C Cytosine

C-2 position Carbon -2 position

CAZy Carbohydrate Active enZyme

cDNA Complementary DNA

C-terminus Carbon terminus

dATP deoxyadenosine Triphosphate

dCTP deoxyacytidine Triphosphate

dGTP deoxyguanosine Triphosphate

dNTPs Deoxynucleotide Triphosphates

dTTP deoxytymidine triphosphate

Del deletion

E coli Escherichia coli

EDTA Ethylenediaminetetraacetic acid

Fuc Fucose

FUT fucosyltransferases

G Guanine

Gal Galactose

GalNAc N-acetylgalactosamine

GalNAcα3 N-acetylgalactosylamine

Galα3 galactosyl

GDP guanosine diphosphate

iv

TABLE OF CONTENTS CONTENTS Page

322 Sample collection------------------------------------------------------------------- 26

323 Ethical Considerations------------------------------------------------------------- 26

324 Data Analysis-------------------------------------------------------------------------- 26

33Blood ABO-typing---------------------------------------------------------------------- 26

331 Forward blood group----------------------------------------------------------------- 26

34 ABO Genotyping------------------------------------------------------------------------ 27

341 DNA Extraction---------------------------------------------------------------------- 27

342 Detection of extracted DNA-------------------------------------------------------- 28

35 PCR reactions---------------------------------------------------------------------------- 28

351 Temperature cycling program------------------------------------------------------ 29

352 Expected PCR results---------------------------------------------------------------- 29

CHAPTER 4

RESULTS----------------------------------------------------------------------------------------

31

41 Study Population------------------------------------------------------------------------- 31

42 Phenotypic Frequency of ABO Blood Groups--------------------------------------- 31

43 The Allele Frequencies of ABO Antigens----------------------------------------- 32

44 PCR Results------------------------------------------------------------------------------- 33

441 Quality of the isolated DNA-------------------------------------------------------- 33

442 Blood group genotyping by allele specific PCR-------------------------------- 33

443 Genotype Frequencies--------------------------------------------------------------- 36

CHAPTER 5

DISCUSSION-----------------------------------------------------------------------------------

39

CHAPTER 6

CONCLUSION and RECOMMENDATIONS-------------------------------------------

43

CHAPTER 7

REFERENCES----------------------------------------------------------------------------------

45

v

List of Tables Table Page

Table 21 Some of Human blood group systems recognized by the ISBT----- 6

Table 22 Serological reaction patterns------------------------------------------------- 15

Table 23 Characteristics of some more frequent B weak phenotypes----------------- 16

Table 31 PCR primers sequences used for ABO genotype---------------------- 24

Table 32 Sample Anti A Anti B reaction for forward blood grouping------- 27

Table 32 Composition of PCR master mix----------------------------------------- 29

Table 41 Phenotypic frequencies of various blood groups in the studied

population-------------------------------------------------------------------

31

Table 42 Observed and expected genotypes for the 201 samples -------------- 32

Table 43 PCR products according to the genotype-------------------------------- 34

Table 44 The frequency of recognized genotypes using multiplex AS-PCR

method for population residing in Gaza Strip--------------------------

37

Table 51 Frequency of ABO alleles in Gaza Strip in comparison to some

other countries--------------------------------------------------------------

41

vi

List of Figures Figure Page

Figure 11 Schematic representation of the genomic organization of the ABO

locus-------------------------------------------------------------------------- 2

Figure 21 Biosynthesis of ABO Antigens------------------------------------------- 8

Figure 22 H antigen structure--------------------------------------------------------- 9

Figure 23 A B and O(H) blood group structures and their synthesis----------- 10

Figure 24 A and B antigen structure------------------------------------------------- 10

Figure 41 Distribution of the subjects according to gender----------------------- 13

Figure 42 Gel electrophoresis for DNA extracted from human blood

samples----------------------------------------------------------------------

33

Figure 43 The electrophoresis pattern of recognized genotypes using the

multiplex AS-PCR method------------------------------------------------

36

vii

ABBREVIATIONS

A Adenine

aa Amino acid

Ala Alanine

Arg Arginine

Asn Asparagine

Asp Aspartic acid

AS-PCR Allele Specific ndash Polymerase Chain Reaction

BGMUT Blood Group antigen gene Mutation database

bp Base pair

degC Celsius

C Cytosine

C-2 position Carbon -2 position

CAZy Carbohydrate Active enZyme

cDNA Complementary DNA

C-terminus Carbon terminus

dATP deoxyadenosine Triphosphate

dCTP deoxyacytidine Triphosphate

dGTP deoxyguanosine Triphosphate

dNTPs Deoxynucleotide Triphosphates

dTTP deoxytymidine triphosphate

Del deletion

E coli Escherichia coli

EDTA Ethylenediaminetetraacetic acid

Fuc Fucose

FUT fucosyltransferases

G Guanine

Gal Galactose

GalNAc N-acetylgalactosamine

GalNAcα3 N-acetylgalactosylamine

Galα3 galactosyl

GDP guanosine diphosphate

v

List of Tables Table Page

Table 21 Some of Human blood group systems recognized by the ISBT----- 6

Table 22 Serological reaction patterns------------------------------------------------- 15

Table 23 Characteristics of some more frequent B weak phenotypes----------------- 16

Table 31 PCR primers sequences used for ABO genotype---------------------- 24

Table 32 Sample Anti A Anti B reaction for forward blood grouping------- 27

Table 32 Composition of PCR master mix----------------------------------------- 29

Table 41 Phenotypic frequencies of various blood groups in the studied

population-------------------------------------------------------------------

31

Table 42 Observed and expected genotypes for the 201 samples -------------- 32

Table 43 PCR products according to the genotype-------------------------------- 34

Table 44 The frequency of recognized genotypes using multiplex AS-PCR

method for population residing in Gaza Strip--------------------------

37

Table 51 Frequency of ABO alleles in Gaza Strip in comparison to some

other countries--------------------------------------------------------------

41

vi

List of Figures Figure Page

Figure 11 Schematic representation of the genomic organization of the ABO

locus-------------------------------------------------------------------------- 2

Figure 21 Biosynthesis of ABO Antigens------------------------------------------- 8

Figure 22 H antigen structure--------------------------------------------------------- 9

Figure 23 A B and O(H) blood group structures and their synthesis----------- 10

Figure 24 A and B antigen structure------------------------------------------------- 10

Figure 41 Distribution of the subjects according to gender----------------------- 13

Figure 42 Gel electrophoresis for DNA extracted from human blood

samples----------------------------------------------------------------------

33

Figure 43 The electrophoresis pattern of recognized genotypes using the

multiplex AS-PCR method------------------------------------------------

36

vii

ABBREVIATIONS

A Adenine

aa Amino acid

Ala Alanine

Arg Arginine

Asn Asparagine

Asp Aspartic acid

AS-PCR Allele Specific ndash Polymerase Chain Reaction

BGMUT Blood Group antigen gene Mutation database

bp Base pair

degC Celsius

C Cytosine

C-2 position Carbon -2 position

CAZy Carbohydrate Active enZyme

cDNA Complementary DNA

C-terminus Carbon terminus

dATP deoxyadenosine Triphosphate

dCTP deoxyacytidine Triphosphate

dGTP deoxyguanosine Triphosphate

dNTPs Deoxynucleotide Triphosphates

dTTP deoxytymidine triphosphate

Del deletion

E coli Escherichia coli

EDTA Ethylenediaminetetraacetic acid

Fuc Fucose

FUT fucosyltransferases

G Guanine

Gal Galactose

GalNAc N-acetylgalactosamine

GalNAcα3 N-acetylgalactosylamine

Galα3 galactosyl

GDP guanosine diphosphate

vi

List of Figures Figure Page

Figure 11 Schematic representation of the genomic organization of the ABO

locus-------------------------------------------------------------------------- 2

Figure 21 Biosynthesis of ABO Antigens------------------------------------------- 8

Figure 22 H antigen structure--------------------------------------------------------- 9

Figure 23 A B and O(H) blood group structures and their synthesis----------- 10

Figure 24 A and B antigen structure------------------------------------------------- 10

Figure 41 Distribution of the subjects according to gender----------------------- 13

Figure 42 Gel electrophoresis for DNA extracted from human blood

samples----------------------------------------------------------------------

33

Figure 43 The electrophoresis pattern of recognized genotypes using the

multiplex AS-PCR method------------------------------------------------

36

vii

ABBREVIATIONS

A Adenine

aa Amino acid

Ala Alanine

Arg Arginine

Asn Asparagine

Asp Aspartic acid

AS-PCR Allele Specific ndash Polymerase Chain Reaction

BGMUT Blood Group antigen gene Mutation database

bp Base pair

degC Celsius

C Cytosine

C-2 position Carbon -2 position

CAZy Carbohydrate Active enZyme

cDNA Complementary DNA

C-terminus Carbon terminus

dATP deoxyadenosine Triphosphate

dCTP deoxyacytidine Triphosphate

dGTP deoxyguanosine Triphosphate

dNTPs Deoxynucleotide Triphosphates

dTTP deoxytymidine triphosphate

Del deletion

E coli Escherichia coli

EDTA Ethylenediaminetetraacetic acid

Fuc Fucose

FUT fucosyltransferases

G Guanine

Gal Galactose

GalNAc N-acetylgalactosamine

GalNAcα3 N-acetylgalactosylamine

Galα3 galactosyl

GDP guanosine diphosphate

vii

ABBREVIATIONS

A Adenine

aa Amino acid

Ala Alanine

Arg Arginine

Asn Asparagine

Asp Aspartic acid

AS-PCR Allele Specific ndash Polymerase Chain Reaction

BGMUT Blood Group antigen gene Mutation database

bp Base pair

degC Celsius

C Cytosine

C-2 position Carbon -2 position

CAZy Carbohydrate Active enZyme

cDNA Complementary DNA

C-terminus Carbon terminus

dATP deoxyadenosine Triphosphate

dCTP deoxyacytidine Triphosphate

dGTP deoxyguanosine Triphosphate

dNTPs Deoxynucleotide Triphosphates

dTTP deoxytymidine triphosphate

Del deletion

E coli Escherichia coli

EDTA Ethylenediaminetetraacetic acid

Fuc Fucose

FUT fucosyltransferases

G Guanine

Gal Galactose

GalNAc N-acetylgalactosamine

GalNAcα3 N-acetylgalactosylamine

Galα3 galactosyl

GDP guanosine diphosphate

viii

Glc glucose

GlcNAc N-acetylglucosamine

Glu Glutamic acid

Gly Glycine

GTA Glycosyltransferases A

GTB Glycosyltransferases B

GTs Glycosyltransferases

Ile Isoleucine

ISBT International Society of Blood Transfusion

kb Kilo base

Leu Leucine

Met Methionine

mf Mixed field

ml Milliliter

Mn++ Manganese

microl Micro liter

nt nucleotide

MTHFR Methylenetetrahydrofolate reductase

PCR Polymerase chain reaction

PCR-SSCP PCR- Single-Strand Conformation Polymorphism

Phe Phenylalanine

pmol picomole

Pro Proline

RBCs Red Blood Cells

RFLP Restriction Fragment Length Polymorphism

rpm Round per minute

Ser Serine

SNP Single ndash Nucleotide polymorphism

SSP Sequence-Specific Primers

SPSS Statistical Package for Social Sciences

T Thymine

Trp Tryptophan

UV Ultra Violet

ix

UDP uridine diphosphate

UDP-Gal uridine diphosphate galactose

UDP-GalNAc uridine diphosphate N-acetylgalactosamine

Val Valine

VTE Venous thromboembolism

vWF von Willebrand factor

w weak

x

To my family especially my parents

for their continuous encouragement

and unlimited support to all those who

enliven my days and brighten my ways

To whom who change my life

xi

Acknowledgments

This work has been carried out in the Genetic Diagnosis Laboratory at the

Islamic university of Gaza Palestine

First and for most I am thankful to his almighty God for this work To those

people who directly or indirectly helped a lot to make this work perfect

I wish to express my gratitude and deepest thanks to his Excellency

Professor Fadel A Sharif to his initiating planning supervision and scientific

guidance of this work He stand with me step by step and he was very keen to

teach me every thing right

My thanks and appreciation to V Dean for Academic AffairsndashCollege of science

and technology Mr Niddal S Abu hujayer for his great help and support

I would like to extend my thanks to the staff of the college of science and

technology especially the Medical Sciences department Khan Younis where I

am working for their support

I am grateful too for the support and advise from Mr Ahmad S Silmi for

his assistance advice and support

I am deeply grateful to Mr Mohammad J Ashour for his helpful and

friendly support during the laboratory work

Finally I want to thank my family parents brothers and sisters who

they always stood beside me and gave me encouragement all the time

3

CHAPTER 1

INTRODUCTION

2

CHAPTER 1

INTRODUCTION

11 Background

In clinical practice the ABO blood group system is one of the most important

since the A and B epitopes may provoke a strong immune reaction With the

introduction of blood typing and cross-matching techniques blood transfusion became

not only a simple but also a much safer procedure Furthermore although ABO typing

reduced the occurrence of transfusion reactions they still occurred indicating the

presence of other genetic differences in blood groups of importance in transfusion

medicine as well as in the later emerging field of organ transplantation(1)

Human ABO locus is located in chromosome 9q341-q342 (2-5) and consists of 7 exons

distributed over 18 kb of genomic DNA ranging in size from 28 to 688 base pairs (bp)

and 6 introns with 554 to 12982 bp (6-8) (Figure 11) Exon 7 contains most of the

largest coding sequence whereas exon 6 contains the deletion found in most O alleles

(9)

Figure 11 Schematic representation of the genomic organization of the ABO gene (10)

1

Molecular genetic studies of human ABO genes have demonstrated that ABO genes

have two critical single base substitutions in the last coding exon that result in amino

acid substitutions responsible for the different donor nucleotide sugar substrate

specificity between A- and B-transferases A single base deletion in exon 6 was

ascribed to shift the reading frame of codons and to abolish the transferase activity of A-

transferase in most O alleles (45911)

ABO genotyping is important not only for blood transfusion but also for tissuecell and

organ transplantations Also ABO genotypes are important evidence at crime scenes

and for personal identification in forensic investigations and paternity testing

To our knowledge this is the first study in Gaza Strip investigating the ABO gene

polymorphism In this study multiplex allele-specific PCR is used for determining the

ABO genotypes and the corresponding allele frequency in a group of 201 unrelated

Palestinians residing in Gaza Strip

4

12 Objectives of the Study

121 General Objective

To determine the major ABO alleles and genotypes frequencies in a Palestinian

population residing in Gaza Strip

122 Specific objectives

1- To employ PCR and Allele-specific (AS)-PCR for molecular genotyping of ABO

blood system

2- To correlate ABO genotypes with phenotypes in blood samples of Gaza Strip

population

3- To compare the frequency of ABO genotypes with other populations

5

CHAPTER 2

LITIRATURE REVIEW

6

CHAPTER 2

LITIRATURE REVIEW

21 Background

The International Society of Blood Transfusion (ISBT) Working Committee on

Terminology for Red Cell Surface Antigens was set up in 1980 to establish and define a

meaningful nomenclature for different blood groups (12) Every valid blood group

antigen is given a six digit identification number There are 29 different systems to date

and the first three digits represent the systems (001-029)

The symbol for a gene or cluster of genes controlling a blood group system is

often the italicized symbol for the system The ABO genotypes should consequently be

written in italicized capital letters (Table 21)

Table 21 Some of Human blood group systems recognized by the ISBT (12)

No Name Symbol No of

antigens

Gene name(s) Chromosome

001 ABO ABO 4 ABO 9

002 MNS MNS 43 GYPA GYPB GYPE 4

003 P P1 1 P1 22

004 Rh RH 49 RHD RHCE 1

005 Lutheran LU 19 LU 19

006 Kell KEL 25 KEL 7

007 Lewis LE 6 FUT3 19

008 Duffy FY 6 DARC 1

009 Kidd JK 3 SLC14A1 18

22 Biosynthesis of ABH antigens

The biochemical basis of the ABO and H antigens is well understood due to

intensive studies during the 1950s and 1960s by the pioneering work on ovarian cyst

fluids (which contain large amounts of water-soluble blood-group-active glycoproteins)

by Morgan amp Watkins and Kabat The ABO antigens are not limited to erythroid

tissues but are also found in different tissues and on some epithelial cells (13)The

antigens are also present in the secretory fluids in the majority of humans therefore they

7

can sometimes be noted as histo-blood group antigens (14) ABH antigens are

carbohydrate structures These oligosaccharide chains are generally conjugated with

polypeptides to form glycoproteins Oligosaccharides are synthesized in a stepwise

fashion the addition of each monosaccharide being catalyzed by a specific

glycosyltransferase enzyme (15)

The glycoproteins contain a peptide backbone to which multiple oligosaccharide

chains are attached through an alkali-labile glycosidic bond (1617) to the hydroxyl

group of serine or threonine (18) Most of the oligosaccharide chains are linked to the

backbone through an N-acetylgalactosamine residue The carbohydrate moiety of the

ABH glycoproteins consists primarily of four sugars D-galactose L-fucose N-acetyl-

D-galactosamine and N-acetyl-D-glucosamine (19) The amino acid compositions of the

different blood group glycoproteins are similar to each other and unrelated to blood-

group specificity

Expression of H A and B antigens is dependent on the presence of specific

monosaccharides attached to various precursor disaccharides at the non-reducing end of

a carbohydrate chain (14) A transferase product of A allele transfers the

monosaccharide N acetylgalactosamine from the donor substrate uridine diphosphate

(UDP)-N-acetylgalactosamine to the fucosylated galactosyl residue of the H antigen to

produce an active structure The B transferase product of B allele transfers galactose

from UDP-galactose to the fucosylated galactose of H to produce a B-active structure

(20)Individuals with the gene for Glycosyltransferases A have blood group A those

with the gene for Glycosyltransferases B have blood group B those with genes for both

enzymes or a cis-acting form of GTA or GTB have blood type AB and those with a

mutated inactive form of enzyme have blood group O (Figure 21 )

8

Figure 21 Biosynthesis of ABO Antigens (2)

The major alleles at the ABO locus are A B and O and to-date a number of ABO

blood groups variants have been reported with approximately 250 different alleles

registered in the Blood Group antigen gene Mutation database BGMUT (21)

221 H antigen

In nature there exists at least four H antigens on glycolipids and glycoproteins

that are recognized by GTA and GTB (22)The most common are the type I and the type

II H antigens (23)H antigen is produced when an α12-Lfucosyltransferase catalyses the

transfer of L-fucose from a guanosine diphosphate (GDP)-L-fucose donor to the C-2

position of the terminal galactose of one of the precursor structures (Figure 21) Two

α12-L-fucosyltransferases produced by two genes FUT1 (H) and FUT2 (Se) catalyze

9

the biosynthesis of H-active structures in different tissues (24) mainly in epithelial cells

and body fluids such as saliva

The H antigen (Figure 22) is the natural precursor of A and B antigens and its

fucose residue is required for A and B glycosyltransferases to recognize it as the

acceptor and transfer GalNAc or Gal to its terminal Gal Depending on the disaccharide

precursor core chain on which ABH determinants are synthesized they can be further

divided into different types

Figure 22 H antigen structure

Everybody expresses H antigen on his red cells but only about 80 of

Europeans have H antigen in their body secretions These people are called ABH

secretors because if they have an A andor B gene they also secrete A andor B

antigens The remaining 20 are called ABH non-secretors as they do not secrete H A

or B regardless of ABO genotype (2526)

222 A and B antigens

A and B antigens can be produced by the presence of the appropriate A- or B

transferase (Figure 21) The A gene product is an α13-N-acetyl galactosaminyl-

transferase which transfers N-acetyl-galactosamine from a uridine diphosphate (UDP)-

N acetylgalactosamine donor to the fucosylated galactosyl residue of H antigen The B

gene product an α 13-D-galactosyltransferase transfers D-galactose from UDP-

galactose to the fucosylated galactose of H A and B are alleles at the ABO locus on

chromosome 9 A third allele O does not produce an active enzyme and in persons

homozygous for O the H antigen remains unmodified (Figure 23)

31

Figure 23 Summary of A B and O(H) blood group structures and their synthesis(28)

The A and B antigens are carbohydrate molecules built stepwise from

saccharides such as galactosamine (GalNAc) glucosamine (GlcNAc) fucose (Fuc)

galactose (Gal) and glucose (Glc) H-antigen is the requisite precursor and

galactosylamine (GalNAcα3) or galactosyl (Galα3) with α1-3 linkage onto H antigen

become the A and B antigens respectively (514) (Figure 24)

Figure 24 A and B antigen structure

The majority of ABO antigens on red cells are linked to glycoproteins

(approximately 70) thus they very much influence the blood group activity on the red

cells At each step in the biosynthesis of ABO antigens and carbohydrate chains

synthesis is facilitated by glycosyltransferases which are competing for available

precursors and substrates This represents approximately 80 of the total complement

of red cell ABH determinants Another 5 times 105 ABH determinants localize to the red

33

cell glucose transport protein (Band 45) Small numbers of ABH antigens are also

expressed by other red cell glycoproteins (28)

23 ABO Glycosyltransferases

Glycosyltransferases (GTs) constitute a large family of enzymes that are

involved in the biosynthesis of oligosaccharides polysaccharides and glycoconjugates

(29)Particularly abundant are the GTs that transfer a sugar residue from an activated

nucleotide sugar donor to specific acceptor molecules forming glycosidic bonds

Glycosyltransferases are classified into 87 different families based on substrateproduct

stereochemistry according to the CAZy database (30)

Knowledge of the sequences of the ABO genes (9) have established that

mammalian Glycosyltransferases A and Glycosyltransferases B are type II integral

membrane proteins containing 354 amino acids and are localized in the lumen of the

Golgi apparatus These enzymes typically have a short amino-terminal cytoplasmic tail

a hydrophobic membrane domain a short protease-sensitive stem region and a large

catalytic domain that includes the carboxy terminus(31) GTA and GTB are very similar

in the coding regions and the soluble enzyme can be found in serum (32)urine (33)and

milk (34)The enzyme contains an acceptor recognition domain that binds H antigen and

a donor recognition domain that binds UDP-GalNAc or UDP-Gal GTA and GTB

require the metal ion Mn++ (manganese) for activity (35)

Glycosyltransferases are antigenic structures Human antibodies to blood group

transferases are often produced following organ transplantation (36-39)

24 ABO Subgroups

241 A and B Subgroups

An ABO blood group subtype is called a subgroup andor a variant Subgroups

of ABO are distinguished by decreased amounts of A B or O (H) antigens on red blood

cells The most common are subgroups of A and B Blood type A appears to have the

most variation in subgroups The two most common subgroups of blood group A are A1

and A2 expressing on average 1 million and 250 000 A determinants respectively (40)

32

2411 A Subgroups

The A1 (A101) allele is the reference allele often denoted as the consensus

sequence in an ABO genotype context (9)but has additionally eight variant alleles

There are variants of A1 alleles one of which is very common in Asian populations with

a 467CrarrT (A102) polymorphism resulting in the substitution Pro156Leu (41)Two

minor A1 alleles (A103 and A104) have been described and differ as follows The first

one also has the 467CrarrT point mutation but an additional silent mutation 567CrarrT

the second allele contains a silent polymorphism in nucleotide 297ArarrG (42) A105 is

like A102 with the same mutation in exon 7 467CrarrT but analysis of intron 6 showed

additional single-nucleotide polymorphism (SNP) compared to A102 Another A1 allele

(A106) contains both 297ArarrG and 467CrarrT (43)

The A2 subgroup is the most common A phenotype after the A1 subgroup The

main genetic difference between A1 and A2 alleles is one point mutation in exon 7

467CrarrT (Pro156Leu) and a deletion of one of the three cytosines at nt 1059-1061

(CCC to CC) The latter mutation results in an extension of the reading frame by 64

nucleotides This deletion occurs in the codon before the translation stop codon (TGA)

resulting in a gene product with an extra 21 amino acid at its C-terminus The

glycosyltransferases encoded by the A2 allele have lower efficiency leading to a weaker

A phenotype The enzyme activity is decreased by 30-50 times compared to A1

(22)Other variants of A2 alleles have subsequently been elucidated (A202-206) Three

A2-like alleles were shown to have three different single mutations near the 3acute end of

exon 7 by Ogasawara A2-2 (A202) contains 1054CrarrT (Arg352Trp) A2-3 (A203) with

1054CrarrG (Arg352Gly) (42) and A2-4 (A205) with both 467CrarrT and an additional

new mutation 1009ArarrG (Arg337Gly) (44) A204 with four common B-related base

substitutions (297ArarrG 526CrarrG 657CrarrT and 703GrarrA) seems to be a hybrid with

two extra substitutions 771CrarrT (silent mutation) and 829GrarrA (Val277Met) One

other rare A2 subgroup A2-5 (A206) carried only the single deletion (1061delC)

(4546)

31

2412 B Subgroups

Subtypes of blood type B are classified by the quantity of B antigen and the

amount of B antigen decreases in the order B B3 Bx Bm Bel B1 allele (B101)

showed seven single nucleotide substitutions 297 526 657 703 796 803 and 930

throughout exon 6 and 7 and later one extra mutation outside the coding region at the 3acute

end at nt 1096 (33) The four amino acids substitutions governed by nt 526CrarrG

(Arg176Gly) 703GrarrA (Gly235Ser) 796CrarrA (Leu266Met) and 803GrarrC

(Gly268Ala) discriminate GTA from GTB (4147) while the substitutions 297 657 and

930 are silent

ABO glycosyltransferases can accordingly be described by using the letters A

and B to illustrate the derivation of the amino acid at these four residues GTA would be

represented by AAAA indicating the presence of ArgGlyLeuGly and similarly BBBB

would describe the GTB with GlySerMetAla at residues 176235266 and 268

respectively The substitutions at positions 266 and 268 were shown to be responsible

for the nucleotidedonor specificity of the transferases (47) and the other residues may

have a role in acceptor binding and turnover (48)Some other variants have been

reported afterwards that differ from B1 (B101) by lacking the 930 substitution for B2

(B102) the 657 substitution for B3 (B103) (47) the 526 substitution for B4 (B107) (44)

and 297 for B108 (43)

242 O Subgroups

The blood group O demonstrates the absence of A and B antigens on the RBC

surface in forward blood typing Reverse blood typing indicates the presence of both

anti-A and -B in the plasma The first O allele (O1-1 [O01]) was shown to be identical

to the consensus A allele (A101) except for a nucleotide deletion 261delG in exon 6

This results in a shift in the reading frame giving rise to a truncated protein that alters

the protein sequence after amino acid 88 A stop codon halts translation after amino acid

117 and the resulting protein is enzymatically inactive (41) Some other O1-1-like

alleles that are characterized by the presence of the 261delG and at least one additional

point mutation A second kind of O allele has the same inactivating deletion (261delG)

as the original O allele (O1-1 [O01]) but in addition has nine point mutations spread

34

throughout exons 3 to 7 (4145) and a further 13 mutations have been found amongst

the intron 6 (50)

Additional polymorphisms associated with blood group O have been found up to

approximately 4300 bp from ABO exon 7 (51) some of which correlated with O1 and

O1v alleles (52) Other O alleles not due to 261delG also exist that are caused by other

inactivating mutations along the reading frame The ( O2-1 [O03]) was the first allele

described of this type (5354) which has a critical mutation (802G rarr A) causing an

amino acid change (Gly268Arg) that prevents the enzyme from utilizing the nucleotide

sugar donor (55) This O allele comprises approximately 2 to 5 percent of O alleles in

Caucasian persons but seems to be absent or at least very rare in other populations

(5456) Other rare O alleles such as O3 [O08] that does not have 261delG but instead

contains both the common A2 allele polymorphisms 467CrarrT and C-deletion at nt

1059-1061 and an insertion of an extra guanosine in the 7-guanosine sequence at nt

798-804 (45) Two other rare O alleles lacking 261delG were reported in the Japanese

population by Ogasawara O301 [O14] has the missense mutation 893CrarrT (Ala268Val)

on an A102 background whereas O302 [O15] has the nonsense mutation 927CrarrA

(Tyr309Stop) on an A101 background (43)

243 Weak subgroups

In addition to the major phenotypes characterized by either strong or absent

haemagglutination with anti-A-A1 and -B reagents the ABO blood group system also

includes phenotypes in which erythrocytes react weakly with the anti-A and ndashB

reagents for example A3 Ax Afinn Ael B3 Bx Bv Bel cis-AB (57)

It is difficult to determine clearly their specific ABO subgroup by conventional

serological methods The weak subgroups are important in more than one way First

they risk complicating patient and donor blood group determination At worst the

wrong group can be assigned if eg a weak antigen is missed Second they allow us to

characterize and understand glycosyltransferase mechanisms by studying the results of

mutations in the underlying alleles

35

2431 Weak A alleles

The RBCs from individuals with the A3 phenotype agglutinate strongly with

anti-A and anti-AB in vitro but show a large number of free cells One A3B individual

had a novel point mutation 871GrarrA (Asp291Asn) on the A1 [A101] (58) and this

allele was named A301 Ax alleles are responsible for the rare subgroup Ax and the

RBCs typically show a weak positive reaction with anti-AB and anti-H There are four

base substitutions involved in these alleles 646TrarrA (Phe216Ile) 681GrarrA (silent)

771CrarrT (silent) 829GrarrA (Val277Met) The encoded transferase is expected to be 37

aa longer than the normal consensus allele and 16 aa longer than A2-encoded

transferase The serological characteristics of some other minor phenotypes included

among A subgroups eg Aend Am Ay Afinn Abantu and the collective description Aw are

more unclear Table 22 shows the serological reaction patterns of weak A alleles

Table 22 Serological reaction patterns

A negative reaction is noted by 0 and positive reactions are denoted from + (very weak

agglutination) to 4+ (maximal agglutination)mf mixed field agglutination

2432 Weak B alleles

B variants are much more uncommon than A variants this may reflect the

relatively low frequency of the B blood group in many populations These phenotypes

Subgroup

of A

RBC reactions with Anti-A1

in serum

Anti-A Anti-AB Anti-A1 Anti-H

A1 4+ 4+ 4+ 0 No

A2 4+ 4+ 0 4+ Sometimes

Aint 4+ 4+ 2+3+ 2+3+ No

A3 2++mf 2++ mf 0 4+ No

Ax 0+ 2++ 0 4+ Often

Ael 0 0 0 4+ Sometimes

Aend + + 0 4+ Sometimes

Afinn + + 0 4+ Yes

Abantu +(+) +(+) 0 4+ Yes

Am 0+ 0+ 0 4+ No

Ay 0 0 0 4+ No

36

often appear to result from missense mutations at the ABO locus causing single aa

changes in the GTB Characteristics of some B variants are summarized in Table 23

(57)

Table 23 Characteristics of some more frequent B weak phenotypes

mf mixed field w very weak agglutination

The B3 phenotype have missense mutation 1054CrarrT (Arg352Trp) on a B1

(B101) background and named B301 (58) A Bx allele responsible for the Bx phenotype

had a point mutation at nt 871GrarrA (Asp291Asn) (59) which was also found in an A3

sample (58) The Bel phenotype was divided into two suballeles Bel-1 and Bel-2 (Bel01

and Bel02) which had substitutions at 641TrarrG (Met214Arg) and 669GrarrT

(Glu223Asp) respectively (59) these missense mutations reduce the enzymatic

activities of the GTB Ogasawara et al (1996) also found another B3 allele B302 which

differs from B consensus by two nucleotide substitutions 646TrarrA (Phe216Ile) and

657TrarrC

CisAB alleles rare phenotype was described by Seyfried in 1964 It represents a

very interesting phenomenon that proved that it is possible for a child with blood group

O to have a parent with blood group AB Seyfried hypothesized that both A and B

determinants of the AB blood group could be located on the same chromosome Later

on this hypothesis confirmed in that the existence of an exceptional ABO allele

encoding a glycosyltransferase is indeed able to produce both A and B enzymes at the

same time (57) Cis-ABO1 was sequenced and showed the substitution 803GrarrC

(Gly268Ala) on the A1-2 (A102) background and thus can be described as AAAB (60)

Another allele named cis-ABO2 was discovered when a Vietnamese man who was to

undergo organ transplantation showed irregular blood grouping results The sequencing

Subgroup

of B

RBC reactions with Anti-B

in serum

Anti-A Anti-AB Anti-B Anti-H

B 0 ++++ ++++ ++ None

B3 1 mf mf +++ None

Bx 1 w w +++ Yes

Bel 0 w 0w +++ None

Bm 0 0 0 +++ Sometimes

37

showed that his ABO genes were nearly identical to the normal B allele except for a

796ArarrC (Met266Leu) substitution (61)

25 ABO antibodies

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after

stimulation by a specific antigen The antibody formed binds to the specific antigen in

order to mark the antigen for destruction The type of antigenic exposure occurring in

the body determines if the antibody is a naturally occurring or immune antibody The

term lsquonaturally occurringrsquo is used for blood group antibodies produced in individuals

who have never been transfused with red cells carrying the relevant antigen or been

pregnant with a fetus carrying the relevant antigen Naturally occurring antibodies can

be formed after exposure to environmental agents that are similar to red cell antigens

such as bacteria dust or pollen (62)

Most of these antibodies are not clinically significant with the exception of ABO

antibodies It is possible that food and environmental antigens (bacterial viral or plant

antigens) have epitopes similar enough to A and B glycoprotein antigens The

antibodies created against these environmental antigens in the first years of life can

cross-react with ABO-incompatible red blood cells (RBCs) that it comes in contact with

during blood transfusion later in life Anti-A antibodies are hypothesized to originate

from immune response towards influenza virus whose epitopes are similar enough to

the α-D-N-galactosamine on the A glycoprotein to be able to elicit a cross-reaction

Anti-B antibodies are hypothesized to originate from antibodies produced against

Gram-negative bacteria such as E coli cross-reacting with the α-D-galactose on the B

glycoprotein (49) Anti-A and anti-B antibodies (called isohaemagglutinins) which are

not present in the newborn appear in the first years of life They are isoantibodies that

are produced by an individual against antigens produced by members of the same

species (isoantigens) Anti-A and anti-B antibodies are usually IgM type which are not

able to pass through the placenta to the fetal blood circulation and react best at room

temperature or lower The ABO antibodies may also be found in various body fluids

including saliva milk cervical secretions tears and cysts These antibodies are

detected at about 3 months and increase their titer until the 5th

to 10th

year of life

ix

UDP uridine diphosphate

UDP-Gal uridine diphosphate galactose

UDP-GalNAc uridine diphosphate N-acetylgalactosamine

Val Valine

VTE Venous thromboembolism

vWF von Willebrand factor

w weak

x

To my family especially my parents

for their continuous encouragement

and unlimited support to all those who

enliven my days and brighten my ways

To whom who change my life

xi

Acknowledgments

This work has been carried out in the Genetic Diagnosis Laboratory at the

Islamic university of Gaza Palestine

First and for most I am thankful to his almighty God for this work To those

people who directly or indirectly helped a lot to make this work perfect

I wish to express my gratitude and deepest thanks to his Excellency

Professor Fadel A Sharif to his initiating planning supervision and scientific

guidance of this work He stand with me step by step and he was very keen to

teach me every thing right

My thanks and appreciation to V Dean for Academic AffairsndashCollege of science

and technology Mr Niddal S Abu hujayer for his great help and support

I would like to extend my thanks to the staff of the college of science and

technology especially the Medical Sciences department Khan Younis where I

am working for their support

I am grateful too for the support and advise from Mr Ahmad S Silmi for

his assistance advice and support

I am deeply grateful to Mr Mohammad J Ashour for his helpful and

friendly support during the laboratory work

Finally I want to thank my family parents brothers and sisters who

they always stood beside me and gave me encouragement all the time

3

CHAPTER 1

INTRODUCTION

2

CHAPTER 1

INTRODUCTION

11 Background

In clinical practice the ABO blood group system is one of the most important

since the A and B epitopes may provoke a strong immune reaction With the

introduction of blood typing and cross-matching techniques blood transfusion became

not only a simple but also a much safer procedure Furthermore although ABO typing

reduced the occurrence of transfusion reactions they still occurred indicating the

presence of other genetic differences in blood groups of importance in transfusion

medicine as well as in the later emerging field of organ transplantation(1)

Human ABO locus is located in chromosome 9q341-q342 (2-5) and consists of 7 exons

distributed over 18 kb of genomic DNA ranging in size from 28 to 688 base pairs (bp)

and 6 introns with 554 to 12982 bp (6-8) (Figure 11) Exon 7 contains most of the

largest coding sequence whereas exon 6 contains the deletion found in most O alleles

(9)

Figure 11 Schematic representation of the genomic organization of the ABO gene (10)

1

Molecular genetic studies of human ABO genes have demonstrated that ABO genes

have two critical single base substitutions in the last coding exon that result in amino

acid substitutions responsible for the different donor nucleotide sugar substrate

specificity between A- and B-transferases A single base deletion in exon 6 was

ascribed to shift the reading frame of codons and to abolish the transferase activity of A-

transferase in most O alleles (45911)

ABO genotyping is important not only for blood transfusion but also for tissuecell and

organ transplantations Also ABO genotypes are important evidence at crime scenes

and for personal identification in forensic investigations and paternity testing

To our knowledge this is the first study in Gaza Strip investigating the ABO gene

polymorphism In this study multiplex allele-specific PCR is used for determining the

ABO genotypes and the corresponding allele frequency in a group of 201 unrelated

Palestinians residing in Gaza Strip

4

12 Objectives of the Study

121 General Objective

To determine the major ABO alleles and genotypes frequencies in a Palestinian

population residing in Gaza Strip

122 Specific objectives

1- To employ PCR and Allele-specific (AS)-PCR for molecular genotyping of ABO

blood system

2- To correlate ABO genotypes with phenotypes in blood samples of Gaza Strip

population

3- To compare the frequency of ABO genotypes with other populations

5

CHAPTER 2

LITIRATURE REVIEW

6

CHAPTER 2

LITIRATURE REVIEW

21 Background

The International Society of Blood Transfusion (ISBT) Working Committee on

Terminology for Red Cell Surface Antigens was set up in 1980 to establish and define a

meaningful nomenclature for different blood groups (12) Every valid blood group

antigen is given a six digit identification number There are 29 different systems to date

and the first three digits represent the systems (001-029)

The symbol for a gene or cluster of genes controlling a blood group system is

often the italicized symbol for the system The ABO genotypes should consequently be

written in italicized capital letters (Table 21)

Table 21 Some of Human blood group systems recognized by the ISBT (12)

No Name Symbol No of

antigens

Gene name(s) Chromosome

001 ABO ABO 4 ABO 9

002 MNS MNS 43 GYPA GYPB GYPE 4

003 P P1 1 P1 22

004 Rh RH 49 RHD RHCE 1

005 Lutheran LU 19 LU 19

006 Kell KEL 25 KEL 7

007 Lewis LE 6 FUT3 19

008 Duffy FY 6 DARC 1

009 Kidd JK 3 SLC14A1 18

22 Biosynthesis of ABH antigens

The biochemical basis of the ABO and H antigens is well understood due to

intensive studies during the 1950s and 1960s by the pioneering work on ovarian cyst

fluids (which contain large amounts of water-soluble blood-group-active glycoproteins)

by Morgan amp Watkins and Kabat The ABO antigens are not limited to erythroid

tissues but are also found in different tissues and on some epithelial cells (13)The

antigens are also present in the secretory fluids in the majority of humans therefore they

7

can sometimes be noted as histo-blood group antigens (14) ABH antigens are

carbohydrate structures These oligosaccharide chains are generally conjugated with

polypeptides to form glycoproteins Oligosaccharides are synthesized in a stepwise

fashion the addition of each monosaccharide being catalyzed by a specific

glycosyltransferase enzyme (15)

The glycoproteins contain a peptide backbone to which multiple oligosaccharide

chains are attached through an alkali-labile glycosidic bond (1617) to the hydroxyl

group of serine or threonine (18) Most of the oligosaccharide chains are linked to the

backbone through an N-acetylgalactosamine residue The carbohydrate moiety of the

ABH glycoproteins consists primarily of four sugars D-galactose L-fucose N-acetyl-

D-galactosamine and N-acetyl-D-glucosamine (19) The amino acid compositions of the

different blood group glycoproteins are similar to each other and unrelated to blood-

group specificity

Expression of H A and B antigens is dependent on the presence of specific

monosaccharides attached to various precursor disaccharides at the non-reducing end of

a carbohydrate chain (14) A transferase product of A allele transfers the

monosaccharide N acetylgalactosamine from the donor substrate uridine diphosphate

(UDP)-N-acetylgalactosamine to the fucosylated galactosyl residue of the H antigen to

produce an active structure The B transferase product of B allele transfers galactose

from UDP-galactose to the fucosylated galactose of H to produce a B-active structure

(20)Individuals with the gene for Glycosyltransferases A have blood group A those

with the gene for Glycosyltransferases B have blood group B those with genes for both

enzymes or a cis-acting form of GTA or GTB have blood type AB and those with a

mutated inactive form of enzyme have blood group O (Figure 21 )

8

Figure 21 Biosynthesis of ABO Antigens (2)

The major alleles at the ABO locus are A B and O and to-date a number of ABO

blood groups variants have been reported with approximately 250 different alleles

registered in the Blood Group antigen gene Mutation database BGMUT (21)

221 H antigen

In nature there exists at least four H antigens on glycolipids and glycoproteins

that are recognized by GTA and GTB (22)The most common are the type I and the type

II H antigens (23)H antigen is produced when an α12-Lfucosyltransferase catalyses the

transfer of L-fucose from a guanosine diphosphate (GDP)-L-fucose donor to the C-2

position of the terminal galactose of one of the precursor structures (Figure 21) Two

α12-L-fucosyltransferases produced by two genes FUT1 (H) and FUT2 (Se) catalyze

9

the biosynthesis of H-active structures in different tissues (24) mainly in epithelial cells

and body fluids such as saliva

The H antigen (Figure 22) is the natural precursor of A and B antigens and its

fucose residue is required for A and B glycosyltransferases to recognize it as the

acceptor and transfer GalNAc or Gal to its terminal Gal Depending on the disaccharide

precursor core chain on which ABH determinants are synthesized they can be further

divided into different types

Figure 22 H antigen structure

Everybody expresses H antigen on his red cells but only about 80 of

Europeans have H antigen in their body secretions These people are called ABH

secretors because if they have an A andor B gene they also secrete A andor B

antigens The remaining 20 are called ABH non-secretors as they do not secrete H A

or B regardless of ABO genotype (2526)

222 A and B antigens

A and B antigens can be produced by the presence of the appropriate A- or B

transferase (Figure 21) The A gene product is an α13-N-acetyl galactosaminyl-

transferase which transfers N-acetyl-galactosamine from a uridine diphosphate (UDP)-

N acetylgalactosamine donor to the fucosylated galactosyl residue of H antigen The B

gene product an α 13-D-galactosyltransferase transfers D-galactose from UDP-

galactose to the fucosylated galactose of H A and B are alleles at the ABO locus on

chromosome 9 A third allele O does not produce an active enzyme and in persons

homozygous for O the H antigen remains unmodified (Figure 23)

31

Figure 23 Summary of A B and O(H) blood group structures and their synthesis(28)

The A and B antigens are carbohydrate molecules built stepwise from

saccharides such as galactosamine (GalNAc) glucosamine (GlcNAc) fucose (Fuc)

galactose (Gal) and glucose (Glc) H-antigen is the requisite precursor and

galactosylamine (GalNAcα3) or galactosyl (Galα3) with α1-3 linkage onto H antigen

become the A and B antigens respectively (514) (Figure 24)

Figure 24 A and B antigen structure

The majority of ABO antigens on red cells are linked to glycoproteins

(approximately 70) thus they very much influence the blood group activity on the red

cells At each step in the biosynthesis of ABO antigens and carbohydrate chains

synthesis is facilitated by glycosyltransferases which are competing for available

precursors and substrates This represents approximately 80 of the total complement

of red cell ABH determinants Another 5 times 105 ABH determinants localize to the red

33

cell glucose transport protein (Band 45) Small numbers of ABH antigens are also

expressed by other red cell glycoproteins (28)

23 ABO Glycosyltransferases

Glycosyltransferases (GTs) constitute a large family of enzymes that are

involved in the biosynthesis of oligosaccharides polysaccharides and glycoconjugates

(29)Particularly abundant are the GTs that transfer a sugar residue from an activated

nucleotide sugar donor to specific acceptor molecules forming glycosidic bonds

Glycosyltransferases are classified into 87 different families based on substrateproduct

stereochemistry according to the CAZy database (30)

Knowledge of the sequences of the ABO genes (9) have established that

mammalian Glycosyltransferases A and Glycosyltransferases B are type II integral

membrane proteins containing 354 amino acids and are localized in the lumen of the

Golgi apparatus These enzymes typically have a short amino-terminal cytoplasmic tail

a hydrophobic membrane domain a short protease-sensitive stem region and a large

catalytic domain that includes the carboxy terminus(31) GTA and GTB are very similar

in the coding regions and the soluble enzyme can be found in serum (32)urine (33)and

milk (34)The enzyme contains an acceptor recognition domain that binds H antigen and

a donor recognition domain that binds UDP-GalNAc or UDP-Gal GTA and GTB

require the metal ion Mn++ (manganese) for activity (35)

Glycosyltransferases are antigenic structures Human antibodies to blood group

transferases are often produced following organ transplantation (36-39)

24 ABO Subgroups

241 A and B Subgroups

An ABO blood group subtype is called a subgroup andor a variant Subgroups

of ABO are distinguished by decreased amounts of A B or O (H) antigens on red blood

cells The most common are subgroups of A and B Blood type A appears to have the

most variation in subgroups The two most common subgroups of blood group A are A1

and A2 expressing on average 1 million and 250 000 A determinants respectively (40)

32

2411 A Subgroups

The A1 (A101) allele is the reference allele often denoted as the consensus

sequence in an ABO genotype context (9)but has additionally eight variant alleles

There are variants of A1 alleles one of which is very common in Asian populations with

a 467CrarrT (A102) polymorphism resulting in the substitution Pro156Leu (41)Two

minor A1 alleles (A103 and A104) have been described and differ as follows The first

one also has the 467CrarrT point mutation but an additional silent mutation 567CrarrT

the second allele contains a silent polymorphism in nucleotide 297ArarrG (42) A105 is

like A102 with the same mutation in exon 7 467CrarrT but analysis of intron 6 showed

additional single-nucleotide polymorphism (SNP) compared to A102 Another A1 allele

(A106) contains both 297ArarrG and 467CrarrT (43)

The A2 subgroup is the most common A phenotype after the A1 subgroup The

main genetic difference between A1 and A2 alleles is one point mutation in exon 7

467CrarrT (Pro156Leu) and a deletion of one of the three cytosines at nt 1059-1061

(CCC to CC) The latter mutation results in an extension of the reading frame by 64

nucleotides This deletion occurs in the codon before the translation stop codon (TGA)

resulting in a gene product with an extra 21 amino acid at its C-terminus The

glycosyltransferases encoded by the A2 allele have lower efficiency leading to a weaker

A phenotype The enzyme activity is decreased by 30-50 times compared to A1

(22)Other variants of A2 alleles have subsequently been elucidated (A202-206) Three

A2-like alleles were shown to have three different single mutations near the 3acute end of

exon 7 by Ogasawara A2-2 (A202) contains 1054CrarrT (Arg352Trp) A2-3 (A203) with

1054CrarrG (Arg352Gly) (42) and A2-4 (A205) with both 467CrarrT and an additional

new mutation 1009ArarrG (Arg337Gly) (44) A204 with four common B-related base

substitutions (297ArarrG 526CrarrG 657CrarrT and 703GrarrA) seems to be a hybrid with

two extra substitutions 771CrarrT (silent mutation) and 829GrarrA (Val277Met) One

other rare A2 subgroup A2-5 (A206) carried only the single deletion (1061delC)

(4546)

31

2412 B Subgroups

Subtypes of blood type B are classified by the quantity of B antigen and the

amount of B antigen decreases in the order B B3 Bx Bm Bel B1 allele (B101)

showed seven single nucleotide substitutions 297 526 657 703 796 803 and 930

throughout exon 6 and 7 and later one extra mutation outside the coding region at the 3acute

end at nt 1096 (33) The four amino acids substitutions governed by nt 526CrarrG

(Arg176Gly) 703GrarrA (Gly235Ser) 796CrarrA (Leu266Met) and 803GrarrC

(Gly268Ala) discriminate GTA from GTB (4147) while the substitutions 297 657 and

930 are silent

ABO glycosyltransferases can accordingly be described by using the letters A

and B to illustrate the derivation of the amino acid at these four residues GTA would be

represented by AAAA indicating the presence of ArgGlyLeuGly and similarly BBBB

would describe the GTB with GlySerMetAla at residues 176235266 and 268

respectively The substitutions at positions 266 and 268 were shown to be responsible

for the nucleotidedonor specificity of the transferases (47) and the other residues may

have a role in acceptor binding and turnover (48)Some other variants have been

reported afterwards that differ from B1 (B101) by lacking the 930 substitution for B2

(B102) the 657 substitution for B3 (B103) (47) the 526 substitution for B4 (B107) (44)

and 297 for B108 (43)

242 O Subgroups

The blood group O demonstrates the absence of A and B antigens on the RBC

surface in forward blood typing Reverse blood typing indicates the presence of both

anti-A and -B in the plasma The first O allele (O1-1 [O01]) was shown to be identical

to the consensus A allele (A101) except for a nucleotide deletion 261delG in exon 6

This results in a shift in the reading frame giving rise to a truncated protein that alters

the protein sequence after amino acid 88 A stop codon halts translation after amino acid

117 and the resulting protein is enzymatically inactive (41) Some other O1-1-like

alleles that are characterized by the presence of the 261delG and at least one additional

point mutation A second kind of O allele has the same inactivating deletion (261delG)

as the original O allele (O1-1 [O01]) but in addition has nine point mutations spread

34

throughout exons 3 to 7 (4145) and a further 13 mutations have been found amongst

the intron 6 (50)

Additional polymorphisms associated with blood group O have been found up to

approximately 4300 bp from ABO exon 7 (51) some of which correlated with O1 and

O1v alleles (52) Other O alleles not due to 261delG also exist that are caused by other

inactivating mutations along the reading frame The ( O2-1 [O03]) was the first allele

described of this type (5354) which has a critical mutation (802G rarr A) causing an

amino acid change (Gly268Arg) that prevents the enzyme from utilizing the nucleotide

sugar donor (55) This O allele comprises approximately 2 to 5 percent of O alleles in

Caucasian persons but seems to be absent or at least very rare in other populations

(5456) Other rare O alleles such as O3 [O08] that does not have 261delG but instead

contains both the common A2 allele polymorphisms 467CrarrT and C-deletion at nt

1059-1061 and an insertion of an extra guanosine in the 7-guanosine sequence at nt

798-804 (45) Two other rare O alleles lacking 261delG were reported in the Japanese

population by Ogasawara O301 [O14] has the missense mutation 893CrarrT (Ala268Val)

on an A102 background whereas O302 [O15] has the nonsense mutation 927CrarrA

(Tyr309Stop) on an A101 background (43)

243 Weak subgroups

In addition to the major phenotypes characterized by either strong or absent

haemagglutination with anti-A-A1 and -B reagents the ABO blood group system also

includes phenotypes in which erythrocytes react weakly with the anti-A and ndashB

reagents for example A3 Ax Afinn Ael B3 Bx Bv Bel cis-AB (57)

It is difficult to determine clearly their specific ABO subgroup by conventional

serological methods The weak subgroups are important in more than one way First

they risk complicating patient and donor blood group determination At worst the

wrong group can be assigned if eg a weak antigen is missed Second they allow us to

characterize and understand glycosyltransferase mechanisms by studying the results of

mutations in the underlying alleles

35

2431 Weak A alleles

The RBCs from individuals with the A3 phenotype agglutinate strongly with

anti-A and anti-AB in vitro but show a large number of free cells One A3B individual

had a novel point mutation 871GrarrA (Asp291Asn) on the A1 [A101] (58) and this

allele was named A301 Ax alleles are responsible for the rare subgroup Ax and the

RBCs typically show a weak positive reaction with anti-AB and anti-H There are four

base substitutions involved in these alleles 646TrarrA (Phe216Ile) 681GrarrA (silent)

771CrarrT (silent) 829GrarrA (Val277Met) The encoded transferase is expected to be 37

aa longer than the normal consensus allele and 16 aa longer than A2-encoded

transferase The serological characteristics of some other minor phenotypes included

among A subgroups eg Aend Am Ay Afinn Abantu and the collective description Aw are

more unclear Table 22 shows the serological reaction patterns of weak A alleles

Table 22 Serological reaction patterns

A negative reaction is noted by 0 and positive reactions are denoted from + (very weak

agglutination) to 4+ (maximal agglutination)mf mixed field agglutination

2432 Weak B alleles

B variants are much more uncommon than A variants this may reflect the

relatively low frequency of the B blood group in many populations These phenotypes

Subgroup

of A

RBC reactions with Anti-A1

in serum

Anti-A Anti-AB Anti-A1 Anti-H

A1 4+ 4+ 4+ 0 No

A2 4+ 4+ 0 4+ Sometimes

Aint 4+ 4+ 2+3+ 2+3+ No

A3 2++mf 2++ mf 0 4+ No

Ax 0+ 2++ 0 4+ Often

Ael 0 0 0 4+ Sometimes

Aend + + 0 4+ Sometimes

Afinn + + 0 4+ Yes

Abantu +(+) +(+) 0 4+ Yes

Am 0+ 0+ 0 4+ No

Ay 0 0 0 4+ No

36

often appear to result from missense mutations at the ABO locus causing single aa

changes in the GTB Characteristics of some B variants are summarized in Table 23

(57)

Table 23 Characteristics of some more frequent B weak phenotypes

mf mixed field w very weak agglutination

The B3 phenotype have missense mutation 1054CrarrT (Arg352Trp) on a B1

(B101) background and named B301 (58) A Bx allele responsible for the Bx phenotype

had a point mutation at nt 871GrarrA (Asp291Asn) (59) which was also found in an A3

sample (58) The Bel phenotype was divided into two suballeles Bel-1 and Bel-2 (Bel01

and Bel02) which had substitutions at 641TrarrG (Met214Arg) and 669GrarrT

(Glu223Asp) respectively (59) these missense mutations reduce the enzymatic

activities of the GTB Ogasawara et al (1996) also found another B3 allele B302 which

differs from B consensus by two nucleotide substitutions 646TrarrA (Phe216Ile) and

657TrarrC

CisAB alleles rare phenotype was described by Seyfried in 1964 It represents a

very interesting phenomenon that proved that it is possible for a child with blood group

O to have a parent with blood group AB Seyfried hypothesized that both A and B

determinants of the AB blood group could be located on the same chromosome Later

on this hypothesis confirmed in that the existence of an exceptional ABO allele

encoding a glycosyltransferase is indeed able to produce both A and B enzymes at the

same time (57) Cis-ABO1 was sequenced and showed the substitution 803GrarrC

(Gly268Ala) on the A1-2 (A102) background and thus can be described as AAAB (60)

Another allele named cis-ABO2 was discovered when a Vietnamese man who was to

undergo organ transplantation showed irregular blood grouping results The sequencing

Subgroup

of B

RBC reactions with Anti-B

in serum

Anti-A Anti-AB Anti-B Anti-H

B 0 ++++ ++++ ++ None

B3 1 mf mf +++ None

Bx 1 w w +++ Yes

Bel 0 w 0w +++ None

Bm 0 0 0 +++ Sometimes

37

showed that his ABO genes were nearly identical to the normal B allele except for a

796ArarrC (Met266Leu) substitution (61)

25 ABO antibodies

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after

stimulation by a specific antigen The antibody formed binds to the specific antigen in

order to mark the antigen for destruction The type of antigenic exposure occurring in

the body determines if the antibody is a naturally occurring or immune antibody The

term lsquonaturally occurringrsquo is used for blood group antibodies produced in individuals

who have never been transfused with red cells carrying the relevant antigen or been

pregnant with a fetus carrying the relevant antigen Naturally occurring antibodies can

be formed after exposure to environmental agents that are similar to red cell antigens

such as bacteria dust or pollen (62)

Most of these antibodies are not clinically significant with the exception of ABO

antibodies It is possible that food and environmental antigens (bacterial viral or plant

antigens) have epitopes similar enough to A and B glycoprotein antigens The

antibodies created against these environmental antigens in the first years of life can

cross-react with ABO-incompatible red blood cells (RBCs) that it comes in contact with

during blood transfusion later in life Anti-A antibodies are hypothesized to originate

from immune response towards influenza virus whose epitopes are similar enough to

the α-D-N-galactosamine on the A glycoprotein to be able to elicit a cross-reaction

Anti-B antibodies are hypothesized to originate from antibodies produced against

Gram-negative bacteria such as E coli cross-reacting with the α-D-galactose on the B

glycoprotein (49) Anti-A and anti-B antibodies (called isohaemagglutinins) which are

not present in the newborn appear in the first years of life They are isoantibodies that

are produced by an individual against antigens produced by members of the same

species (isoantigens) Anti-A and anti-B antibodies are usually IgM type which are not

able to pass through the placenta to the fetal blood circulation and react best at room

temperature or lower The ABO antibodies may also be found in various body fluids

including saliva milk cervical secretions tears and cysts These antibodies are

detected at about 3 months and increase their titer until the 5th

to 10th

year of life

x

To my family especially my parents

for their continuous encouragement

and unlimited support to all those who

enliven my days and brighten my ways

To whom who change my life

xi

Acknowledgments

This work has been carried out in the Genetic Diagnosis Laboratory at the

Islamic university of Gaza Palestine

First and for most I am thankful to his almighty God for this work To those

people who directly or indirectly helped a lot to make this work perfect

I wish to express my gratitude and deepest thanks to his Excellency

Professor Fadel A Sharif to his initiating planning supervision and scientific

guidance of this work He stand with me step by step and he was very keen to

teach me every thing right

My thanks and appreciation to V Dean for Academic AffairsndashCollege of science

and technology Mr Niddal S Abu hujayer for his great help and support

I would like to extend my thanks to the staff of the college of science and

technology especially the Medical Sciences department Khan Younis where I

am working for their support

I am grateful too for the support and advise from Mr Ahmad S Silmi for

his assistance advice and support

I am deeply grateful to Mr Mohammad J Ashour for his helpful and

friendly support during the laboratory work

Finally I want to thank my family parents brothers and sisters who

they always stood beside me and gave me encouragement all the time

3

CHAPTER 1

INTRODUCTION

2

CHAPTER 1

INTRODUCTION

11 Background

In clinical practice the ABO blood group system is one of the most important

since the A and B epitopes may provoke a strong immune reaction With the

introduction of blood typing and cross-matching techniques blood transfusion became

not only a simple but also a much safer procedure Furthermore although ABO typing

reduced the occurrence of transfusion reactions they still occurred indicating the

presence of other genetic differences in blood groups of importance in transfusion

medicine as well as in the later emerging field of organ transplantation(1)

Human ABO locus is located in chromosome 9q341-q342 (2-5) and consists of 7 exons

distributed over 18 kb of genomic DNA ranging in size from 28 to 688 base pairs (bp)

and 6 introns with 554 to 12982 bp (6-8) (Figure 11) Exon 7 contains most of the

largest coding sequence whereas exon 6 contains the deletion found in most O alleles

(9)

Figure 11 Schematic representation of the genomic organization of the ABO gene (10)

1

Molecular genetic studies of human ABO genes have demonstrated that ABO genes

have two critical single base substitutions in the last coding exon that result in amino

acid substitutions responsible for the different donor nucleotide sugar substrate

specificity between A- and B-transferases A single base deletion in exon 6 was

ascribed to shift the reading frame of codons and to abolish the transferase activity of A-

transferase in most O alleles (45911)

ABO genotyping is important not only for blood transfusion but also for tissuecell and

organ transplantations Also ABO genotypes are important evidence at crime scenes

and for personal identification in forensic investigations and paternity testing

To our knowledge this is the first study in Gaza Strip investigating the ABO gene

polymorphism In this study multiplex allele-specific PCR is used for determining the

ABO genotypes and the corresponding allele frequency in a group of 201 unrelated

Palestinians residing in Gaza Strip

4

12 Objectives of the Study

121 General Objective

To determine the major ABO alleles and genotypes frequencies in a Palestinian

population residing in Gaza Strip

122 Specific objectives

1- To employ PCR and Allele-specific (AS)-PCR for molecular genotyping of ABO

blood system

2- To correlate ABO genotypes with phenotypes in blood samples of Gaza Strip

population

3- To compare the frequency of ABO genotypes with other populations

5

CHAPTER 2

LITIRATURE REVIEW

6

CHAPTER 2

LITIRATURE REVIEW

21 Background

The International Society of Blood Transfusion (ISBT) Working Committee on

Terminology for Red Cell Surface Antigens was set up in 1980 to establish and define a

meaningful nomenclature for different blood groups (12) Every valid blood group

antigen is given a six digit identification number There are 29 different systems to date

and the first three digits represent the systems (001-029)

The symbol for a gene or cluster of genes controlling a blood group system is

often the italicized symbol for the system The ABO genotypes should consequently be

written in italicized capital letters (Table 21)

Table 21 Some of Human blood group systems recognized by the ISBT (12)

No Name Symbol No of

antigens

Gene name(s) Chromosome

001 ABO ABO 4 ABO 9

002 MNS MNS 43 GYPA GYPB GYPE 4

003 P P1 1 P1 22

004 Rh RH 49 RHD RHCE 1

005 Lutheran LU 19 LU 19

006 Kell KEL 25 KEL 7

007 Lewis LE 6 FUT3 19

008 Duffy FY 6 DARC 1

009 Kidd JK 3 SLC14A1 18

22 Biosynthesis of ABH antigens

The biochemical basis of the ABO and H antigens is well understood due to

intensive studies during the 1950s and 1960s by the pioneering work on ovarian cyst

fluids (which contain large amounts of water-soluble blood-group-active glycoproteins)

by Morgan amp Watkins and Kabat The ABO antigens are not limited to erythroid

tissues but are also found in different tissues and on some epithelial cells (13)The

antigens are also present in the secretory fluids in the majority of humans therefore they

7

can sometimes be noted as histo-blood group antigens (14) ABH antigens are

carbohydrate structures These oligosaccharide chains are generally conjugated with

polypeptides to form glycoproteins Oligosaccharides are synthesized in a stepwise

fashion the addition of each monosaccharide being catalyzed by a specific

glycosyltransferase enzyme (15)

The glycoproteins contain a peptide backbone to which multiple oligosaccharide

chains are attached through an alkali-labile glycosidic bond (1617) to the hydroxyl

group of serine or threonine (18) Most of the oligosaccharide chains are linked to the

backbone through an N-acetylgalactosamine residue The carbohydrate moiety of the

ABH glycoproteins consists primarily of four sugars D-galactose L-fucose N-acetyl-

D-galactosamine and N-acetyl-D-glucosamine (19) The amino acid compositions of the

different blood group glycoproteins are similar to each other and unrelated to blood-

group specificity

Expression of H A and B antigens is dependent on the presence of specific

monosaccharides attached to various precursor disaccharides at the non-reducing end of

a carbohydrate chain (14) A transferase product of A allele transfers the

monosaccharide N acetylgalactosamine from the donor substrate uridine diphosphate

(UDP)-N-acetylgalactosamine to the fucosylated galactosyl residue of the H antigen to

produce an active structure The B transferase product of B allele transfers galactose

from UDP-galactose to the fucosylated galactose of H to produce a B-active structure

(20)Individuals with the gene for Glycosyltransferases A have blood group A those

with the gene for Glycosyltransferases B have blood group B those with genes for both

enzymes or a cis-acting form of GTA or GTB have blood type AB and those with a

mutated inactive form of enzyme have blood group O (Figure 21 )

8

Figure 21 Biosynthesis of ABO Antigens (2)

The major alleles at the ABO locus are A B and O and to-date a number of ABO

blood groups variants have been reported with approximately 250 different alleles

registered in the Blood Group antigen gene Mutation database BGMUT (21)

221 H antigen

In nature there exists at least four H antigens on glycolipids and glycoproteins

that are recognized by GTA and GTB (22)The most common are the type I and the type

II H antigens (23)H antigen is produced when an α12-Lfucosyltransferase catalyses the

transfer of L-fucose from a guanosine diphosphate (GDP)-L-fucose donor to the C-2

position of the terminal galactose of one of the precursor structures (Figure 21) Two

α12-L-fucosyltransferases produced by two genes FUT1 (H) and FUT2 (Se) catalyze

9

the biosynthesis of H-active structures in different tissues (24) mainly in epithelial cells

and body fluids such as saliva

The H antigen (Figure 22) is the natural precursor of A and B antigens and its

fucose residue is required for A and B glycosyltransferases to recognize it as the

acceptor and transfer GalNAc or Gal to its terminal Gal Depending on the disaccharide

precursor core chain on which ABH determinants are synthesized they can be further

divided into different types

Figure 22 H antigen structure

Everybody expresses H antigen on his red cells but only about 80 of

Europeans have H antigen in their body secretions These people are called ABH

secretors because if they have an A andor B gene they also secrete A andor B

antigens The remaining 20 are called ABH non-secretors as they do not secrete H A

or B regardless of ABO genotype (2526)

222 A and B antigens

A and B antigens can be produced by the presence of the appropriate A- or B

transferase (Figure 21) The A gene product is an α13-N-acetyl galactosaminyl-

transferase which transfers N-acetyl-galactosamine from a uridine diphosphate (UDP)-

N acetylgalactosamine donor to the fucosylated galactosyl residue of H antigen The B

gene product an α 13-D-galactosyltransferase transfers D-galactose from UDP-

galactose to the fucosylated galactose of H A and B are alleles at the ABO locus on

chromosome 9 A third allele O does not produce an active enzyme and in persons

homozygous for O the H antigen remains unmodified (Figure 23)

31

Figure 23 Summary of A B and O(H) blood group structures and their synthesis(28)

The A and B antigens are carbohydrate molecules built stepwise from

saccharides such as galactosamine (GalNAc) glucosamine (GlcNAc) fucose (Fuc)

galactose (Gal) and glucose (Glc) H-antigen is the requisite precursor and

galactosylamine (GalNAcα3) or galactosyl (Galα3) with α1-3 linkage onto H antigen

become the A and B antigens respectively (514) (Figure 24)

Figure 24 A and B antigen structure

The majority of ABO antigens on red cells are linked to glycoproteins

(approximately 70) thus they very much influence the blood group activity on the red

cells At each step in the biosynthesis of ABO antigens and carbohydrate chains

synthesis is facilitated by glycosyltransferases which are competing for available

precursors and substrates This represents approximately 80 of the total complement

of red cell ABH determinants Another 5 times 105 ABH determinants localize to the red

33

cell glucose transport protein (Band 45) Small numbers of ABH antigens are also

expressed by other red cell glycoproteins (28)

23 ABO Glycosyltransferases

Glycosyltransferases (GTs) constitute a large family of enzymes that are

involved in the biosynthesis of oligosaccharides polysaccharides and glycoconjugates

(29)Particularly abundant are the GTs that transfer a sugar residue from an activated

nucleotide sugar donor to specific acceptor molecules forming glycosidic bonds

Glycosyltransferases are classified into 87 different families based on substrateproduct

stereochemistry according to the CAZy database (30)

Knowledge of the sequences of the ABO genes (9) have established that

mammalian Glycosyltransferases A and Glycosyltransferases B are type II integral

membrane proteins containing 354 amino acids and are localized in the lumen of the

Golgi apparatus These enzymes typically have a short amino-terminal cytoplasmic tail

a hydrophobic membrane domain a short protease-sensitive stem region and a large

catalytic domain that includes the carboxy terminus(31) GTA and GTB are very similar

in the coding regions and the soluble enzyme can be found in serum (32)urine (33)and

milk (34)The enzyme contains an acceptor recognition domain that binds H antigen and

a donor recognition domain that binds UDP-GalNAc or UDP-Gal GTA and GTB

require the metal ion Mn++ (manganese) for activity (35)

Glycosyltransferases are antigenic structures Human antibodies to blood group

transferases are often produced following organ transplantation (36-39)

24 ABO Subgroups

241 A and B Subgroups

An ABO blood group subtype is called a subgroup andor a variant Subgroups

of ABO are distinguished by decreased amounts of A B or O (H) antigens on red blood

cells The most common are subgroups of A and B Blood type A appears to have the

most variation in subgroups The two most common subgroups of blood group A are A1

and A2 expressing on average 1 million and 250 000 A determinants respectively (40)

32

2411 A Subgroups

The A1 (A101) allele is the reference allele often denoted as the consensus

sequence in an ABO genotype context (9)but has additionally eight variant alleles

There are variants of A1 alleles one of which is very common in Asian populations with

a 467CrarrT (A102) polymorphism resulting in the substitution Pro156Leu (41)Two

minor A1 alleles (A103 and A104) have been described and differ as follows The first

one also has the 467CrarrT point mutation but an additional silent mutation 567CrarrT

the second allele contains a silent polymorphism in nucleotide 297ArarrG (42) A105 is

like A102 with the same mutation in exon 7 467CrarrT but analysis of intron 6 showed

additional single-nucleotide polymorphism (SNP) compared to A102 Another A1 allele

(A106) contains both 297ArarrG and 467CrarrT (43)

The A2 subgroup is the most common A phenotype after the A1 subgroup The

main genetic difference between A1 and A2 alleles is one point mutation in exon 7

467CrarrT (Pro156Leu) and a deletion of one of the three cytosines at nt 1059-1061

(CCC to CC) The latter mutation results in an extension of the reading frame by 64

nucleotides This deletion occurs in the codon before the translation stop codon (TGA)

resulting in a gene product with an extra 21 amino acid at its C-terminus The

glycosyltransferases encoded by the A2 allele have lower efficiency leading to a weaker

A phenotype The enzyme activity is decreased by 30-50 times compared to A1

(22)Other variants of A2 alleles have subsequently been elucidated (A202-206) Three

A2-like alleles were shown to have three different single mutations near the 3acute end of

exon 7 by Ogasawara A2-2 (A202) contains 1054CrarrT (Arg352Trp) A2-3 (A203) with

1054CrarrG (Arg352Gly) (42) and A2-4 (A205) with both 467CrarrT and an additional

new mutation 1009ArarrG (Arg337Gly) (44) A204 with four common B-related base

substitutions (297ArarrG 526CrarrG 657CrarrT and 703GrarrA) seems to be a hybrid with

two extra substitutions 771CrarrT (silent mutation) and 829GrarrA (Val277Met) One

other rare A2 subgroup A2-5 (A206) carried only the single deletion (1061delC)

(4546)

31

2412 B Subgroups

Subtypes of blood type B are classified by the quantity of B antigen and the

amount of B antigen decreases in the order B B3 Bx Bm Bel B1 allele (B101)

showed seven single nucleotide substitutions 297 526 657 703 796 803 and 930

throughout exon 6 and 7 and later one extra mutation outside the coding region at the 3acute

end at nt 1096 (33) The four amino acids substitutions governed by nt 526CrarrG

(Arg176Gly) 703GrarrA (Gly235Ser) 796CrarrA (Leu266Met) and 803GrarrC

(Gly268Ala) discriminate GTA from GTB (4147) while the substitutions 297 657 and

930 are silent

ABO glycosyltransferases can accordingly be described by using the letters A

and B to illustrate the derivation of the amino acid at these four residues GTA would be

represented by AAAA indicating the presence of ArgGlyLeuGly and similarly BBBB

would describe the GTB with GlySerMetAla at residues 176235266 and 268

respectively The substitutions at positions 266 and 268 were shown to be responsible

for the nucleotidedonor specificity of the transferases (47) and the other residues may

have a role in acceptor binding and turnover (48)Some other variants have been

reported afterwards that differ from B1 (B101) by lacking the 930 substitution for B2

(B102) the 657 substitution for B3 (B103) (47) the 526 substitution for B4 (B107) (44)

and 297 for B108 (43)

242 O Subgroups

The blood group O demonstrates the absence of A and B antigens on the RBC

surface in forward blood typing Reverse blood typing indicates the presence of both

anti-A and -B in the plasma The first O allele (O1-1 [O01]) was shown to be identical

to the consensus A allele (A101) except for a nucleotide deletion 261delG in exon 6

This results in a shift in the reading frame giving rise to a truncated protein that alters

the protein sequence after amino acid 88 A stop codon halts translation after amino acid

117 and the resulting protein is enzymatically inactive (41) Some other O1-1-like

alleles that are characterized by the presence of the 261delG and at least one additional

point mutation A second kind of O allele has the same inactivating deletion (261delG)

as the original O allele (O1-1 [O01]) but in addition has nine point mutations spread

34

throughout exons 3 to 7 (4145) and a further 13 mutations have been found amongst

the intron 6 (50)

Additional polymorphisms associated with blood group O have been found up to

approximately 4300 bp from ABO exon 7 (51) some of which correlated with O1 and

O1v alleles (52) Other O alleles not due to 261delG also exist that are caused by other

inactivating mutations along the reading frame The ( O2-1 [O03]) was the first allele

described of this type (5354) which has a critical mutation (802G rarr A) causing an

amino acid change (Gly268Arg) that prevents the enzyme from utilizing the nucleotide

sugar donor (55) This O allele comprises approximately 2 to 5 percent of O alleles in

Caucasian persons but seems to be absent or at least very rare in other populations

(5456) Other rare O alleles such as O3 [O08] that does not have 261delG but instead

contains both the common A2 allele polymorphisms 467CrarrT and C-deletion at nt

1059-1061 and an insertion of an extra guanosine in the 7-guanosine sequence at nt

798-804 (45) Two other rare O alleles lacking 261delG were reported in the Japanese

population by Ogasawara O301 [O14] has the missense mutation 893CrarrT (Ala268Val)

on an A102 background whereas O302 [O15] has the nonsense mutation 927CrarrA

(Tyr309Stop) on an A101 background (43)

243 Weak subgroups

In addition to the major phenotypes characterized by either strong or absent

haemagglutination with anti-A-A1 and -B reagents the ABO blood group system also

includes phenotypes in which erythrocytes react weakly with the anti-A and ndashB

reagents for example A3 Ax Afinn Ael B3 Bx Bv Bel cis-AB (57)

It is difficult to determine clearly their specific ABO subgroup by conventional

serological methods The weak subgroups are important in more than one way First

they risk complicating patient and donor blood group determination At worst the

wrong group can be assigned if eg a weak antigen is missed Second they allow us to

characterize and understand glycosyltransferase mechanisms by studying the results of

mutations in the underlying alleles

35

2431 Weak A alleles

The RBCs from individuals with the A3 phenotype agglutinate strongly with

anti-A and anti-AB in vitro but show a large number of free cells One A3B individual

had a novel point mutation 871GrarrA (Asp291Asn) on the A1 [A101] (58) and this

allele was named A301 Ax alleles are responsible for the rare subgroup Ax and the

RBCs typically show a weak positive reaction with anti-AB and anti-H There are four

base substitutions involved in these alleles 646TrarrA (Phe216Ile) 681GrarrA (silent)

771CrarrT (silent) 829GrarrA (Val277Met) The encoded transferase is expected to be 37

aa longer than the normal consensus allele and 16 aa longer than A2-encoded

transferase The serological characteristics of some other minor phenotypes included

among A subgroups eg Aend Am Ay Afinn Abantu and the collective description Aw are

more unclear Table 22 shows the serological reaction patterns of weak A alleles

Table 22 Serological reaction patterns

A negative reaction is noted by 0 and positive reactions are denoted from + (very weak

agglutination) to 4+ (maximal agglutination)mf mixed field agglutination

2432 Weak B alleles

B variants are much more uncommon than A variants this may reflect the

relatively low frequency of the B blood group in many populations These phenotypes

Subgroup

of A

RBC reactions with Anti-A1

in serum

Anti-A Anti-AB Anti-A1 Anti-H

A1 4+ 4+ 4+ 0 No

A2 4+ 4+ 0 4+ Sometimes

Aint 4+ 4+ 2+3+ 2+3+ No

A3 2++mf 2++ mf 0 4+ No

Ax 0+ 2++ 0 4+ Often

Ael 0 0 0 4+ Sometimes

Aend + + 0 4+ Sometimes

Afinn + + 0 4+ Yes

Abantu +(+) +(+) 0 4+ Yes

Am 0+ 0+ 0 4+ No

Ay 0 0 0 4+ No

36

often appear to result from missense mutations at the ABO locus causing single aa

changes in the GTB Characteristics of some B variants are summarized in Table 23

(57)

Table 23 Characteristics of some more frequent B weak phenotypes

mf mixed field w very weak agglutination

The B3 phenotype have missense mutation 1054CrarrT (Arg352Trp) on a B1

(B101) background and named B301 (58) A Bx allele responsible for the Bx phenotype

had a point mutation at nt 871GrarrA (Asp291Asn) (59) which was also found in an A3

sample (58) The Bel phenotype was divided into two suballeles Bel-1 and Bel-2 (Bel01

and Bel02) which had substitutions at 641TrarrG (Met214Arg) and 669GrarrT

(Glu223Asp) respectively (59) these missense mutations reduce the enzymatic

activities of the GTB Ogasawara et al (1996) also found another B3 allele B302 which

differs from B consensus by two nucleotide substitutions 646TrarrA (Phe216Ile) and

657TrarrC

CisAB alleles rare phenotype was described by Seyfried in 1964 It represents a

very interesting phenomenon that proved that it is possible for a child with blood group

O to have a parent with blood group AB Seyfried hypothesized that both A and B

determinants of the AB blood group could be located on the same chromosome Later

on this hypothesis confirmed in that the existence of an exceptional ABO allele

encoding a glycosyltransferase is indeed able to produce both A and B enzymes at the

same time (57) Cis-ABO1 was sequenced and showed the substitution 803GrarrC

(Gly268Ala) on the A1-2 (A102) background and thus can be described as AAAB (60)

Another allele named cis-ABO2 was discovered when a Vietnamese man who was to

undergo organ transplantation showed irregular blood grouping results The sequencing

Subgroup

of B

RBC reactions with Anti-B

in serum

Anti-A Anti-AB Anti-B Anti-H

B 0 ++++ ++++ ++ None

B3 1 mf mf +++ None

Bx 1 w w +++ Yes

Bel 0 w 0w +++ None

Bm 0 0 0 +++ Sometimes

37

showed that his ABO genes were nearly identical to the normal B allele except for a

796ArarrC (Met266Leu) substitution (61)

25 ABO antibodies

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after

stimulation by a specific antigen The antibody formed binds to the specific antigen in

order to mark the antigen for destruction The type of antigenic exposure occurring in

the body determines if the antibody is a naturally occurring or immune antibody The

term lsquonaturally occurringrsquo is used for blood group antibodies produced in individuals

who have never been transfused with red cells carrying the relevant antigen or been

pregnant with a fetus carrying the relevant antigen Naturally occurring antibodies can

be formed after exposure to environmental agents that are similar to red cell antigens

such as bacteria dust or pollen (62)

Most of these antibodies are not clinically significant with the exception of ABO

antibodies It is possible that food and environmental antigens (bacterial viral or plant

antigens) have epitopes similar enough to A and B glycoprotein antigens The

antibodies created against these environmental antigens in the first years of life can

cross-react with ABO-incompatible red blood cells (RBCs) that it comes in contact with

during blood transfusion later in life Anti-A antibodies are hypothesized to originate

from immune response towards influenza virus whose epitopes are similar enough to

the α-D-N-galactosamine on the A glycoprotein to be able to elicit a cross-reaction

Anti-B antibodies are hypothesized to originate from antibodies produced against

Gram-negative bacteria such as E coli cross-reacting with the α-D-galactose on the B

glycoprotein (49) Anti-A and anti-B antibodies (called isohaemagglutinins) which are

not present in the newborn appear in the first years of life They are isoantibodies that

are produced by an individual against antigens produced by members of the same

species (isoantigens) Anti-A and anti-B antibodies are usually IgM type which are not

able to pass through the placenta to the fetal blood circulation and react best at room

temperature or lower The ABO antibodies may also be found in various body fluids

including saliva milk cervical secretions tears and cysts These antibodies are

detected at about 3 months and increase their titer until the 5th

to 10th

year of life

xi

Acknowledgments

This work has been carried out in the Genetic Diagnosis Laboratory at the

Islamic university of Gaza Palestine

First and for most I am thankful to his almighty God for this work To those

people who directly or indirectly helped a lot to make this work perfect

I wish to express my gratitude and deepest thanks to his Excellency

Professor Fadel A Sharif to his initiating planning supervision and scientific

guidance of this work He stand with me step by step and he was very keen to

teach me every thing right

My thanks and appreciation to V Dean for Academic AffairsndashCollege of science

and technology Mr Niddal S Abu hujayer for his great help and support

I would like to extend my thanks to the staff of the college of science and

technology especially the Medical Sciences department Khan Younis where I

am working for their support

I am grateful too for the support and advise from Mr Ahmad S Silmi for

his assistance advice and support

I am deeply grateful to Mr Mohammad J Ashour for his helpful and

friendly support during the laboratory work

Finally I want to thank my family parents brothers and sisters who

they always stood beside me and gave me encouragement all the time

3

CHAPTER 1

INTRODUCTION

2

CHAPTER 1

INTRODUCTION

11 Background

In clinical practice the ABO blood group system is one of the most important

since the A and B epitopes may provoke a strong immune reaction With the

introduction of blood typing and cross-matching techniques blood transfusion became

not only a simple but also a much safer procedure Furthermore although ABO typing

reduced the occurrence of transfusion reactions they still occurred indicating the

presence of other genetic differences in blood groups of importance in transfusion

medicine as well as in the later emerging field of organ transplantation(1)

Human ABO locus is located in chromosome 9q341-q342 (2-5) and consists of 7 exons

distributed over 18 kb of genomic DNA ranging in size from 28 to 688 base pairs (bp)

and 6 introns with 554 to 12982 bp (6-8) (Figure 11) Exon 7 contains most of the

largest coding sequence whereas exon 6 contains the deletion found in most O alleles

(9)

Figure 11 Schematic representation of the genomic organization of the ABO gene (10)

1

Molecular genetic studies of human ABO genes have demonstrated that ABO genes

have two critical single base substitutions in the last coding exon that result in amino

acid substitutions responsible for the different donor nucleotide sugar substrate

specificity between A- and B-transferases A single base deletion in exon 6 was

ascribed to shift the reading frame of codons and to abolish the transferase activity of A-

transferase in most O alleles (45911)

ABO genotyping is important not only for blood transfusion but also for tissuecell and

organ transplantations Also ABO genotypes are important evidence at crime scenes

and for personal identification in forensic investigations and paternity testing

To our knowledge this is the first study in Gaza Strip investigating the ABO gene

polymorphism In this study multiplex allele-specific PCR is used for determining the

ABO genotypes and the corresponding allele frequency in a group of 201 unrelated

Palestinians residing in Gaza Strip

4

12 Objectives of the Study

121 General Objective

To determine the major ABO alleles and genotypes frequencies in a Palestinian

population residing in Gaza Strip

122 Specific objectives

1- To employ PCR and Allele-specific (AS)-PCR for molecular genotyping of ABO

blood system

2- To correlate ABO genotypes with phenotypes in blood samples of Gaza Strip

population

3- To compare the frequency of ABO genotypes with other populations

5

CHAPTER 2

LITIRATURE REVIEW

6

CHAPTER 2

LITIRATURE REVIEW

21 Background

The International Society of Blood Transfusion (ISBT) Working Committee on

Terminology for Red Cell Surface Antigens was set up in 1980 to establish and define a

meaningful nomenclature for different blood groups (12) Every valid blood group

antigen is given a six digit identification number There are 29 different systems to date

and the first three digits represent the systems (001-029)

The symbol for a gene or cluster of genes controlling a blood group system is

often the italicized symbol for the system The ABO genotypes should consequently be

written in italicized capital letters (Table 21)

Table 21 Some of Human blood group systems recognized by the ISBT (12)

No Name Symbol No of

antigens

Gene name(s) Chromosome

001 ABO ABO 4 ABO 9

002 MNS MNS 43 GYPA GYPB GYPE 4

003 P P1 1 P1 22

004 Rh RH 49 RHD RHCE 1

005 Lutheran LU 19 LU 19

006 Kell KEL 25 KEL 7

007 Lewis LE 6 FUT3 19

008 Duffy FY 6 DARC 1

009 Kidd JK 3 SLC14A1 18

22 Biosynthesis of ABH antigens

The biochemical basis of the ABO and H antigens is well understood due to

intensive studies during the 1950s and 1960s by the pioneering work on ovarian cyst

fluids (which contain large amounts of water-soluble blood-group-active glycoproteins)

by Morgan amp Watkins and Kabat The ABO antigens are not limited to erythroid

tissues but are also found in different tissues and on some epithelial cells (13)The

antigens are also present in the secretory fluids in the majority of humans therefore they

7

can sometimes be noted as histo-blood group antigens (14) ABH antigens are

carbohydrate structures These oligosaccharide chains are generally conjugated with

polypeptides to form glycoproteins Oligosaccharides are synthesized in a stepwise

fashion the addition of each monosaccharide being catalyzed by a specific

glycosyltransferase enzyme (15)

The glycoproteins contain a peptide backbone to which multiple oligosaccharide

chains are attached through an alkali-labile glycosidic bond (1617) to the hydroxyl

group of serine or threonine (18) Most of the oligosaccharide chains are linked to the

backbone through an N-acetylgalactosamine residue The carbohydrate moiety of the

ABH glycoproteins consists primarily of four sugars D-galactose L-fucose N-acetyl-

D-galactosamine and N-acetyl-D-glucosamine (19) The amino acid compositions of the

different blood group glycoproteins are similar to each other and unrelated to blood-

group specificity

Expression of H A and B antigens is dependent on the presence of specific

monosaccharides attached to various precursor disaccharides at the non-reducing end of

a carbohydrate chain (14) A transferase product of A allele transfers the

monosaccharide N acetylgalactosamine from the donor substrate uridine diphosphate

(UDP)-N-acetylgalactosamine to the fucosylated galactosyl residue of the H antigen to

produce an active structure The B transferase product of B allele transfers galactose

from UDP-galactose to the fucosylated galactose of H to produce a B-active structure

(20)Individuals with the gene for Glycosyltransferases A have blood group A those

with the gene for Glycosyltransferases B have blood group B those with genes for both

enzymes or a cis-acting form of GTA or GTB have blood type AB and those with a

mutated inactive form of enzyme have blood group O (Figure 21 )

8

Figure 21 Biosynthesis of ABO Antigens (2)

The major alleles at the ABO locus are A B and O and to-date a number of ABO

blood groups variants have been reported with approximately 250 different alleles

registered in the Blood Group antigen gene Mutation database BGMUT (21)

221 H antigen

In nature there exists at least four H antigens on glycolipids and glycoproteins

that are recognized by GTA and GTB (22)The most common are the type I and the type

II H antigens (23)H antigen is produced when an α12-Lfucosyltransferase catalyses the

transfer of L-fucose from a guanosine diphosphate (GDP)-L-fucose donor to the C-2

position of the terminal galactose of one of the precursor structures (Figure 21) Two

α12-L-fucosyltransferases produced by two genes FUT1 (H) and FUT2 (Se) catalyze

9

the biosynthesis of H-active structures in different tissues (24) mainly in epithelial cells

and body fluids such as saliva

The H antigen (Figure 22) is the natural precursor of A and B antigens and its

fucose residue is required for A and B glycosyltransferases to recognize it as the

acceptor and transfer GalNAc or Gal to its terminal Gal Depending on the disaccharide

precursor core chain on which ABH determinants are synthesized they can be further

divided into different types

Figure 22 H antigen structure

Everybody expresses H antigen on his red cells but only about 80 of

Europeans have H antigen in their body secretions These people are called ABH

secretors because if they have an A andor B gene they also secrete A andor B

antigens The remaining 20 are called ABH non-secretors as they do not secrete H A

or B regardless of ABO genotype (2526)

222 A and B antigens

A and B antigens can be produced by the presence of the appropriate A- or B

transferase (Figure 21) The A gene product is an α13-N-acetyl galactosaminyl-

transferase which transfers N-acetyl-galactosamine from a uridine diphosphate (UDP)-

N acetylgalactosamine donor to the fucosylated galactosyl residue of H antigen The B

gene product an α 13-D-galactosyltransferase transfers D-galactose from UDP-

galactose to the fucosylated galactose of H A and B are alleles at the ABO locus on

chromosome 9 A third allele O does not produce an active enzyme and in persons

homozygous for O the H antigen remains unmodified (Figure 23)

31

Figure 23 Summary of A B and O(H) blood group structures and their synthesis(28)

The A and B antigens are carbohydrate molecules built stepwise from

saccharides such as galactosamine (GalNAc) glucosamine (GlcNAc) fucose (Fuc)

galactose (Gal) and glucose (Glc) H-antigen is the requisite precursor and

galactosylamine (GalNAcα3) or galactosyl (Galα3) with α1-3 linkage onto H antigen

become the A and B antigens respectively (514) (Figure 24)

Figure 24 A and B antigen structure

The majority of ABO antigens on red cells are linked to glycoproteins

(approximately 70) thus they very much influence the blood group activity on the red

cells At each step in the biosynthesis of ABO antigens and carbohydrate chains

synthesis is facilitated by glycosyltransferases which are competing for available

precursors and substrates This represents approximately 80 of the total complement

of red cell ABH determinants Another 5 times 105 ABH determinants localize to the red

33

cell glucose transport protein (Band 45) Small numbers of ABH antigens are also

expressed by other red cell glycoproteins (28)

23 ABO Glycosyltransferases

Glycosyltransferases (GTs) constitute a large family of enzymes that are

involved in the biosynthesis of oligosaccharides polysaccharides and glycoconjugates

(29)Particularly abundant are the GTs that transfer a sugar residue from an activated

nucleotide sugar donor to specific acceptor molecules forming glycosidic bonds

Glycosyltransferases are classified into 87 different families based on substrateproduct

stereochemistry according to the CAZy database (30)

Knowledge of the sequences of the ABO genes (9) have established that

mammalian Glycosyltransferases A and Glycosyltransferases B are type II integral

membrane proteins containing 354 amino acids and are localized in the lumen of the

Golgi apparatus These enzymes typically have a short amino-terminal cytoplasmic tail

a hydrophobic membrane domain a short protease-sensitive stem region and a large

catalytic domain that includes the carboxy terminus(31) GTA and GTB are very similar

in the coding regions and the soluble enzyme can be found in serum (32)urine (33)and

milk (34)The enzyme contains an acceptor recognition domain that binds H antigen and

a donor recognition domain that binds UDP-GalNAc or UDP-Gal GTA and GTB

require the metal ion Mn++ (manganese) for activity (35)

Glycosyltransferases are antigenic structures Human antibodies to blood group

transferases are often produced following organ transplantation (36-39)

24 ABO Subgroups

241 A and B Subgroups

An ABO blood group subtype is called a subgroup andor a variant Subgroups

of ABO are distinguished by decreased amounts of A B or O (H) antigens on red blood

cells The most common are subgroups of A and B Blood type A appears to have the

most variation in subgroups The two most common subgroups of blood group A are A1

and A2 expressing on average 1 million and 250 000 A determinants respectively (40)

32

2411 A Subgroups

The A1 (A101) allele is the reference allele often denoted as the consensus

sequence in an ABO genotype context (9)but has additionally eight variant alleles

There are variants of A1 alleles one of which is very common in Asian populations with

a 467CrarrT (A102) polymorphism resulting in the substitution Pro156Leu (41)Two

minor A1 alleles (A103 and A104) have been described and differ as follows The first

one also has the 467CrarrT point mutation but an additional silent mutation 567CrarrT

the second allele contains a silent polymorphism in nucleotide 297ArarrG (42) A105 is

like A102 with the same mutation in exon 7 467CrarrT but analysis of intron 6 showed

additional single-nucleotide polymorphism (SNP) compared to A102 Another A1 allele

(A106) contains both 297ArarrG and 467CrarrT (43)

The A2 subgroup is the most common A phenotype after the A1 subgroup The

main genetic difference between A1 and A2 alleles is one point mutation in exon 7

467CrarrT (Pro156Leu) and a deletion of one of the three cytosines at nt 1059-1061

(CCC to CC) The latter mutation results in an extension of the reading frame by 64

nucleotides This deletion occurs in the codon before the translation stop codon (TGA)

resulting in a gene product with an extra 21 amino acid at its C-terminus The

glycosyltransferases encoded by the A2 allele have lower efficiency leading to a weaker

A phenotype The enzyme activity is decreased by 30-50 times compared to A1

(22)Other variants of A2 alleles have subsequently been elucidated (A202-206) Three

A2-like alleles were shown to have three different single mutations near the 3acute end of

exon 7 by Ogasawara A2-2 (A202) contains 1054CrarrT (Arg352Trp) A2-3 (A203) with

1054CrarrG (Arg352Gly) (42) and A2-4 (A205) with both 467CrarrT and an additional

new mutation 1009ArarrG (Arg337Gly) (44) A204 with four common B-related base

substitutions (297ArarrG 526CrarrG 657CrarrT and 703GrarrA) seems to be a hybrid with

two extra substitutions 771CrarrT (silent mutation) and 829GrarrA (Val277Met) One

other rare A2 subgroup A2-5 (A206) carried only the single deletion (1061delC)

(4546)

31

2412 B Subgroups

Subtypes of blood type B are classified by the quantity of B antigen and the

amount of B antigen decreases in the order B B3 Bx Bm Bel B1 allele (B101)

showed seven single nucleotide substitutions 297 526 657 703 796 803 and 930

throughout exon 6 and 7 and later one extra mutation outside the coding region at the 3acute

end at nt 1096 (33) The four amino acids substitutions governed by nt 526CrarrG

(Arg176Gly) 703GrarrA (Gly235Ser) 796CrarrA (Leu266Met) and 803GrarrC

(Gly268Ala) discriminate GTA from GTB (4147) while the substitutions 297 657 and

930 are silent

ABO glycosyltransferases can accordingly be described by using the letters A

and B to illustrate the derivation of the amino acid at these four residues GTA would be

represented by AAAA indicating the presence of ArgGlyLeuGly and similarly BBBB

would describe the GTB with GlySerMetAla at residues 176235266 and 268

respectively The substitutions at positions 266 and 268 were shown to be responsible

for the nucleotidedonor specificity of the transferases (47) and the other residues may

have a role in acceptor binding and turnover (48)Some other variants have been

reported afterwards that differ from B1 (B101) by lacking the 930 substitution for B2

(B102) the 657 substitution for B3 (B103) (47) the 526 substitution for B4 (B107) (44)

and 297 for B108 (43)

242 O Subgroups

The blood group O demonstrates the absence of A and B antigens on the RBC

surface in forward blood typing Reverse blood typing indicates the presence of both

anti-A and -B in the plasma The first O allele (O1-1 [O01]) was shown to be identical

to the consensus A allele (A101) except for a nucleotide deletion 261delG in exon 6

This results in a shift in the reading frame giving rise to a truncated protein that alters

the protein sequence after amino acid 88 A stop codon halts translation after amino acid

117 and the resulting protein is enzymatically inactive (41) Some other O1-1-like

alleles that are characterized by the presence of the 261delG and at least one additional

point mutation A second kind of O allele has the same inactivating deletion (261delG)

as the original O allele (O1-1 [O01]) but in addition has nine point mutations spread

34

throughout exons 3 to 7 (4145) and a further 13 mutations have been found amongst

the intron 6 (50)

Additional polymorphisms associated with blood group O have been found up to

approximately 4300 bp from ABO exon 7 (51) some of which correlated with O1 and

O1v alleles (52) Other O alleles not due to 261delG also exist that are caused by other

inactivating mutations along the reading frame The ( O2-1 [O03]) was the first allele

described of this type (5354) which has a critical mutation (802G rarr A) causing an

amino acid change (Gly268Arg) that prevents the enzyme from utilizing the nucleotide

sugar donor (55) This O allele comprises approximately 2 to 5 percent of O alleles in

Caucasian persons but seems to be absent or at least very rare in other populations

(5456) Other rare O alleles such as O3 [O08] that does not have 261delG but instead

contains both the common A2 allele polymorphisms 467CrarrT and C-deletion at nt

1059-1061 and an insertion of an extra guanosine in the 7-guanosine sequence at nt

798-804 (45) Two other rare O alleles lacking 261delG were reported in the Japanese

population by Ogasawara O301 [O14] has the missense mutation 893CrarrT (Ala268Val)

on an A102 background whereas O302 [O15] has the nonsense mutation 927CrarrA

(Tyr309Stop) on an A101 background (43)

243 Weak subgroups

In addition to the major phenotypes characterized by either strong or absent

haemagglutination with anti-A-A1 and -B reagents the ABO blood group system also

includes phenotypes in which erythrocytes react weakly with the anti-A and ndashB

reagents for example A3 Ax Afinn Ael B3 Bx Bv Bel cis-AB (57)

It is difficult to determine clearly their specific ABO subgroup by conventional

serological methods The weak subgroups are important in more than one way First

they risk complicating patient and donor blood group determination At worst the

wrong group can be assigned if eg a weak antigen is missed Second they allow us to

characterize and understand glycosyltransferase mechanisms by studying the results of

mutations in the underlying alleles

35

2431 Weak A alleles

The RBCs from individuals with the A3 phenotype agglutinate strongly with

anti-A and anti-AB in vitro but show a large number of free cells One A3B individual

had a novel point mutation 871GrarrA (Asp291Asn) on the A1 [A101] (58) and this

allele was named A301 Ax alleles are responsible for the rare subgroup Ax and the

RBCs typically show a weak positive reaction with anti-AB and anti-H There are four

base substitutions involved in these alleles 646TrarrA (Phe216Ile) 681GrarrA (silent)

771CrarrT (silent) 829GrarrA (Val277Met) The encoded transferase is expected to be 37

aa longer than the normal consensus allele and 16 aa longer than A2-encoded

transferase The serological characteristics of some other minor phenotypes included

among A subgroups eg Aend Am Ay Afinn Abantu and the collective description Aw are

more unclear Table 22 shows the serological reaction patterns of weak A alleles

Table 22 Serological reaction patterns

A negative reaction is noted by 0 and positive reactions are denoted from + (very weak

agglutination) to 4+ (maximal agglutination)mf mixed field agglutination

2432 Weak B alleles

B variants are much more uncommon than A variants this may reflect the

relatively low frequency of the B blood group in many populations These phenotypes

Subgroup

of A

RBC reactions with Anti-A1

in serum

Anti-A Anti-AB Anti-A1 Anti-H

A1 4+ 4+ 4+ 0 No

A2 4+ 4+ 0 4+ Sometimes

Aint 4+ 4+ 2+3+ 2+3+ No

A3 2++mf 2++ mf 0 4+ No

Ax 0+ 2++ 0 4+ Often

Ael 0 0 0 4+ Sometimes

Aend + + 0 4+ Sometimes

Afinn + + 0 4+ Yes

Abantu +(+) +(+) 0 4+ Yes

Am 0+ 0+ 0 4+ No

Ay 0 0 0 4+ No

36

often appear to result from missense mutations at the ABO locus causing single aa

changes in the GTB Characteristics of some B variants are summarized in Table 23

(57)

Table 23 Characteristics of some more frequent B weak phenotypes

mf mixed field w very weak agglutination

The B3 phenotype have missense mutation 1054CrarrT (Arg352Trp) on a B1

(B101) background and named B301 (58) A Bx allele responsible for the Bx phenotype

had a point mutation at nt 871GrarrA (Asp291Asn) (59) which was also found in an A3

sample (58) The Bel phenotype was divided into two suballeles Bel-1 and Bel-2 (Bel01

and Bel02) which had substitutions at 641TrarrG (Met214Arg) and 669GrarrT

(Glu223Asp) respectively (59) these missense mutations reduce the enzymatic

activities of the GTB Ogasawara et al (1996) also found another B3 allele B302 which

differs from B consensus by two nucleotide substitutions 646TrarrA (Phe216Ile) and

657TrarrC

CisAB alleles rare phenotype was described by Seyfried in 1964 It represents a

very interesting phenomenon that proved that it is possible for a child with blood group

O to have a parent with blood group AB Seyfried hypothesized that both A and B

determinants of the AB blood group could be located on the same chromosome Later

on this hypothesis confirmed in that the existence of an exceptional ABO allele

encoding a glycosyltransferase is indeed able to produce both A and B enzymes at the

same time (57) Cis-ABO1 was sequenced and showed the substitution 803GrarrC

(Gly268Ala) on the A1-2 (A102) background and thus can be described as AAAB (60)

Another allele named cis-ABO2 was discovered when a Vietnamese man who was to

undergo organ transplantation showed irregular blood grouping results The sequencing

Subgroup

of B

RBC reactions with Anti-B

in serum

Anti-A Anti-AB Anti-B Anti-H

B 0 ++++ ++++ ++ None

B3 1 mf mf +++ None

Bx 1 w w +++ Yes

Bel 0 w 0w +++ None

Bm 0 0 0 +++ Sometimes

37

showed that his ABO genes were nearly identical to the normal B allele except for a

796ArarrC (Met266Leu) substitution (61)

25 ABO antibodies

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after

stimulation by a specific antigen The antibody formed binds to the specific antigen in

order to mark the antigen for destruction The type of antigenic exposure occurring in

the body determines if the antibody is a naturally occurring or immune antibody The

term lsquonaturally occurringrsquo is used for blood group antibodies produced in individuals

who have never been transfused with red cells carrying the relevant antigen or been

pregnant with a fetus carrying the relevant antigen Naturally occurring antibodies can

be formed after exposure to environmental agents that are similar to red cell antigens

such as bacteria dust or pollen (62)

Most of these antibodies are not clinically significant with the exception of ABO

antibodies It is possible that food and environmental antigens (bacterial viral or plant

antigens) have epitopes similar enough to A and B glycoprotein antigens The

antibodies created against these environmental antigens in the first years of life can

cross-react with ABO-incompatible red blood cells (RBCs) that it comes in contact with

during blood transfusion later in life Anti-A antibodies are hypothesized to originate

from immune response towards influenza virus whose epitopes are similar enough to

the α-D-N-galactosamine on the A glycoprotein to be able to elicit a cross-reaction

Anti-B antibodies are hypothesized to originate from antibodies produced against

Gram-negative bacteria such as E coli cross-reacting with the α-D-galactose on the B

glycoprotein (49) Anti-A and anti-B antibodies (called isohaemagglutinins) which are

not present in the newborn appear in the first years of life They are isoantibodies that

are produced by an individual against antigens produced by members of the same

species (isoantigens) Anti-A and anti-B antibodies are usually IgM type which are not

able to pass through the placenta to the fetal blood circulation and react best at room

temperature or lower The ABO antibodies may also be found in various body fluids

including saliva milk cervical secretions tears and cysts These antibodies are

detected at about 3 months and increase their titer until the 5th

to 10th

year of life

3

CHAPTER 1

INTRODUCTION

2

CHAPTER 1

INTRODUCTION

11 Background

In clinical practice the ABO blood group system is one of the most important

since the A and B epitopes may provoke a strong immune reaction With the

introduction of blood typing and cross-matching techniques blood transfusion became

not only a simple but also a much safer procedure Furthermore although ABO typing

reduced the occurrence of transfusion reactions they still occurred indicating the

presence of other genetic differences in blood groups of importance in transfusion

medicine as well as in the later emerging field of organ transplantation(1)

Human ABO locus is located in chromosome 9q341-q342 (2-5) and consists of 7 exons

distributed over 18 kb of genomic DNA ranging in size from 28 to 688 base pairs (bp)

and 6 introns with 554 to 12982 bp (6-8) (Figure 11) Exon 7 contains most of the

largest coding sequence whereas exon 6 contains the deletion found in most O alleles

(9)

Figure 11 Schematic representation of the genomic organization of the ABO gene (10)

1

Molecular genetic studies of human ABO genes have demonstrated that ABO genes

have two critical single base substitutions in the last coding exon that result in amino

acid substitutions responsible for the different donor nucleotide sugar substrate

specificity between A- and B-transferases A single base deletion in exon 6 was

ascribed to shift the reading frame of codons and to abolish the transferase activity of A-

transferase in most O alleles (45911)

ABO genotyping is important not only for blood transfusion but also for tissuecell and

organ transplantations Also ABO genotypes are important evidence at crime scenes

and for personal identification in forensic investigations and paternity testing

To our knowledge this is the first study in Gaza Strip investigating the ABO gene

polymorphism In this study multiplex allele-specific PCR is used for determining the

ABO genotypes and the corresponding allele frequency in a group of 201 unrelated

Palestinians residing in Gaza Strip

4

12 Objectives of the Study

121 General Objective

To determine the major ABO alleles and genotypes frequencies in a Palestinian

population residing in Gaza Strip

122 Specific objectives

1- To employ PCR and Allele-specific (AS)-PCR for molecular genotyping of ABO

blood system

2- To correlate ABO genotypes with phenotypes in blood samples of Gaza Strip

population

3- To compare the frequency of ABO genotypes with other populations

5

CHAPTER 2

LITIRATURE REVIEW

6

CHAPTER 2

LITIRATURE REVIEW

21 Background

The International Society of Blood Transfusion (ISBT) Working Committee on

Terminology for Red Cell Surface Antigens was set up in 1980 to establish and define a

meaningful nomenclature for different blood groups (12) Every valid blood group

antigen is given a six digit identification number There are 29 different systems to date

and the first three digits represent the systems (001-029)

The symbol for a gene or cluster of genes controlling a blood group system is

often the italicized symbol for the system The ABO genotypes should consequently be

written in italicized capital letters (Table 21)

Table 21 Some of Human blood group systems recognized by the ISBT (12)

No Name Symbol No of

antigens

Gene name(s) Chromosome

001 ABO ABO 4 ABO 9

002 MNS MNS 43 GYPA GYPB GYPE 4

003 P P1 1 P1 22

004 Rh RH 49 RHD RHCE 1

005 Lutheran LU 19 LU 19

006 Kell KEL 25 KEL 7

007 Lewis LE 6 FUT3 19

008 Duffy FY 6 DARC 1

009 Kidd JK 3 SLC14A1 18

22 Biosynthesis of ABH antigens

The biochemical basis of the ABO and H antigens is well understood due to

intensive studies during the 1950s and 1960s by the pioneering work on ovarian cyst

fluids (which contain large amounts of water-soluble blood-group-active glycoproteins)

by Morgan amp Watkins and Kabat The ABO antigens are not limited to erythroid

tissues but are also found in different tissues and on some epithelial cells (13)The

antigens are also present in the secretory fluids in the majority of humans therefore they

7

can sometimes be noted as histo-blood group antigens (14) ABH antigens are

carbohydrate structures These oligosaccharide chains are generally conjugated with

polypeptides to form glycoproteins Oligosaccharides are synthesized in a stepwise

fashion the addition of each monosaccharide being catalyzed by a specific

glycosyltransferase enzyme (15)

The glycoproteins contain a peptide backbone to which multiple oligosaccharide

chains are attached through an alkali-labile glycosidic bond (1617) to the hydroxyl

group of serine or threonine (18) Most of the oligosaccharide chains are linked to the

backbone through an N-acetylgalactosamine residue The carbohydrate moiety of the

ABH glycoproteins consists primarily of four sugars D-galactose L-fucose N-acetyl-

D-galactosamine and N-acetyl-D-glucosamine (19) The amino acid compositions of the

different blood group glycoproteins are similar to each other and unrelated to blood-

group specificity

Expression of H A and B antigens is dependent on the presence of specific

monosaccharides attached to various precursor disaccharides at the non-reducing end of

a carbohydrate chain (14) A transferase product of A allele transfers the

monosaccharide N acetylgalactosamine from the donor substrate uridine diphosphate

(UDP)-N-acetylgalactosamine to the fucosylated galactosyl residue of the H antigen to

produce an active structure The B transferase product of B allele transfers galactose

from UDP-galactose to the fucosylated galactose of H to produce a B-active structure

(20)Individuals with the gene for Glycosyltransferases A have blood group A those

with the gene for Glycosyltransferases B have blood group B those with genes for both

enzymes or a cis-acting form of GTA or GTB have blood type AB and those with a

mutated inactive form of enzyme have blood group O (Figure 21 )

8

Figure 21 Biosynthesis of ABO Antigens (2)

The major alleles at the ABO locus are A B and O and to-date a number of ABO

blood groups variants have been reported with approximately 250 different alleles

registered in the Blood Group antigen gene Mutation database BGMUT (21)

221 H antigen

In nature there exists at least four H antigens on glycolipids and glycoproteins

that are recognized by GTA and GTB (22)The most common are the type I and the type

II H antigens (23)H antigen is produced when an α12-Lfucosyltransferase catalyses the

transfer of L-fucose from a guanosine diphosphate (GDP)-L-fucose donor to the C-2

position of the terminal galactose of one of the precursor structures (Figure 21) Two

α12-L-fucosyltransferases produced by two genes FUT1 (H) and FUT2 (Se) catalyze

9

the biosynthesis of H-active structures in different tissues (24) mainly in epithelial cells

and body fluids such as saliva

The H antigen (Figure 22) is the natural precursor of A and B antigens and its

fucose residue is required for A and B glycosyltransferases to recognize it as the

acceptor and transfer GalNAc or Gal to its terminal Gal Depending on the disaccharide

precursor core chain on which ABH determinants are synthesized they can be further

divided into different types

Figure 22 H antigen structure

Everybody expresses H antigen on his red cells but only about 80 of

Europeans have H antigen in their body secretions These people are called ABH

secretors because if they have an A andor B gene they also secrete A andor B

antigens The remaining 20 are called ABH non-secretors as they do not secrete H A

or B regardless of ABO genotype (2526)

222 A and B antigens

A and B antigens can be produced by the presence of the appropriate A- or B

transferase (Figure 21) The A gene product is an α13-N-acetyl galactosaminyl-

transferase which transfers N-acetyl-galactosamine from a uridine diphosphate (UDP)-

N acetylgalactosamine donor to the fucosylated galactosyl residue of H antigen The B

gene product an α 13-D-galactosyltransferase transfers D-galactose from UDP-

galactose to the fucosylated galactose of H A and B are alleles at the ABO locus on

chromosome 9 A third allele O does not produce an active enzyme and in persons

homozygous for O the H antigen remains unmodified (Figure 23)

31

Figure 23 Summary of A B and O(H) blood group structures and their synthesis(28)

The A and B antigens are carbohydrate molecules built stepwise from

saccharides such as galactosamine (GalNAc) glucosamine (GlcNAc) fucose (Fuc)

galactose (Gal) and glucose (Glc) H-antigen is the requisite precursor and

galactosylamine (GalNAcα3) or galactosyl (Galα3) with α1-3 linkage onto H antigen

become the A and B antigens respectively (514) (Figure 24)

Figure 24 A and B antigen structure

The majority of ABO antigens on red cells are linked to glycoproteins

(approximately 70) thus they very much influence the blood group activity on the red

cells At each step in the biosynthesis of ABO antigens and carbohydrate chains

synthesis is facilitated by glycosyltransferases which are competing for available

precursors and substrates This represents approximately 80 of the total complement

of red cell ABH determinants Another 5 times 105 ABH determinants localize to the red

33

cell glucose transport protein (Band 45) Small numbers of ABH antigens are also

expressed by other red cell glycoproteins (28)

23 ABO Glycosyltransferases

Glycosyltransferases (GTs) constitute a large family of enzymes that are

involved in the biosynthesis of oligosaccharides polysaccharides and glycoconjugates

(29)Particularly abundant are the GTs that transfer a sugar residue from an activated

nucleotide sugar donor to specific acceptor molecules forming glycosidic bonds

Glycosyltransferases are classified into 87 different families based on substrateproduct

stereochemistry according to the CAZy database (30)

Knowledge of the sequences of the ABO genes (9) have established that

mammalian Glycosyltransferases A and Glycosyltransferases B are type II integral

membrane proteins containing 354 amino acids and are localized in the lumen of the

Golgi apparatus These enzymes typically have a short amino-terminal cytoplasmic tail

a hydrophobic membrane domain a short protease-sensitive stem region and a large

catalytic domain that includes the carboxy terminus(31) GTA and GTB are very similar

in the coding regions and the soluble enzyme can be found in serum (32)urine (33)and

milk (34)The enzyme contains an acceptor recognition domain that binds H antigen and

a donor recognition domain that binds UDP-GalNAc or UDP-Gal GTA and GTB

require the metal ion Mn++ (manganese) for activity (35)

Glycosyltransferases are antigenic structures Human antibodies to blood group

transferases are often produced following organ transplantation (36-39)

24 ABO Subgroups

241 A and B Subgroups

An ABO blood group subtype is called a subgroup andor a variant Subgroups

of ABO are distinguished by decreased amounts of A B or O (H) antigens on red blood

cells The most common are subgroups of A and B Blood type A appears to have the

most variation in subgroups The two most common subgroups of blood group A are A1

and A2 expressing on average 1 million and 250 000 A determinants respectively (40)

32

2411 A Subgroups

The A1 (A101) allele is the reference allele often denoted as the consensus

sequence in an ABO genotype context (9)but has additionally eight variant alleles

There are variants of A1 alleles one of which is very common in Asian populations with

a 467CrarrT (A102) polymorphism resulting in the substitution Pro156Leu (41)Two

minor A1 alleles (A103 and A104) have been described and differ as follows The first

one also has the 467CrarrT point mutation but an additional silent mutation 567CrarrT

the second allele contains a silent polymorphism in nucleotide 297ArarrG (42) A105 is

like A102 with the same mutation in exon 7 467CrarrT but analysis of intron 6 showed

additional single-nucleotide polymorphism (SNP) compared to A102 Another A1 allele

(A106) contains both 297ArarrG and 467CrarrT (43)

The A2 subgroup is the most common A phenotype after the A1 subgroup The

main genetic difference between A1 and A2 alleles is one point mutation in exon 7

467CrarrT (Pro156Leu) and a deletion of one of the three cytosines at nt 1059-1061

(CCC to CC) The latter mutation results in an extension of the reading frame by 64

nucleotides This deletion occurs in the codon before the translation stop codon (TGA)

resulting in a gene product with an extra 21 amino acid at its C-terminus The

glycosyltransferases encoded by the A2 allele have lower efficiency leading to a weaker

A phenotype The enzyme activity is decreased by 30-50 times compared to A1

(22)Other variants of A2 alleles have subsequently been elucidated (A202-206) Three

A2-like alleles were shown to have three different single mutations near the 3acute end of

exon 7 by Ogasawara A2-2 (A202) contains 1054CrarrT (Arg352Trp) A2-3 (A203) with

1054CrarrG (Arg352Gly) (42) and A2-4 (A205) with both 467CrarrT and an additional

new mutation 1009ArarrG (Arg337Gly) (44) A204 with four common B-related base

substitutions (297ArarrG 526CrarrG 657CrarrT and 703GrarrA) seems to be a hybrid with

two extra substitutions 771CrarrT (silent mutation) and 829GrarrA (Val277Met) One

other rare A2 subgroup A2-5 (A206) carried only the single deletion (1061delC)

(4546)

31

2412 B Subgroups

Subtypes of blood type B are classified by the quantity of B antigen and the

amount of B antigen decreases in the order B B3 Bx Bm Bel B1 allele (B101)

showed seven single nucleotide substitutions 297 526 657 703 796 803 and 930

throughout exon 6 and 7 and later one extra mutation outside the coding region at the 3acute

end at nt 1096 (33) The four amino acids substitutions governed by nt 526CrarrG

(Arg176Gly) 703GrarrA (Gly235Ser) 796CrarrA (Leu266Met) and 803GrarrC

(Gly268Ala) discriminate GTA from GTB (4147) while the substitutions 297 657 and

930 are silent

ABO glycosyltransferases can accordingly be described by using the letters A

and B to illustrate the derivation of the amino acid at these four residues GTA would be

represented by AAAA indicating the presence of ArgGlyLeuGly and similarly BBBB

would describe the GTB with GlySerMetAla at residues 176235266 and 268

respectively The substitutions at positions 266 and 268 were shown to be responsible

for the nucleotidedonor specificity of the transferases (47) and the other residues may

have a role in acceptor binding and turnover (48)Some other variants have been

reported afterwards that differ from B1 (B101) by lacking the 930 substitution for B2

(B102) the 657 substitution for B3 (B103) (47) the 526 substitution for B4 (B107) (44)

and 297 for B108 (43)

242 O Subgroups

The blood group O demonstrates the absence of A and B antigens on the RBC

surface in forward blood typing Reverse blood typing indicates the presence of both

anti-A and -B in the plasma The first O allele (O1-1 [O01]) was shown to be identical

to the consensus A allele (A101) except for a nucleotide deletion 261delG in exon 6

This results in a shift in the reading frame giving rise to a truncated protein that alters

the protein sequence after amino acid 88 A stop codon halts translation after amino acid

117 and the resulting protein is enzymatically inactive (41) Some other O1-1-like

alleles that are characterized by the presence of the 261delG and at least one additional

point mutation A second kind of O allele has the same inactivating deletion (261delG)

as the original O allele (O1-1 [O01]) but in addition has nine point mutations spread

34

throughout exons 3 to 7 (4145) and a further 13 mutations have been found amongst

the intron 6 (50)

Additional polymorphisms associated with blood group O have been found up to

approximately 4300 bp from ABO exon 7 (51) some of which correlated with O1 and

O1v alleles (52) Other O alleles not due to 261delG also exist that are caused by other

inactivating mutations along the reading frame The ( O2-1 [O03]) was the first allele

described of this type (5354) which has a critical mutation (802G rarr A) causing an

amino acid change (Gly268Arg) that prevents the enzyme from utilizing the nucleotide

sugar donor (55) This O allele comprises approximately 2 to 5 percent of O alleles in

Caucasian persons but seems to be absent or at least very rare in other populations

(5456) Other rare O alleles such as O3 [O08] that does not have 261delG but instead

contains both the common A2 allele polymorphisms 467CrarrT and C-deletion at nt

1059-1061 and an insertion of an extra guanosine in the 7-guanosine sequence at nt

798-804 (45) Two other rare O alleles lacking 261delG were reported in the Japanese

population by Ogasawara O301 [O14] has the missense mutation 893CrarrT (Ala268Val)

on an A102 background whereas O302 [O15] has the nonsense mutation 927CrarrA

(Tyr309Stop) on an A101 background (43)

243 Weak subgroups

In addition to the major phenotypes characterized by either strong or absent

haemagglutination with anti-A-A1 and -B reagents the ABO blood group system also

includes phenotypes in which erythrocytes react weakly with the anti-A and ndashB

reagents for example A3 Ax Afinn Ael B3 Bx Bv Bel cis-AB (57)

It is difficult to determine clearly their specific ABO subgroup by conventional

serological methods The weak subgroups are important in more than one way First

they risk complicating patient and donor blood group determination At worst the

wrong group can be assigned if eg a weak antigen is missed Second they allow us to

characterize and understand glycosyltransferase mechanisms by studying the results of

mutations in the underlying alleles

35

2431 Weak A alleles

The RBCs from individuals with the A3 phenotype agglutinate strongly with

anti-A and anti-AB in vitro but show a large number of free cells One A3B individual

had a novel point mutation 871GrarrA (Asp291Asn) on the A1 [A101] (58) and this

allele was named A301 Ax alleles are responsible for the rare subgroup Ax and the

RBCs typically show a weak positive reaction with anti-AB and anti-H There are four

base substitutions involved in these alleles 646TrarrA (Phe216Ile) 681GrarrA (silent)

771CrarrT (silent) 829GrarrA (Val277Met) The encoded transferase is expected to be 37

aa longer than the normal consensus allele and 16 aa longer than A2-encoded

transferase The serological characteristics of some other minor phenotypes included

among A subgroups eg Aend Am Ay Afinn Abantu and the collective description Aw are

more unclear Table 22 shows the serological reaction patterns of weak A alleles

Table 22 Serological reaction patterns

A negative reaction is noted by 0 and positive reactions are denoted from + (very weak

agglutination) to 4+ (maximal agglutination)mf mixed field agglutination

2432 Weak B alleles

B variants are much more uncommon than A variants this may reflect the

relatively low frequency of the B blood group in many populations These phenotypes

Subgroup

of A

RBC reactions with Anti-A1

in serum

Anti-A Anti-AB Anti-A1 Anti-H

A1 4+ 4+ 4+ 0 No

A2 4+ 4+ 0 4+ Sometimes

Aint 4+ 4+ 2+3+ 2+3+ No

A3 2++mf 2++ mf 0 4+ No

Ax 0+ 2++ 0 4+ Often

Ael 0 0 0 4+ Sometimes

Aend + + 0 4+ Sometimes

Afinn + + 0 4+ Yes

Abantu +(+) +(+) 0 4+ Yes

Am 0+ 0+ 0 4+ No

Ay 0 0 0 4+ No

36

often appear to result from missense mutations at the ABO locus causing single aa

changes in the GTB Characteristics of some B variants are summarized in Table 23

(57)

Table 23 Characteristics of some more frequent B weak phenotypes

mf mixed field w very weak agglutination

The B3 phenotype have missense mutation 1054CrarrT (Arg352Trp) on a B1

(B101) background and named B301 (58) A Bx allele responsible for the Bx phenotype

had a point mutation at nt 871GrarrA (Asp291Asn) (59) which was also found in an A3

sample (58) The Bel phenotype was divided into two suballeles Bel-1 and Bel-2 (Bel01

and Bel02) which had substitutions at 641TrarrG (Met214Arg) and 669GrarrT

(Glu223Asp) respectively (59) these missense mutations reduce the enzymatic

activities of the GTB Ogasawara et al (1996) also found another B3 allele B302 which

differs from B consensus by two nucleotide substitutions 646TrarrA (Phe216Ile) and

657TrarrC

CisAB alleles rare phenotype was described by Seyfried in 1964 It represents a

very interesting phenomenon that proved that it is possible for a child with blood group

O to have a parent with blood group AB Seyfried hypothesized that both A and B

determinants of the AB blood group could be located on the same chromosome Later

on this hypothesis confirmed in that the existence of an exceptional ABO allele

encoding a glycosyltransferase is indeed able to produce both A and B enzymes at the

same time (57) Cis-ABO1 was sequenced and showed the substitution 803GrarrC

(Gly268Ala) on the A1-2 (A102) background and thus can be described as AAAB (60)

Another allele named cis-ABO2 was discovered when a Vietnamese man who was to

undergo organ transplantation showed irregular blood grouping results The sequencing

Subgroup

of B

RBC reactions with Anti-B

in serum

Anti-A Anti-AB Anti-B Anti-H

B 0 ++++ ++++ ++ None

B3 1 mf mf +++ None

Bx 1 w w +++ Yes

Bel 0 w 0w +++ None

Bm 0 0 0 +++ Sometimes

37

showed that his ABO genes were nearly identical to the normal B allele except for a

796ArarrC (Met266Leu) substitution (61)

25 ABO antibodies

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after

stimulation by a specific antigen The antibody formed binds to the specific antigen in

order to mark the antigen for destruction The type of antigenic exposure occurring in

the body determines if the antibody is a naturally occurring or immune antibody The

term lsquonaturally occurringrsquo is used for blood group antibodies produced in individuals

who have never been transfused with red cells carrying the relevant antigen or been

pregnant with a fetus carrying the relevant antigen Naturally occurring antibodies can

be formed after exposure to environmental agents that are similar to red cell antigens

such as bacteria dust or pollen (62)

Most of these antibodies are not clinically significant with the exception of ABO

antibodies It is possible that food and environmental antigens (bacterial viral or plant

antigens) have epitopes similar enough to A and B glycoprotein antigens The

antibodies created against these environmental antigens in the first years of life can

cross-react with ABO-incompatible red blood cells (RBCs) that it comes in contact with

during blood transfusion later in life Anti-A antibodies are hypothesized to originate

from immune response towards influenza virus whose epitopes are similar enough to

the α-D-N-galactosamine on the A glycoprotein to be able to elicit a cross-reaction

Anti-B antibodies are hypothesized to originate from antibodies produced against

Gram-negative bacteria such as E coli cross-reacting with the α-D-galactose on the B

glycoprotein (49) Anti-A and anti-B antibodies (called isohaemagglutinins) which are

not present in the newborn appear in the first years of life They are isoantibodies that

are produced by an individual against antigens produced by members of the same

species (isoantigens) Anti-A and anti-B antibodies are usually IgM type which are not

able to pass through the placenta to the fetal blood circulation and react best at room

temperature or lower The ABO antibodies may also be found in various body fluids

including saliva milk cervical secretions tears and cysts These antibodies are

detected at about 3 months and increase their titer until the 5th

to 10th

year of life

2

CHAPTER 1

INTRODUCTION

11 Background

In clinical practice the ABO blood group system is one of the most important

since the A and B epitopes may provoke a strong immune reaction With the

introduction of blood typing and cross-matching techniques blood transfusion became

not only a simple but also a much safer procedure Furthermore although ABO typing

reduced the occurrence of transfusion reactions they still occurred indicating the

presence of other genetic differences in blood groups of importance in transfusion

medicine as well as in the later emerging field of organ transplantation(1)

Human ABO locus is located in chromosome 9q341-q342 (2-5) and consists of 7 exons

distributed over 18 kb of genomic DNA ranging in size from 28 to 688 base pairs (bp)

and 6 introns with 554 to 12982 bp (6-8) (Figure 11) Exon 7 contains most of the

largest coding sequence whereas exon 6 contains the deletion found in most O alleles

(9)

Figure 11 Schematic representation of the genomic organization of the ABO gene (10)

1

Molecular genetic studies of human ABO genes have demonstrated that ABO genes

have two critical single base substitutions in the last coding exon that result in amino

acid substitutions responsible for the different donor nucleotide sugar substrate

specificity between A- and B-transferases A single base deletion in exon 6 was

ascribed to shift the reading frame of codons and to abolish the transferase activity of A-

transferase in most O alleles (45911)

ABO genotyping is important not only for blood transfusion but also for tissuecell and

organ transplantations Also ABO genotypes are important evidence at crime scenes

and for personal identification in forensic investigations and paternity testing

To our knowledge this is the first study in Gaza Strip investigating the ABO gene

polymorphism In this study multiplex allele-specific PCR is used for determining the

ABO genotypes and the corresponding allele frequency in a group of 201 unrelated

Palestinians residing in Gaza Strip

4

12 Objectives of the Study

121 General Objective

To determine the major ABO alleles and genotypes frequencies in a Palestinian

population residing in Gaza Strip

122 Specific objectives

1- To employ PCR and Allele-specific (AS)-PCR for molecular genotyping of ABO

blood system

2- To correlate ABO genotypes with phenotypes in blood samples of Gaza Strip

population

3- To compare the frequency of ABO genotypes with other populations

5

CHAPTER 2

LITIRATURE REVIEW

6

CHAPTER 2

LITIRATURE REVIEW

21 Background

The International Society of Blood Transfusion (ISBT) Working Committee on

Terminology for Red Cell Surface Antigens was set up in 1980 to establish and define a

meaningful nomenclature for different blood groups (12) Every valid blood group

antigen is given a six digit identification number There are 29 different systems to date

and the first three digits represent the systems (001-029)

The symbol for a gene or cluster of genes controlling a blood group system is

often the italicized symbol for the system The ABO genotypes should consequently be

written in italicized capital letters (Table 21)

Table 21 Some of Human blood group systems recognized by the ISBT (12)

No Name Symbol No of

antigens

Gene name(s) Chromosome

001 ABO ABO 4 ABO 9

002 MNS MNS 43 GYPA GYPB GYPE 4

003 P P1 1 P1 22

004 Rh RH 49 RHD RHCE 1

005 Lutheran LU 19 LU 19

006 Kell KEL 25 KEL 7

007 Lewis LE 6 FUT3 19

008 Duffy FY 6 DARC 1

009 Kidd JK 3 SLC14A1 18

22 Biosynthesis of ABH antigens

The biochemical basis of the ABO and H antigens is well understood due to

intensive studies during the 1950s and 1960s by the pioneering work on ovarian cyst

fluids (which contain large amounts of water-soluble blood-group-active glycoproteins)

by Morgan amp Watkins and Kabat The ABO antigens are not limited to erythroid

tissues but are also found in different tissues and on some epithelial cells (13)The

antigens are also present in the secretory fluids in the majority of humans therefore they

7

can sometimes be noted as histo-blood group antigens (14) ABH antigens are

carbohydrate structures These oligosaccharide chains are generally conjugated with

polypeptides to form glycoproteins Oligosaccharides are synthesized in a stepwise

fashion the addition of each monosaccharide being catalyzed by a specific

glycosyltransferase enzyme (15)

The glycoproteins contain a peptide backbone to which multiple oligosaccharide

chains are attached through an alkali-labile glycosidic bond (1617) to the hydroxyl

group of serine or threonine (18) Most of the oligosaccharide chains are linked to the

backbone through an N-acetylgalactosamine residue The carbohydrate moiety of the

ABH glycoproteins consists primarily of four sugars D-galactose L-fucose N-acetyl-

D-galactosamine and N-acetyl-D-glucosamine (19) The amino acid compositions of the

different blood group glycoproteins are similar to each other and unrelated to blood-

group specificity

Expression of H A and B antigens is dependent on the presence of specific

monosaccharides attached to various precursor disaccharides at the non-reducing end of

a carbohydrate chain (14) A transferase product of A allele transfers the

monosaccharide N acetylgalactosamine from the donor substrate uridine diphosphate

(UDP)-N-acetylgalactosamine to the fucosylated galactosyl residue of the H antigen to

produce an active structure The B transferase product of B allele transfers galactose

from UDP-galactose to the fucosylated galactose of H to produce a B-active structure

(20)Individuals with the gene for Glycosyltransferases A have blood group A those

with the gene for Glycosyltransferases B have blood group B those with genes for both

enzymes or a cis-acting form of GTA or GTB have blood type AB and those with a

mutated inactive form of enzyme have blood group O (Figure 21 )

8

Figure 21 Biosynthesis of ABO Antigens (2)

The major alleles at the ABO locus are A B and O and to-date a number of ABO

blood groups variants have been reported with approximately 250 different alleles

registered in the Blood Group antigen gene Mutation database BGMUT (21)

221 H antigen

In nature there exists at least four H antigens on glycolipids and glycoproteins

that are recognized by GTA and GTB (22)The most common are the type I and the type

II H antigens (23)H antigen is produced when an α12-Lfucosyltransferase catalyses the

transfer of L-fucose from a guanosine diphosphate (GDP)-L-fucose donor to the C-2

position of the terminal galactose of one of the precursor structures (Figure 21) Two

α12-L-fucosyltransferases produced by two genes FUT1 (H) and FUT2 (Se) catalyze

9

the biosynthesis of H-active structures in different tissues (24) mainly in epithelial cells

and body fluids such as saliva

The H antigen (Figure 22) is the natural precursor of A and B antigens and its

fucose residue is required for A and B glycosyltransferases to recognize it as the

acceptor and transfer GalNAc or Gal to its terminal Gal Depending on the disaccharide

precursor core chain on which ABH determinants are synthesized they can be further

divided into different types

Figure 22 H antigen structure

Everybody expresses H antigen on his red cells but only about 80 of

Europeans have H antigen in their body secretions These people are called ABH

secretors because if they have an A andor B gene they also secrete A andor B

antigens The remaining 20 are called ABH non-secretors as they do not secrete H A

or B regardless of ABO genotype (2526)

222 A and B antigens

A and B antigens can be produced by the presence of the appropriate A- or B

transferase (Figure 21) The A gene product is an α13-N-acetyl galactosaminyl-

transferase which transfers N-acetyl-galactosamine from a uridine diphosphate (UDP)-

N acetylgalactosamine donor to the fucosylated galactosyl residue of H antigen The B

gene product an α 13-D-galactosyltransferase transfers D-galactose from UDP-

galactose to the fucosylated galactose of H A and B are alleles at the ABO locus on

chromosome 9 A third allele O does not produce an active enzyme and in persons

homozygous for O the H antigen remains unmodified (Figure 23)

31

Figure 23 Summary of A B and O(H) blood group structures and their synthesis(28)

The A and B antigens are carbohydrate molecules built stepwise from

saccharides such as galactosamine (GalNAc) glucosamine (GlcNAc) fucose (Fuc)

galactose (Gal) and glucose (Glc) H-antigen is the requisite precursor and

galactosylamine (GalNAcα3) or galactosyl (Galα3) with α1-3 linkage onto H antigen

become the A and B antigens respectively (514) (Figure 24)

Figure 24 A and B antigen structure

The majority of ABO antigens on red cells are linked to glycoproteins

(approximately 70) thus they very much influence the blood group activity on the red

cells At each step in the biosynthesis of ABO antigens and carbohydrate chains

synthesis is facilitated by glycosyltransferases which are competing for available

precursors and substrates This represents approximately 80 of the total complement

of red cell ABH determinants Another 5 times 105 ABH determinants localize to the red

33

cell glucose transport protein (Band 45) Small numbers of ABH antigens are also

expressed by other red cell glycoproteins (28)

23 ABO Glycosyltransferases

Glycosyltransferases (GTs) constitute a large family of enzymes that are

involved in the biosynthesis of oligosaccharides polysaccharides and glycoconjugates

(29)Particularly abundant are the GTs that transfer a sugar residue from an activated

nucleotide sugar donor to specific acceptor molecules forming glycosidic bonds

Glycosyltransferases are classified into 87 different families based on substrateproduct

stereochemistry according to the CAZy database (30)

Knowledge of the sequences of the ABO genes (9) have established that

mammalian Glycosyltransferases A and Glycosyltransferases B are type II integral

membrane proteins containing 354 amino acids and are localized in the lumen of the

Golgi apparatus These enzymes typically have a short amino-terminal cytoplasmic tail

a hydrophobic membrane domain a short protease-sensitive stem region and a large

catalytic domain that includes the carboxy terminus(31) GTA and GTB are very similar

in the coding regions and the soluble enzyme can be found in serum (32)urine (33)and

milk (34)The enzyme contains an acceptor recognition domain that binds H antigen and

a donor recognition domain that binds UDP-GalNAc or UDP-Gal GTA and GTB

require the metal ion Mn++ (manganese) for activity (35)

Glycosyltransferases are antigenic structures Human antibodies to blood group

transferases are often produced following organ transplantation (36-39)

24 ABO Subgroups

241 A and B Subgroups

An ABO blood group subtype is called a subgroup andor a variant Subgroups

of ABO are distinguished by decreased amounts of A B or O (H) antigens on red blood

cells The most common are subgroups of A and B Blood type A appears to have the

most variation in subgroups The two most common subgroups of blood group A are A1

and A2 expressing on average 1 million and 250 000 A determinants respectively (40)

32

2411 A Subgroups

The A1 (A101) allele is the reference allele often denoted as the consensus

sequence in an ABO genotype context (9)but has additionally eight variant alleles

There are variants of A1 alleles one of which is very common in Asian populations with

a 467CrarrT (A102) polymorphism resulting in the substitution Pro156Leu (41)Two

minor A1 alleles (A103 and A104) have been described and differ as follows The first

one also has the 467CrarrT point mutation but an additional silent mutation 567CrarrT

the second allele contains a silent polymorphism in nucleotide 297ArarrG (42) A105 is

like A102 with the same mutation in exon 7 467CrarrT but analysis of intron 6 showed

additional single-nucleotide polymorphism (SNP) compared to A102 Another A1 allele

(A106) contains both 297ArarrG and 467CrarrT (43)

The A2 subgroup is the most common A phenotype after the A1 subgroup The

main genetic difference between A1 and A2 alleles is one point mutation in exon 7

467CrarrT (Pro156Leu) and a deletion of one of the three cytosines at nt 1059-1061

(CCC to CC) The latter mutation results in an extension of the reading frame by 64

nucleotides This deletion occurs in the codon before the translation stop codon (TGA)

resulting in a gene product with an extra 21 amino acid at its C-terminus The

glycosyltransferases encoded by the A2 allele have lower efficiency leading to a weaker

A phenotype The enzyme activity is decreased by 30-50 times compared to A1

(22)Other variants of A2 alleles have subsequently been elucidated (A202-206) Three

A2-like alleles were shown to have three different single mutations near the 3acute end of

exon 7 by Ogasawara A2-2 (A202) contains 1054CrarrT (Arg352Trp) A2-3 (A203) with

1054CrarrG (Arg352Gly) (42) and A2-4 (A205) with both 467CrarrT and an additional

new mutation 1009ArarrG (Arg337Gly) (44) A204 with four common B-related base

substitutions (297ArarrG 526CrarrG 657CrarrT and 703GrarrA) seems to be a hybrid with

two extra substitutions 771CrarrT (silent mutation) and 829GrarrA (Val277Met) One

other rare A2 subgroup A2-5 (A206) carried only the single deletion (1061delC)

(4546)

31

2412 B Subgroups

Subtypes of blood type B are classified by the quantity of B antigen and the

amount of B antigen decreases in the order B B3 Bx Bm Bel B1 allele (B101)

showed seven single nucleotide substitutions 297 526 657 703 796 803 and 930

throughout exon 6 and 7 and later one extra mutation outside the coding region at the 3acute

end at nt 1096 (33) The four amino acids substitutions governed by nt 526CrarrG

(Arg176Gly) 703GrarrA (Gly235Ser) 796CrarrA (Leu266Met) and 803GrarrC

(Gly268Ala) discriminate GTA from GTB (4147) while the substitutions 297 657 and

930 are silent

ABO glycosyltransferases can accordingly be described by using the letters A

and B to illustrate the derivation of the amino acid at these four residues GTA would be

represented by AAAA indicating the presence of ArgGlyLeuGly and similarly BBBB

would describe the GTB with GlySerMetAla at residues 176235266 and 268

respectively The substitutions at positions 266 and 268 were shown to be responsible

for the nucleotidedonor specificity of the transferases (47) and the other residues may

have a role in acceptor binding and turnover (48)Some other variants have been

reported afterwards that differ from B1 (B101) by lacking the 930 substitution for B2

(B102) the 657 substitution for B3 (B103) (47) the 526 substitution for B4 (B107) (44)

and 297 for B108 (43)

242 O Subgroups

The blood group O demonstrates the absence of A and B antigens on the RBC

surface in forward blood typing Reverse blood typing indicates the presence of both

anti-A and -B in the plasma The first O allele (O1-1 [O01]) was shown to be identical

to the consensus A allele (A101) except for a nucleotide deletion 261delG in exon 6

This results in a shift in the reading frame giving rise to a truncated protein that alters

the protein sequence after amino acid 88 A stop codon halts translation after amino acid

117 and the resulting protein is enzymatically inactive (41) Some other O1-1-like

alleles that are characterized by the presence of the 261delG and at least one additional

point mutation A second kind of O allele has the same inactivating deletion (261delG)

as the original O allele (O1-1 [O01]) but in addition has nine point mutations spread

34

throughout exons 3 to 7 (4145) and a further 13 mutations have been found amongst

the intron 6 (50)

Additional polymorphisms associated with blood group O have been found up to

approximately 4300 bp from ABO exon 7 (51) some of which correlated with O1 and

O1v alleles (52) Other O alleles not due to 261delG also exist that are caused by other

inactivating mutations along the reading frame The ( O2-1 [O03]) was the first allele

described of this type (5354) which has a critical mutation (802G rarr A) causing an

amino acid change (Gly268Arg) that prevents the enzyme from utilizing the nucleotide

sugar donor (55) This O allele comprises approximately 2 to 5 percent of O alleles in

Caucasian persons but seems to be absent or at least very rare in other populations

(5456) Other rare O alleles such as O3 [O08] that does not have 261delG but instead

contains both the common A2 allele polymorphisms 467CrarrT and C-deletion at nt

1059-1061 and an insertion of an extra guanosine in the 7-guanosine sequence at nt

798-804 (45) Two other rare O alleles lacking 261delG were reported in the Japanese

population by Ogasawara O301 [O14] has the missense mutation 893CrarrT (Ala268Val)

on an A102 background whereas O302 [O15] has the nonsense mutation 927CrarrA

(Tyr309Stop) on an A101 background (43)

243 Weak subgroups

In addition to the major phenotypes characterized by either strong or absent

haemagglutination with anti-A-A1 and -B reagents the ABO blood group system also

includes phenotypes in which erythrocytes react weakly with the anti-A and ndashB

reagents for example A3 Ax Afinn Ael B3 Bx Bv Bel cis-AB (57)

It is difficult to determine clearly their specific ABO subgroup by conventional

serological methods The weak subgroups are important in more than one way First

they risk complicating patient and donor blood group determination At worst the

wrong group can be assigned if eg a weak antigen is missed Second they allow us to

characterize and understand glycosyltransferase mechanisms by studying the results of

mutations in the underlying alleles

35

2431 Weak A alleles

The RBCs from individuals with the A3 phenotype agglutinate strongly with

anti-A and anti-AB in vitro but show a large number of free cells One A3B individual

had a novel point mutation 871GrarrA (Asp291Asn) on the A1 [A101] (58) and this

allele was named A301 Ax alleles are responsible for the rare subgroup Ax and the

RBCs typically show a weak positive reaction with anti-AB and anti-H There are four

base substitutions involved in these alleles 646TrarrA (Phe216Ile) 681GrarrA (silent)

771CrarrT (silent) 829GrarrA (Val277Met) The encoded transferase is expected to be 37

aa longer than the normal consensus allele and 16 aa longer than A2-encoded

transferase The serological characteristics of some other minor phenotypes included

among A subgroups eg Aend Am Ay Afinn Abantu and the collective description Aw are

more unclear Table 22 shows the serological reaction patterns of weak A alleles

Table 22 Serological reaction patterns

A negative reaction is noted by 0 and positive reactions are denoted from + (very weak

agglutination) to 4+ (maximal agglutination)mf mixed field agglutination

2432 Weak B alleles

B variants are much more uncommon than A variants this may reflect the

relatively low frequency of the B blood group in many populations These phenotypes

Subgroup

of A

RBC reactions with Anti-A1

in serum

Anti-A Anti-AB Anti-A1 Anti-H

A1 4+ 4+ 4+ 0 No

A2 4+ 4+ 0 4+ Sometimes

Aint 4+ 4+ 2+3+ 2+3+ No

A3 2++mf 2++ mf 0 4+ No

Ax 0+ 2++ 0 4+ Often

Ael 0 0 0 4+ Sometimes

Aend + + 0 4+ Sometimes

Afinn + + 0 4+ Yes

Abantu +(+) +(+) 0 4+ Yes

Am 0+ 0+ 0 4+ No

Ay 0 0 0 4+ No

36

often appear to result from missense mutations at the ABO locus causing single aa

changes in the GTB Characteristics of some B variants are summarized in Table 23

(57)

Table 23 Characteristics of some more frequent B weak phenotypes

mf mixed field w very weak agglutination

The B3 phenotype have missense mutation 1054CrarrT (Arg352Trp) on a B1

(B101) background and named B301 (58) A Bx allele responsible for the Bx phenotype

had a point mutation at nt 871GrarrA (Asp291Asn) (59) which was also found in an A3

sample (58) The Bel phenotype was divided into two suballeles Bel-1 and Bel-2 (Bel01

and Bel02) which had substitutions at 641TrarrG (Met214Arg) and 669GrarrT

(Glu223Asp) respectively (59) these missense mutations reduce the enzymatic

activities of the GTB Ogasawara et al (1996) also found another B3 allele B302 which

differs from B consensus by two nucleotide substitutions 646TrarrA (Phe216Ile) and

657TrarrC

CisAB alleles rare phenotype was described by Seyfried in 1964 It represents a

very interesting phenomenon that proved that it is possible for a child with blood group

O to have a parent with blood group AB Seyfried hypothesized that both A and B

determinants of the AB blood group could be located on the same chromosome Later

on this hypothesis confirmed in that the existence of an exceptional ABO allele

encoding a glycosyltransferase is indeed able to produce both A and B enzymes at the

same time (57) Cis-ABO1 was sequenced and showed the substitution 803GrarrC

(Gly268Ala) on the A1-2 (A102) background and thus can be described as AAAB (60)

Another allele named cis-ABO2 was discovered when a Vietnamese man who was to

undergo organ transplantation showed irregular blood grouping results The sequencing

Subgroup

of B

RBC reactions with Anti-B

in serum

Anti-A Anti-AB Anti-B Anti-H

B 0 ++++ ++++ ++ None

B3 1 mf mf +++ None

Bx 1 w w +++ Yes

Bel 0 w 0w +++ None

Bm 0 0 0 +++ Sometimes

37

showed that his ABO genes were nearly identical to the normal B allele except for a

796ArarrC (Met266Leu) substitution (61)

25 ABO antibodies

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after

stimulation by a specific antigen The antibody formed binds to the specific antigen in

order to mark the antigen for destruction The type of antigenic exposure occurring in

the body determines if the antibody is a naturally occurring or immune antibody The

term lsquonaturally occurringrsquo is used for blood group antibodies produced in individuals

who have never been transfused with red cells carrying the relevant antigen or been

pregnant with a fetus carrying the relevant antigen Naturally occurring antibodies can

be formed after exposure to environmental agents that are similar to red cell antigens

such as bacteria dust or pollen (62)

Most of these antibodies are not clinically significant with the exception of ABO

antibodies It is possible that food and environmental antigens (bacterial viral or plant

antigens) have epitopes similar enough to A and B glycoprotein antigens The

antibodies created against these environmental antigens in the first years of life can

cross-react with ABO-incompatible red blood cells (RBCs) that it comes in contact with

during blood transfusion later in life Anti-A antibodies are hypothesized to originate

from immune response towards influenza virus whose epitopes are similar enough to

the α-D-N-galactosamine on the A glycoprotein to be able to elicit a cross-reaction

Anti-B antibodies are hypothesized to originate from antibodies produced against

Gram-negative bacteria such as E coli cross-reacting with the α-D-galactose on the B

glycoprotein (49) Anti-A and anti-B antibodies (called isohaemagglutinins) which are

not present in the newborn appear in the first years of life They are isoantibodies that

are produced by an individual against antigens produced by members of the same

species (isoantigens) Anti-A and anti-B antibodies are usually IgM type which are not

able to pass through the placenta to the fetal blood circulation and react best at room

temperature or lower The ABO antibodies may also be found in various body fluids

including saliva milk cervical secretions tears and cysts These antibodies are

detected at about 3 months and increase their titer until the 5th

to 10th

year of life

1

Molecular genetic studies of human ABO genes have demonstrated that ABO genes

have two critical single base substitutions in the last coding exon that result in amino

acid substitutions responsible for the different donor nucleotide sugar substrate

specificity between A- and B-transferases A single base deletion in exon 6 was

ascribed to shift the reading frame of codons and to abolish the transferase activity of A-

transferase in most O alleles (45911)

ABO genotyping is important not only for blood transfusion but also for tissuecell and

organ transplantations Also ABO genotypes are important evidence at crime scenes

and for personal identification in forensic investigations and paternity testing

To our knowledge this is the first study in Gaza Strip investigating the ABO gene

polymorphism In this study multiplex allele-specific PCR is used for determining the

ABO genotypes and the corresponding allele frequency in a group of 201 unrelated

Palestinians residing in Gaza Strip

4

12 Objectives of the Study

121 General Objective

To determine the major ABO alleles and genotypes frequencies in a Palestinian

population residing in Gaza Strip

122 Specific objectives

1- To employ PCR and Allele-specific (AS)-PCR for molecular genotyping of ABO

blood system

2- To correlate ABO genotypes with phenotypes in blood samples of Gaza Strip

population

3- To compare the frequency of ABO genotypes with other populations

5

CHAPTER 2

LITIRATURE REVIEW

6

CHAPTER 2

LITIRATURE REVIEW

21 Background

The International Society of Blood Transfusion (ISBT) Working Committee on

Terminology for Red Cell Surface Antigens was set up in 1980 to establish and define a

meaningful nomenclature for different blood groups (12) Every valid blood group

antigen is given a six digit identification number There are 29 different systems to date

and the first three digits represent the systems (001-029)

The symbol for a gene or cluster of genes controlling a blood group system is

often the italicized symbol for the system The ABO genotypes should consequently be

written in italicized capital letters (Table 21)

Table 21 Some of Human blood group systems recognized by the ISBT (12)

No Name Symbol No of

antigens

Gene name(s) Chromosome

001 ABO ABO 4 ABO 9

002 MNS MNS 43 GYPA GYPB GYPE 4

003 P P1 1 P1 22

004 Rh RH 49 RHD RHCE 1

005 Lutheran LU 19 LU 19

006 Kell KEL 25 KEL 7

007 Lewis LE 6 FUT3 19

008 Duffy FY 6 DARC 1

009 Kidd JK 3 SLC14A1 18

22 Biosynthesis of ABH antigens

The biochemical basis of the ABO and H antigens is well understood due to

intensive studies during the 1950s and 1960s by the pioneering work on ovarian cyst

fluids (which contain large amounts of water-soluble blood-group-active glycoproteins)

by Morgan amp Watkins and Kabat The ABO antigens are not limited to erythroid

tissues but are also found in different tissues and on some epithelial cells (13)The

antigens are also present in the secretory fluids in the majority of humans therefore they

7

can sometimes be noted as histo-blood group antigens (14) ABH antigens are

carbohydrate structures These oligosaccharide chains are generally conjugated with

polypeptides to form glycoproteins Oligosaccharides are synthesized in a stepwise

fashion the addition of each monosaccharide being catalyzed by a specific

glycosyltransferase enzyme (15)

The glycoproteins contain a peptide backbone to which multiple oligosaccharide

chains are attached through an alkali-labile glycosidic bond (1617) to the hydroxyl

group of serine or threonine (18) Most of the oligosaccharide chains are linked to the

backbone through an N-acetylgalactosamine residue The carbohydrate moiety of the

ABH glycoproteins consists primarily of four sugars D-galactose L-fucose N-acetyl-

D-galactosamine and N-acetyl-D-glucosamine (19) The amino acid compositions of the

different blood group glycoproteins are similar to each other and unrelated to blood-

group specificity

Expression of H A and B antigens is dependent on the presence of specific

monosaccharides attached to various precursor disaccharides at the non-reducing end of

a carbohydrate chain (14) A transferase product of A allele transfers the

monosaccharide N acetylgalactosamine from the donor substrate uridine diphosphate

(UDP)-N-acetylgalactosamine to the fucosylated galactosyl residue of the H antigen to

produce an active structure The B transferase product of B allele transfers galactose

from UDP-galactose to the fucosylated galactose of H to produce a B-active structure

(20)Individuals with the gene for Glycosyltransferases A have blood group A those

with the gene for Glycosyltransferases B have blood group B those with genes for both

enzymes or a cis-acting form of GTA or GTB have blood type AB and those with a

mutated inactive form of enzyme have blood group O (Figure 21 )

8

Figure 21 Biosynthesis of ABO Antigens (2)

The major alleles at the ABO locus are A B and O and to-date a number of ABO

blood groups variants have been reported with approximately 250 different alleles

registered in the Blood Group antigen gene Mutation database BGMUT (21)

221 H antigen

In nature there exists at least four H antigens on glycolipids and glycoproteins

that are recognized by GTA and GTB (22)The most common are the type I and the type

II H antigens (23)H antigen is produced when an α12-Lfucosyltransferase catalyses the

transfer of L-fucose from a guanosine diphosphate (GDP)-L-fucose donor to the C-2

position of the terminal galactose of one of the precursor structures (Figure 21) Two

α12-L-fucosyltransferases produced by two genes FUT1 (H) and FUT2 (Se) catalyze

9

the biosynthesis of H-active structures in different tissues (24) mainly in epithelial cells

and body fluids such as saliva

The H antigen (Figure 22) is the natural precursor of A and B antigens and its

fucose residue is required for A and B glycosyltransferases to recognize it as the

acceptor and transfer GalNAc or Gal to its terminal Gal Depending on the disaccharide

precursor core chain on which ABH determinants are synthesized they can be further

divided into different types

Figure 22 H antigen structure

Everybody expresses H antigen on his red cells but only about 80 of

Europeans have H antigen in their body secretions These people are called ABH

secretors because if they have an A andor B gene they also secrete A andor B

antigens The remaining 20 are called ABH non-secretors as they do not secrete H A

or B regardless of ABO genotype (2526)

222 A and B antigens

A and B antigens can be produced by the presence of the appropriate A- or B

transferase (Figure 21) The A gene product is an α13-N-acetyl galactosaminyl-

transferase which transfers N-acetyl-galactosamine from a uridine diphosphate (UDP)-

N acetylgalactosamine donor to the fucosylated galactosyl residue of H antigen The B

gene product an α 13-D-galactosyltransferase transfers D-galactose from UDP-

galactose to the fucosylated galactose of H A and B are alleles at the ABO locus on

chromosome 9 A third allele O does not produce an active enzyme and in persons

homozygous for O the H antigen remains unmodified (Figure 23)

31

Figure 23 Summary of A B and O(H) blood group structures and their synthesis(28)

The A and B antigens are carbohydrate molecules built stepwise from

saccharides such as galactosamine (GalNAc) glucosamine (GlcNAc) fucose (Fuc)

galactose (Gal) and glucose (Glc) H-antigen is the requisite precursor and

galactosylamine (GalNAcα3) or galactosyl (Galα3) with α1-3 linkage onto H antigen

become the A and B antigens respectively (514) (Figure 24)

Figure 24 A and B antigen structure

The majority of ABO antigens on red cells are linked to glycoproteins

(approximately 70) thus they very much influence the blood group activity on the red

cells At each step in the biosynthesis of ABO antigens and carbohydrate chains

synthesis is facilitated by glycosyltransferases which are competing for available

precursors and substrates This represents approximately 80 of the total complement

of red cell ABH determinants Another 5 times 105 ABH determinants localize to the red

33

cell glucose transport protein (Band 45) Small numbers of ABH antigens are also

expressed by other red cell glycoproteins (28)

23 ABO Glycosyltransferases

Glycosyltransferases (GTs) constitute a large family of enzymes that are

involved in the biosynthesis of oligosaccharides polysaccharides and glycoconjugates

(29)Particularly abundant are the GTs that transfer a sugar residue from an activated

nucleotide sugar donor to specific acceptor molecules forming glycosidic bonds

Glycosyltransferases are classified into 87 different families based on substrateproduct

stereochemistry according to the CAZy database (30)

Knowledge of the sequences of the ABO genes (9) have established that

mammalian Glycosyltransferases A and Glycosyltransferases B are type II integral

membrane proteins containing 354 amino acids and are localized in the lumen of the

Golgi apparatus These enzymes typically have a short amino-terminal cytoplasmic tail

a hydrophobic membrane domain a short protease-sensitive stem region and a large

catalytic domain that includes the carboxy terminus(31) GTA and GTB are very similar

in the coding regions and the soluble enzyme can be found in serum (32)urine (33)and

milk (34)The enzyme contains an acceptor recognition domain that binds H antigen and

a donor recognition domain that binds UDP-GalNAc or UDP-Gal GTA and GTB

require the metal ion Mn++ (manganese) for activity (35)

Glycosyltransferases are antigenic structures Human antibodies to blood group

transferases are often produced following organ transplantation (36-39)

24 ABO Subgroups

241 A and B Subgroups

An ABO blood group subtype is called a subgroup andor a variant Subgroups

of ABO are distinguished by decreased amounts of A B or O (H) antigens on red blood

cells The most common are subgroups of A and B Blood type A appears to have the

most variation in subgroups The two most common subgroups of blood group A are A1

and A2 expressing on average 1 million and 250 000 A determinants respectively (40)

32

2411 A Subgroups

The A1 (A101) allele is the reference allele often denoted as the consensus

sequence in an ABO genotype context (9)but has additionally eight variant alleles

There are variants of A1 alleles one of which is very common in Asian populations with

a 467CrarrT (A102) polymorphism resulting in the substitution Pro156Leu (41)Two

minor A1 alleles (A103 and A104) have been described and differ as follows The first

one also has the 467CrarrT point mutation but an additional silent mutation 567CrarrT

the second allele contains a silent polymorphism in nucleotide 297ArarrG (42) A105 is

like A102 with the same mutation in exon 7 467CrarrT but analysis of intron 6 showed

additional single-nucleotide polymorphism (SNP) compared to A102 Another A1 allele

(A106) contains both 297ArarrG and 467CrarrT (43)

The A2 subgroup is the most common A phenotype after the A1 subgroup The

main genetic difference between A1 and A2 alleles is one point mutation in exon 7

467CrarrT (Pro156Leu) and a deletion of one of the three cytosines at nt 1059-1061

(CCC to CC) The latter mutation results in an extension of the reading frame by 64

nucleotides This deletion occurs in the codon before the translation stop codon (TGA)

resulting in a gene product with an extra 21 amino acid at its C-terminus The

glycosyltransferases encoded by the A2 allele have lower efficiency leading to a weaker

A phenotype The enzyme activity is decreased by 30-50 times compared to A1

(22)Other variants of A2 alleles have subsequently been elucidated (A202-206) Three

A2-like alleles were shown to have three different single mutations near the 3acute end of

exon 7 by Ogasawara A2-2 (A202) contains 1054CrarrT (Arg352Trp) A2-3 (A203) with

1054CrarrG (Arg352Gly) (42) and A2-4 (A205) with both 467CrarrT and an additional

new mutation 1009ArarrG (Arg337Gly) (44) A204 with four common B-related base

substitutions (297ArarrG 526CrarrG 657CrarrT and 703GrarrA) seems to be a hybrid with

two extra substitutions 771CrarrT (silent mutation) and 829GrarrA (Val277Met) One

other rare A2 subgroup A2-5 (A206) carried only the single deletion (1061delC)

(4546)

31

2412 B Subgroups

Subtypes of blood type B are classified by the quantity of B antigen and the

amount of B antigen decreases in the order B B3 Bx Bm Bel B1 allele (B101)

showed seven single nucleotide substitutions 297 526 657 703 796 803 and 930

throughout exon 6 and 7 and later one extra mutation outside the coding region at the 3acute

end at nt 1096 (33) The four amino acids substitutions governed by nt 526CrarrG

(Arg176Gly) 703GrarrA (Gly235Ser) 796CrarrA (Leu266Met) and 803GrarrC

(Gly268Ala) discriminate GTA from GTB (4147) while the substitutions 297 657 and

930 are silent

ABO glycosyltransferases can accordingly be described by using the letters A

and B to illustrate the derivation of the amino acid at these four residues GTA would be

represented by AAAA indicating the presence of ArgGlyLeuGly and similarly BBBB

would describe the GTB with GlySerMetAla at residues 176235266 and 268

respectively The substitutions at positions 266 and 268 were shown to be responsible

for the nucleotidedonor specificity of the transferases (47) and the other residues may

have a role in acceptor binding and turnover (48)Some other variants have been

reported afterwards that differ from B1 (B101) by lacking the 930 substitution for B2

(B102) the 657 substitution for B3 (B103) (47) the 526 substitution for B4 (B107) (44)

and 297 for B108 (43)

242 O Subgroups

The blood group O demonstrates the absence of A and B antigens on the RBC

surface in forward blood typing Reverse blood typing indicates the presence of both

anti-A and -B in the plasma The first O allele (O1-1 [O01]) was shown to be identical

to the consensus A allele (A101) except for a nucleotide deletion 261delG in exon 6

This results in a shift in the reading frame giving rise to a truncated protein that alters

the protein sequence after amino acid 88 A stop codon halts translation after amino acid

117 and the resulting protein is enzymatically inactive (41) Some other O1-1-like

alleles that are characterized by the presence of the 261delG and at least one additional

point mutation A second kind of O allele has the same inactivating deletion (261delG)

as the original O allele (O1-1 [O01]) but in addition has nine point mutations spread

34

throughout exons 3 to 7 (4145) and a further 13 mutations have been found amongst

the intron 6 (50)

Additional polymorphisms associated with blood group O have been found up to

approximately 4300 bp from ABO exon 7 (51) some of which correlated with O1 and

O1v alleles (52) Other O alleles not due to 261delG also exist that are caused by other

inactivating mutations along the reading frame The ( O2-1 [O03]) was the first allele

described of this type (5354) which has a critical mutation (802G rarr A) causing an

amino acid change (Gly268Arg) that prevents the enzyme from utilizing the nucleotide

sugar donor (55) This O allele comprises approximately 2 to 5 percent of O alleles in

Caucasian persons but seems to be absent or at least very rare in other populations

(5456) Other rare O alleles such as O3 [O08] that does not have 261delG but instead

contains both the common A2 allele polymorphisms 467CrarrT and C-deletion at nt

1059-1061 and an insertion of an extra guanosine in the 7-guanosine sequence at nt

798-804 (45) Two other rare O alleles lacking 261delG were reported in the Japanese

population by Ogasawara O301 [O14] has the missense mutation 893CrarrT (Ala268Val)

on an A102 background whereas O302 [O15] has the nonsense mutation 927CrarrA

(Tyr309Stop) on an A101 background (43)

243 Weak subgroups

In addition to the major phenotypes characterized by either strong or absent

haemagglutination with anti-A-A1 and -B reagents the ABO blood group system also

includes phenotypes in which erythrocytes react weakly with the anti-A and ndashB

reagents for example A3 Ax Afinn Ael B3 Bx Bv Bel cis-AB (57)

It is difficult to determine clearly their specific ABO subgroup by conventional

serological methods The weak subgroups are important in more than one way First

they risk complicating patient and donor blood group determination At worst the

wrong group can be assigned if eg a weak antigen is missed Second they allow us to

characterize and understand glycosyltransferase mechanisms by studying the results of

mutations in the underlying alleles

35

2431 Weak A alleles

The RBCs from individuals with the A3 phenotype agglutinate strongly with

anti-A and anti-AB in vitro but show a large number of free cells One A3B individual

had a novel point mutation 871GrarrA (Asp291Asn) on the A1 [A101] (58) and this

allele was named A301 Ax alleles are responsible for the rare subgroup Ax and the

RBCs typically show a weak positive reaction with anti-AB and anti-H There are four

base substitutions involved in these alleles 646TrarrA (Phe216Ile) 681GrarrA (silent)

771CrarrT (silent) 829GrarrA (Val277Met) The encoded transferase is expected to be 37

aa longer than the normal consensus allele and 16 aa longer than A2-encoded

transferase The serological characteristics of some other minor phenotypes included

among A subgroups eg Aend Am Ay Afinn Abantu and the collective description Aw are

more unclear Table 22 shows the serological reaction patterns of weak A alleles

Table 22 Serological reaction patterns

A negative reaction is noted by 0 and positive reactions are denoted from + (very weak

agglutination) to 4+ (maximal agglutination)mf mixed field agglutination

2432 Weak B alleles

B variants are much more uncommon than A variants this may reflect the

relatively low frequency of the B blood group in many populations These phenotypes

Subgroup

of A

RBC reactions with Anti-A1

in serum

Anti-A Anti-AB Anti-A1 Anti-H

A1 4+ 4+ 4+ 0 No

A2 4+ 4+ 0 4+ Sometimes

Aint 4+ 4+ 2+3+ 2+3+ No

A3 2++mf 2++ mf 0 4+ No

Ax 0+ 2++ 0 4+ Often

Ael 0 0 0 4+ Sometimes

Aend + + 0 4+ Sometimes

Afinn + + 0 4+ Yes

Abantu +(+) +(+) 0 4+ Yes

Am 0+ 0+ 0 4+ No

Ay 0 0 0 4+ No

36

often appear to result from missense mutations at the ABO locus causing single aa

changes in the GTB Characteristics of some B variants are summarized in Table 23

(57)

Table 23 Characteristics of some more frequent B weak phenotypes

mf mixed field w very weak agglutination

The B3 phenotype have missense mutation 1054CrarrT (Arg352Trp) on a B1

(B101) background and named B301 (58) A Bx allele responsible for the Bx phenotype

had a point mutation at nt 871GrarrA (Asp291Asn) (59) which was also found in an A3

sample (58) The Bel phenotype was divided into two suballeles Bel-1 and Bel-2 (Bel01

and Bel02) which had substitutions at 641TrarrG (Met214Arg) and 669GrarrT

(Glu223Asp) respectively (59) these missense mutations reduce the enzymatic

activities of the GTB Ogasawara et al (1996) also found another B3 allele B302 which

differs from B consensus by two nucleotide substitutions 646TrarrA (Phe216Ile) and

657TrarrC

CisAB alleles rare phenotype was described by Seyfried in 1964 It represents a

very interesting phenomenon that proved that it is possible for a child with blood group

O to have a parent with blood group AB Seyfried hypothesized that both A and B

determinants of the AB blood group could be located on the same chromosome Later

on this hypothesis confirmed in that the existence of an exceptional ABO allele

encoding a glycosyltransferase is indeed able to produce both A and B enzymes at the

same time (57) Cis-ABO1 was sequenced and showed the substitution 803GrarrC

(Gly268Ala) on the A1-2 (A102) background and thus can be described as AAAB (60)

Another allele named cis-ABO2 was discovered when a Vietnamese man who was to

undergo organ transplantation showed irregular blood grouping results The sequencing

Subgroup

of B

RBC reactions with Anti-B

in serum

Anti-A Anti-AB Anti-B Anti-H

B 0 ++++ ++++ ++ None

B3 1 mf mf +++ None

Bx 1 w w +++ Yes

Bel 0 w 0w +++ None

Bm 0 0 0 +++ Sometimes

37

showed that his ABO genes were nearly identical to the normal B allele except for a

796ArarrC (Met266Leu) substitution (61)

25 ABO antibodies

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after

stimulation by a specific antigen The antibody formed binds to the specific antigen in

order to mark the antigen for destruction The type of antigenic exposure occurring in

the body determines if the antibody is a naturally occurring or immune antibody The

term lsquonaturally occurringrsquo is used for blood group antibodies produced in individuals

who have never been transfused with red cells carrying the relevant antigen or been

pregnant with a fetus carrying the relevant antigen Naturally occurring antibodies can

be formed after exposure to environmental agents that are similar to red cell antigens

such as bacteria dust or pollen (62)

Most of these antibodies are not clinically significant with the exception of ABO

antibodies It is possible that food and environmental antigens (bacterial viral or plant

antigens) have epitopes similar enough to A and B glycoprotein antigens The

antibodies created against these environmental antigens in the first years of life can

cross-react with ABO-incompatible red blood cells (RBCs) that it comes in contact with

during blood transfusion later in life Anti-A antibodies are hypothesized to originate

from immune response towards influenza virus whose epitopes are similar enough to

the α-D-N-galactosamine on the A glycoprotein to be able to elicit a cross-reaction

Anti-B antibodies are hypothesized to originate from antibodies produced against

Gram-negative bacteria such as E coli cross-reacting with the α-D-galactose on the B

glycoprotein (49) Anti-A and anti-B antibodies (called isohaemagglutinins) which are

not present in the newborn appear in the first years of life They are isoantibodies that

are produced by an individual against antigens produced by members of the same

species (isoantigens) Anti-A and anti-B antibodies are usually IgM type which are not

able to pass through the placenta to the fetal blood circulation and react best at room

temperature or lower The ABO antibodies may also be found in various body fluids

including saliva milk cervical secretions tears and cysts These antibodies are

detected at about 3 months and increase their titer until the 5th

to 10th

year of life

4

12 Objectives of the Study

121 General Objective

To determine the major ABO alleles and genotypes frequencies in a Palestinian

population residing in Gaza Strip

122 Specific objectives

1- To employ PCR and Allele-specific (AS)-PCR for molecular genotyping of ABO

blood system

2- To correlate ABO genotypes with phenotypes in blood samples of Gaza Strip

population

3- To compare the frequency of ABO genotypes with other populations

5

CHAPTER 2

LITIRATURE REVIEW

6

CHAPTER 2

LITIRATURE REVIEW

21 Background

The International Society of Blood Transfusion (ISBT) Working Committee on

Terminology for Red Cell Surface Antigens was set up in 1980 to establish and define a

meaningful nomenclature for different blood groups (12) Every valid blood group

antigen is given a six digit identification number There are 29 different systems to date

and the first three digits represent the systems (001-029)

The symbol for a gene or cluster of genes controlling a blood group system is

often the italicized symbol for the system The ABO genotypes should consequently be

written in italicized capital letters (Table 21)

Table 21 Some of Human blood group systems recognized by the ISBT (12)

No Name Symbol No of

antigens

Gene name(s) Chromosome

001 ABO ABO 4 ABO 9

002 MNS MNS 43 GYPA GYPB GYPE 4

003 P P1 1 P1 22

004 Rh RH 49 RHD RHCE 1

005 Lutheran LU 19 LU 19

006 Kell KEL 25 KEL 7

007 Lewis LE 6 FUT3 19

008 Duffy FY 6 DARC 1

009 Kidd JK 3 SLC14A1 18

22 Biosynthesis of ABH antigens

The biochemical basis of the ABO and H antigens is well understood due to

intensive studies during the 1950s and 1960s by the pioneering work on ovarian cyst

fluids (which contain large amounts of water-soluble blood-group-active glycoproteins)

by Morgan amp Watkins and Kabat The ABO antigens are not limited to erythroid

tissues but are also found in different tissues and on some epithelial cells (13)The

antigens are also present in the secretory fluids in the majority of humans therefore they

7

can sometimes be noted as histo-blood group antigens (14) ABH antigens are

carbohydrate structures These oligosaccharide chains are generally conjugated with

polypeptides to form glycoproteins Oligosaccharides are synthesized in a stepwise

fashion the addition of each monosaccharide being catalyzed by a specific

glycosyltransferase enzyme (15)

The glycoproteins contain a peptide backbone to which multiple oligosaccharide

chains are attached through an alkali-labile glycosidic bond (1617) to the hydroxyl

group of serine or threonine (18) Most of the oligosaccharide chains are linked to the

backbone through an N-acetylgalactosamine residue The carbohydrate moiety of the

ABH glycoproteins consists primarily of four sugars D-galactose L-fucose N-acetyl-

D-galactosamine and N-acetyl-D-glucosamine (19) The amino acid compositions of the

different blood group glycoproteins are similar to each other and unrelated to blood-

group specificity

Expression of H A and B antigens is dependent on the presence of specific

monosaccharides attached to various precursor disaccharides at the non-reducing end of

a carbohydrate chain (14) A transferase product of A allele transfers the

monosaccharide N acetylgalactosamine from the donor substrate uridine diphosphate

(UDP)-N-acetylgalactosamine to the fucosylated galactosyl residue of the H antigen to

produce an active structure The B transferase product of B allele transfers galactose

from UDP-galactose to the fucosylated galactose of H to produce a B-active structure

(20)Individuals with the gene for Glycosyltransferases A have blood group A those

with the gene for Glycosyltransferases B have blood group B those with genes for both

enzymes or a cis-acting form of GTA or GTB have blood type AB and those with a

mutated inactive form of enzyme have blood group O (Figure 21 )

8

Figure 21 Biosynthesis of ABO Antigens (2)

The major alleles at the ABO locus are A B and O and to-date a number of ABO

blood groups variants have been reported with approximately 250 different alleles

registered in the Blood Group antigen gene Mutation database BGMUT (21)

221 H antigen

In nature there exists at least four H antigens on glycolipids and glycoproteins

that are recognized by GTA and GTB (22)The most common are the type I and the type

II H antigens (23)H antigen is produced when an α12-Lfucosyltransferase catalyses the

transfer of L-fucose from a guanosine diphosphate (GDP)-L-fucose donor to the C-2

position of the terminal galactose of one of the precursor structures (Figure 21) Two

α12-L-fucosyltransferases produced by two genes FUT1 (H) and FUT2 (Se) catalyze

9

the biosynthesis of H-active structures in different tissues (24) mainly in epithelial cells

and body fluids such as saliva

The H antigen (Figure 22) is the natural precursor of A and B antigens and its

fucose residue is required for A and B glycosyltransferases to recognize it as the

acceptor and transfer GalNAc or Gal to its terminal Gal Depending on the disaccharide

precursor core chain on which ABH determinants are synthesized they can be further

divided into different types

Figure 22 H antigen structure

Everybody expresses H antigen on his red cells but only about 80 of

Europeans have H antigen in their body secretions These people are called ABH

secretors because if they have an A andor B gene they also secrete A andor B

antigens The remaining 20 are called ABH non-secretors as they do not secrete H A

or B regardless of ABO genotype (2526)

222 A and B antigens

A and B antigens can be produced by the presence of the appropriate A- or B

transferase (Figure 21) The A gene product is an α13-N-acetyl galactosaminyl-

transferase which transfers N-acetyl-galactosamine from a uridine diphosphate (UDP)-

N acetylgalactosamine donor to the fucosylated galactosyl residue of H antigen The B

gene product an α 13-D-galactosyltransferase transfers D-galactose from UDP-

galactose to the fucosylated galactose of H A and B are alleles at the ABO locus on

chromosome 9 A third allele O does not produce an active enzyme and in persons

homozygous for O the H antigen remains unmodified (Figure 23)

31

Figure 23 Summary of A B and O(H) blood group structures and their synthesis(28)

The A and B antigens are carbohydrate molecules built stepwise from

saccharides such as galactosamine (GalNAc) glucosamine (GlcNAc) fucose (Fuc)

galactose (Gal) and glucose (Glc) H-antigen is the requisite precursor and

galactosylamine (GalNAcα3) or galactosyl (Galα3) with α1-3 linkage onto H antigen

become the A and B antigens respectively (514) (Figure 24)

Figure 24 A and B antigen structure

The majority of ABO antigens on red cells are linked to glycoproteins

(approximately 70) thus they very much influence the blood group activity on the red

cells At each step in the biosynthesis of ABO antigens and carbohydrate chains

synthesis is facilitated by glycosyltransferases which are competing for available

precursors and substrates This represents approximately 80 of the total complement

of red cell ABH determinants Another 5 times 105 ABH determinants localize to the red

33

cell glucose transport protein (Band 45) Small numbers of ABH antigens are also

expressed by other red cell glycoproteins (28)

23 ABO Glycosyltransferases

Glycosyltransferases (GTs) constitute a large family of enzymes that are

involved in the biosynthesis of oligosaccharides polysaccharides and glycoconjugates

(29)Particularly abundant are the GTs that transfer a sugar residue from an activated

nucleotide sugar donor to specific acceptor molecules forming glycosidic bonds

Glycosyltransferases are classified into 87 different families based on substrateproduct

stereochemistry according to the CAZy database (30)

Knowledge of the sequences of the ABO genes (9) have established that

mammalian Glycosyltransferases A and Glycosyltransferases B are type II integral

membrane proteins containing 354 amino acids and are localized in the lumen of the

Golgi apparatus These enzymes typically have a short amino-terminal cytoplasmic tail

a hydrophobic membrane domain a short protease-sensitive stem region and a large

catalytic domain that includes the carboxy terminus(31) GTA and GTB are very similar

in the coding regions and the soluble enzyme can be found in serum (32)urine (33)and

milk (34)The enzyme contains an acceptor recognition domain that binds H antigen and

a donor recognition domain that binds UDP-GalNAc or UDP-Gal GTA and GTB

require the metal ion Mn++ (manganese) for activity (35)

Glycosyltransferases are antigenic structures Human antibodies to blood group

transferases are often produced following organ transplantation (36-39)

24 ABO Subgroups

241 A and B Subgroups

An ABO blood group subtype is called a subgroup andor a variant Subgroups

of ABO are distinguished by decreased amounts of A B or O (H) antigens on red blood

cells The most common are subgroups of A and B Blood type A appears to have the

most variation in subgroups The two most common subgroups of blood group A are A1

and A2 expressing on average 1 million and 250 000 A determinants respectively (40)

32

2411 A Subgroups

The A1 (A101) allele is the reference allele often denoted as the consensus

sequence in an ABO genotype context (9)but has additionally eight variant alleles

There are variants of A1 alleles one of which is very common in Asian populations with

a 467CrarrT (A102) polymorphism resulting in the substitution Pro156Leu (41)Two

minor A1 alleles (A103 and A104) have been described and differ as follows The first

one also has the 467CrarrT point mutation but an additional silent mutation 567CrarrT

the second allele contains a silent polymorphism in nucleotide 297ArarrG (42) A105 is

like A102 with the same mutation in exon 7 467CrarrT but analysis of intron 6 showed

additional single-nucleotide polymorphism (SNP) compared to A102 Another A1 allele

(A106) contains both 297ArarrG and 467CrarrT (43)

The A2 subgroup is the most common A phenotype after the A1 subgroup The

main genetic difference between A1 and A2 alleles is one point mutation in exon 7

467CrarrT (Pro156Leu) and a deletion of one of the three cytosines at nt 1059-1061

(CCC to CC) The latter mutation results in an extension of the reading frame by 64

nucleotides This deletion occurs in the codon before the translation stop codon (TGA)

resulting in a gene product with an extra 21 amino acid at its C-terminus The

glycosyltransferases encoded by the A2 allele have lower efficiency leading to a weaker

A phenotype The enzyme activity is decreased by 30-50 times compared to A1

(22)Other variants of A2 alleles have subsequently been elucidated (A202-206) Three

A2-like alleles were shown to have three different single mutations near the 3acute end of

exon 7 by Ogasawara A2-2 (A202) contains 1054CrarrT (Arg352Trp) A2-3 (A203) with

1054CrarrG (Arg352Gly) (42) and A2-4 (A205) with both 467CrarrT and an additional

new mutation 1009ArarrG (Arg337Gly) (44) A204 with four common B-related base

substitutions (297ArarrG 526CrarrG 657CrarrT and 703GrarrA) seems to be a hybrid with

two extra substitutions 771CrarrT (silent mutation) and 829GrarrA (Val277Met) One

other rare A2 subgroup A2-5 (A206) carried only the single deletion (1061delC)

(4546)

31

2412 B Subgroups

Subtypes of blood type B are classified by the quantity of B antigen and the

amount of B antigen decreases in the order B B3 Bx Bm Bel B1 allele (B101)

showed seven single nucleotide substitutions 297 526 657 703 796 803 and 930

throughout exon 6 and 7 and later one extra mutation outside the coding region at the 3acute

end at nt 1096 (33) The four amino acids substitutions governed by nt 526CrarrG

(Arg176Gly) 703GrarrA (Gly235Ser) 796CrarrA (Leu266Met) and 803GrarrC

(Gly268Ala) discriminate GTA from GTB (4147) while the substitutions 297 657 and

930 are silent

ABO glycosyltransferases can accordingly be described by using the letters A

and B to illustrate the derivation of the amino acid at these four residues GTA would be

represented by AAAA indicating the presence of ArgGlyLeuGly and similarly BBBB

would describe the GTB with GlySerMetAla at residues 176235266 and 268

respectively The substitutions at positions 266 and 268 were shown to be responsible

for the nucleotidedonor specificity of the transferases (47) and the other residues may

have a role in acceptor binding and turnover (48)Some other variants have been

reported afterwards that differ from B1 (B101) by lacking the 930 substitution for B2

(B102) the 657 substitution for B3 (B103) (47) the 526 substitution for B4 (B107) (44)

and 297 for B108 (43)

242 O Subgroups

The blood group O demonstrates the absence of A and B antigens on the RBC

surface in forward blood typing Reverse blood typing indicates the presence of both

anti-A and -B in the plasma The first O allele (O1-1 [O01]) was shown to be identical

to the consensus A allele (A101) except for a nucleotide deletion 261delG in exon 6

This results in a shift in the reading frame giving rise to a truncated protein that alters

the protein sequence after amino acid 88 A stop codon halts translation after amino acid

117 and the resulting protein is enzymatically inactive (41) Some other O1-1-like

alleles that are characterized by the presence of the 261delG and at least one additional

point mutation A second kind of O allele has the same inactivating deletion (261delG)

as the original O allele (O1-1 [O01]) but in addition has nine point mutations spread

34

throughout exons 3 to 7 (4145) and a further 13 mutations have been found amongst

the intron 6 (50)

Additional polymorphisms associated with blood group O have been found up to

approximately 4300 bp from ABO exon 7 (51) some of which correlated with O1 and

O1v alleles (52) Other O alleles not due to 261delG also exist that are caused by other

inactivating mutations along the reading frame The ( O2-1 [O03]) was the first allele

described of this type (5354) which has a critical mutation (802G rarr A) causing an

amino acid change (Gly268Arg) that prevents the enzyme from utilizing the nucleotide

sugar donor (55) This O allele comprises approximately 2 to 5 percent of O alleles in

Caucasian persons but seems to be absent or at least very rare in other populations

(5456) Other rare O alleles such as O3 [O08] that does not have 261delG but instead

contains both the common A2 allele polymorphisms 467CrarrT and C-deletion at nt

1059-1061 and an insertion of an extra guanosine in the 7-guanosine sequence at nt

798-804 (45) Two other rare O alleles lacking 261delG were reported in the Japanese

population by Ogasawara O301 [O14] has the missense mutation 893CrarrT (Ala268Val)

on an A102 background whereas O302 [O15] has the nonsense mutation 927CrarrA

(Tyr309Stop) on an A101 background (43)

243 Weak subgroups

In addition to the major phenotypes characterized by either strong or absent

haemagglutination with anti-A-A1 and -B reagents the ABO blood group system also

includes phenotypes in which erythrocytes react weakly with the anti-A and ndashB

reagents for example A3 Ax Afinn Ael B3 Bx Bv Bel cis-AB (57)

It is difficult to determine clearly their specific ABO subgroup by conventional

serological methods The weak subgroups are important in more than one way First

they risk complicating patient and donor blood group determination At worst the

wrong group can be assigned if eg a weak antigen is missed Second they allow us to

characterize and understand glycosyltransferase mechanisms by studying the results of

mutations in the underlying alleles

35

2431 Weak A alleles

The RBCs from individuals with the A3 phenotype agglutinate strongly with

anti-A and anti-AB in vitro but show a large number of free cells One A3B individual

had a novel point mutation 871GrarrA (Asp291Asn) on the A1 [A101] (58) and this

allele was named A301 Ax alleles are responsible for the rare subgroup Ax and the

RBCs typically show a weak positive reaction with anti-AB and anti-H There are four

base substitutions involved in these alleles 646TrarrA (Phe216Ile) 681GrarrA (silent)

771CrarrT (silent) 829GrarrA (Val277Met) The encoded transferase is expected to be 37

aa longer than the normal consensus allele and 16 aa longer than A2-encoded

transferase The serological characteristics of some other minor phenotypes included

among A subgroups eg Aend Am Ay Afinn Abantu and the collective description Aw are

more unclear Table 22 shows the serological reaction patterns of weak A alleles

Table 22 Serological reaction patterns

A negative reaction is noted by 0 and positive reactions are denoted from + (very weak

agglutination) to 4+ (maximal agglutination)mf mixed field agglutination

2432 Weak B alleles

B variants are much more uncommon than A variants this may reflect the

relatively low frequency of the B blood group in many populations These phenotypes

Subgroup

of A

RBC reactions with Anti-A1

in serum

Anti-A Anti-AB Anti-A1 Anti-H

A1 4+ 4+ 4+ 0 No

A2 4+ 4+ 0 4+ Sometimes

Aint 4+ 4+ 2+3+ 2+3+ No

A3 2++mf 2++ mf 0 4+ No

Ax 0+ 2++ 0 4+ Often

Ael 0 0 0 4+ Sometimes

Aend + + 0 4+ Sometimes

Afinn + + 0 4+ Yes

Abantu +(+) +(+) 0 4+ Yes

Am 0+ 0+ 0 4+ No

Ay 0 0 0 4+ No

36

often appear to result from missense mutations at the ABO locus causing single aa

changes in the GTB Characteristics of some B variants are summarized in Table 23

(57)

Table 23 Characteristics of some more frequent B weak phenotypes

mf mixed field w very weak agglutination

The B3 phenotype have missense mutation 1054CrarrT (Arg352Trp) on a B1

(B101) background and named B301 (58) A Bx allele responsible for the Bx phenotype

had a point mutation at nt 871GrarrA (Asp291Asn) (59) which was also found in an A3

sample (58) The Bel phenotype was divided into two suballeles Bel-1 and Bel-2 (Bel01

and Bel02) which had substitutions at 641TrarrG (Met214Arg) and 669GrarrT

(Glu223Asp) respectively (59) these missense mutations reduce the enzymatic

activities of the GTB Ogasawara et al (1996) also found another B3 allele B302 which

differs from B consensus by two nucleotide substitutions 646TrarrA (Phe216Ile) and

657TrarrC

CisAB alleles rare phenotype was described by Seyfried in 1964 It represents a

very interesting phenomenon that proved that it is possible for a child with blood group

O to have a parent with blood group AB Seyfried hypothesized that both A and B

determinants of the AB blood group could be located on the same chromosome Later

on this hypothesis confirmed in that the existence of an exceptional ABO allele

encoding a glycosyltransferase is indeed able to produce both A and B enzymes at the

same time (57) Cis-ABO1 was sequenced and showed the substitution 803GrarrC

(Gly268Ala) on the A1-2 (A102) background and thus can be described as AAAB (60)

Another allele named cis-ABO2 was discovered when a Vietnamese man who was to

undergo organ transplantation showed irregular blood grouping results The sequencing

Subgroup

of B

RBC reactions with Anti-B

in serum

Anti-A Anti-AB Anti-B Anti-H

B 0 ++++ ++++ ++ None

B3 1 mf mf +++ None

Bx 1 w w +++ Yes

Bel 0 w 0w +++ None

Bm 0 0 0 +++ Sometimes

37

showed that his ABO genes were nearly identical to the normal B allele except for a

796ArarrC (Met266Leu) substitution (61)

25 ABO antibodies

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after

stimulation by a specific antigen The antibody formed binds to the specific antigen in

order to mark the antigen for destruction The type of antigenic exposure occurring in

the body determines if the antibody is a naturally occurring or immune antibody The

term lsquonaturally occurringrsquo is used for blood group antibodies produced in individuals

who have never been transfused with red cells carrying the relevant antigen or been

pregnant with a fetus carrying the relevant antigen Naturally occurring antibodies can

be formed after exposure to environmental agents that are similar to red cell antigens

such as bacteria dust or pollen (62)

Most of these antibodies are not clinically significant with the exception of ABO

antibodies It is possible that food and environmental antigens (bacterial viral or plant

antigens) have epitopes similar enough to A and B glycoprotein antigens The

antibodies created against these environmental antigens in the first years of life can

cross-react with ABO-incompatible red blood cells (RBCs) that it comes in contact with

during blood transfusion later in life Anti-A antibodies are hypothesized to originate

from immune response towards influenza virus whose epitopes are similar enough to

the α-D-N-galactosamine on the A glycoprotein to be able to elicit a cross-reaction

Anti-B antibodies are hypothesized to originate from antibodies produced against

Gram-negative bacteria such as E coli cross-reacting with the α-D-galactose on the B

glycoprotein (49) Anti-A and anti-B antibodies (called isohaemagglutinins) which are

not present in the newborn appear in the first years of life They are isoantibodies that

are produced by an individual against antigens produced by members of the same

species (isoantigens) Anti-A and anti-B antibodies are usually IgM type which are not

able to pass through the placenta to the fetal blood circulation and react best at room

temperature or lower The ABO antibodies may also be found in various body fluids

including saliva milk cervical secretions tears and cysts These antibodies are

detected at about 3 months and increase their titer until the 5th

to 10th

year of life

5

CHAPTER 2

LITIRATURE REVIEW

6

CHAPTER 2

LITIRATURE REVIEW

21 Background

The International Society of Blood Transfusion (ISBT) Working Committee on

Terminology for Red Cell Surface Antigens was set up in 1980 to establish and define a

meaningful nomenclature for different blood groups (12) Every valid blood group

antigen is given a six digit identification number There are 29 different systems to date

and the first three digits represent the systems (001-029)

The symbol for a gene or cluster of genes controlling a blood group system is

often the italicized symbol for the system The ABO genotypes should consequently be

written in italicized capital letters (Table 21)

Table 21 Some of Human blood group systems recognized by the ISBT (12)

No Name Symbol No of

antigens

Gene name(s) Chromosome

001 ABO ABO 4 ABO 9

002 MNS MNS 43 GYPA GYPB GYPE 4

003 P P1 1 P1 22

004 Rh RH 49 RHD RHCE 1

005 Lutheran LU 19 LU 19

006 Kell KEL 25 KEL 7

007 Lewis LE 6 FUT3 19

008 Duffy FY 6 DARC 1

009 Kidd JK 3 SLC14A1 18

22 Biosynthesis of ABH antigens

The biochemical basis of the ABO and H antigens is well understood due to

intensive studies during the 1950s and 1960s by the pioneering work on ovarian cyst

fluids (which contain large amounts of water-soluble blood-group-active glycoproteins)

by Morgan amp Watkins and Kabat The ABO antigens are not limited to erythroid

tissues but are also found in different tissues and on some epithelial cells (13)The

antigens are also present in the secretory fluids in the majority of humans therefore they

7

can sometimes be noted as histo-blood group antigens (14) ABH antigens are

carbohydrate structures These oligosaccharide chains are generally conjugated with

polypeptides to form glycoproteins Oligosaccharides are synthesized in a stepwise

fashion the addition of each monosaccharide being catalyzed by a specific

glycosyltransferase enzyme (15)

The glycoproteins contain a peptide backbone to which multiple oligosaccharide

chains are attached through an alkali-labile glycosidic bond (1617) to the hydroxyl

group of serine or threonine (18) Most of the oligosaccharide chains are linked to the

backbone through an N-acetylgalactosamine residue The carbohydrate moiety of the

ABH glycoproteins consists primarily of four sugars D-galactose L-fucose N-acetyl-

D-galactosamine and N-acetyl-D-glucosamine (19) The amino acid compositions of the

different blood group glycoproteins are similar to each other and unrelated to blood-

group specificity

Expression of H A and B antigens is dependent on the presence of specific

monosaccharides attached to various precursor disaccharides at the non-reducing end of

a carbohydrate chain (14) A transferase product of A allele transfers the

monosaccharide N acetylgalactosamine from the donor substrate uridine diphosphate

(UDP)-N-acetylgalactosamine to the fucosylated galactosyl residue of the H antigen to

produce an active structure The B transferase product of B allele transfers galactose

from UDP-galactose to the fucosylated galactose of H to produce a B-active structure

(20)Individuals with the gene for Glycosyltransferases A have blood group A those

with the gene for Glycosyltransferases B have blood group B those with genes for both

enzymes or a cis-acting form of GTA or GTB have blood type AB and those with a

mutated inactive form of enzyme have blood group O (Figure 21 )

8

Figure 21 Biosynthesis of ABO Antigens (2)

The major alleles at the ABO locus are A B and O and to-date a number of ABO

blood groups variants have been reported with approximately 250 different alleles

registered in the Blood Group antigen gene Mutation database BGMUT (21)

221 H antigen

In nature there exists at least four H antigens on glycolipids and glycoproteins

that are recognized by GTA and GTB (22)The most common are the type I and the type

II H antigens (23)H antigen is produced when an α12-Lfucosyltransferase catalyses the

transfer of L-fucose from a guanosine diphosphate (GDP)-L-fucose donor to the C-2

position of the terminal galactose of one of the precursor structures (Figure 21) Two

α12-L-fucosyltransferases produced by two genes FUT1 (H) and FUT2 (Se) catalyze

9

the biosynthesis of H-active structures in different tissues (24) mainly in epithelial cells

and body fluids such as saliva

The H antigen (Figure 22) is the natural precursor of A and B antigens and its

fucose residue is required for A and B glycosyltransferases to recognize it as the

acceptor and transfer GalNAc or Gal to its terminal Gal Depending on the disaccharide

precursor core chain on which ABH determinants are synthesized they can be further

divided into different types

Figure 22 H antigen structure

Everybody expresses H antigen on his red cells but only about 80 of

Europeans have H antigen in their body secretions These people are called ABH

secretors because if they have an A andor B gene they also secrete A andor B

antigens The remaining 20 are called ABH non-secretors as they do not secrete H A

or B regardless of ABO genotype (2526)

222 A and B antigens

A and B antigens can be produced by the presence of the appropriate A- or B

transferase (Figure 21) The A gene product is an α13-N-acetyl galactosaminyl-

transferase which transfers N-acetyl-galactosamine from a uridine diphosphate (UDP)-

N acetylgalactosamine donor to the fucosylated galactosyl residue of H antigen The B

gene product an α 13-D-galactosyltransferase transfers D-galactose from UDP-

galactose to the fucosylated galactose of H A and B are alleles at the ABO locus on

chromosome 9 A third allele O does not produce an active enzyme and in persons

homozygous for O the H antigen remains unmodified (Figure 23)

31

Figure 23 Summary of A B and O(H) blood group structures and their synthesis(28)

The A and B antigens are carbohydrate molecules built stepwise from

saccharides such as galactosamine (GalNAc) glucosamine (GlcNAc) fucose (Fuc)

galactose (Gal) and glucose (Glc) H-antigen is the requisite precursor and

galactosylamine (GalNAcα3) or galactosyl (Galα3) with α1-3 linkage onto H antigen

become the A and B antigens respectively (514) (Figure 24)

Figure 24 A and B antigen structure

The majority of ABO antigens on red cells are linked to glycoproteins

(approximately 70) thus they very much influence the blood group activity on the red

cells At each step in the biosynthesis of ABO antigens and carbohydrate chains

synthesis is facilitated by glycosyltransferases which are competing for available

precursors and substrates This represents approximately 80 of the total complement

of red cell ABH determinants Another 5 times 105 ABH determinants localize to the red

33

cell glucose transport protein (Band 45) Small numbers of ABH antigens are also

expressed by other red cell glycoproteins (28)

23 ABO Glycosyltransferases

Glycosyltransferases (GTs) constitute a large family of enzymes that are

involved in the biosynthesis of oligosaccharides polysaccharides and glycoconjugates

(29)Particularly abundant are the GTs that transfer a sugar residue from an activated

nucleotide sugar donor to specific acceptor molecules forming glycosidic bonds

Glycosyltransferases are classified into 87 different families based on substrateproduct

stereochemistry according to the CAZy database (30)

Knowledge of the sequences of the ABO genes (9) have established that

mammalian Glycosyltransferases A and Glycosyltransferases B are type II integral

membrane proteins containing 354 amino acids and are localized in the lumen of the

Golgi apparatus These enzymes typically have a short amino-terminal cytoplasmic tail

a hydrophobic membrane domain a short protease-sensitive stem region and a large

catalytic domain that includes the carboxy terminus(31) GTA and GTB are very similar

in the coding regions and the soluble enzyme can be found in serum (32)urine (33)and

milk (34)The enzyme contains an acceptor recognition domain that binds H antigen and

a donor recognition domain that binds UDP-GalNAc or UDP-Gal GTA and GTB

require the metal ion Mn++ (manganese) for activity (35)

Glycosyltransferases are antigenic structures Human antibodies to blood group

transferases are often produced following organ transplantation (36-39)

24 ABO Subgroups

241 A and B Subgroups

An ABO blood group subtype is called a subgroup andor a variant Subgroups

of ABO are distinguished by decreased amounts of A B or O (H) antigens on red blood

cells The most common are subgroups of A and B Blood type A appears to have the

most variation in subgroups The two most common subgroups of blood group A are A1

and A2 expressing on average 1 million and 250 000 A determinants respectively (40)

32

2411 A Subgroups

The A1 (A101) allele is the reference allele often denoted as the consensus

sequence in an ABO genotype context (9)but has additionally eight variant alleles

There are variants of A1 alleles one of which is very common in Asian populations with

a 467CrarrT (A102) polymorphism resulting in the substitution Pro156Leu (41)Two

minor A1 alleles (A103 and A104) have been described and differ as follows The first

one also has the 467CrarrT point mutation but an additional silent mutation 567CrarrT

the second allele contains a silent polymorphism in nucleotide 297ArarrG (42) A105 is

like A102 with the same mutation in exon 7 467CrarrT but analysis of intron 6 showed

additional single-nucleotide polymorphism (SNP) compared to A102 Another A1 allele

(A106) contains both 297ArarrG and 467CrarrT (43)

The A2 subgroup is the most common A phenotype after the A1 subgroup The

main genetic difference between A1 and A2 alleles is one point mutation in exon 7

467CrarrT (Pro156Leu) and a deletion of one of the three cytosines at nt 1059-1061

(CCC to CC) The latter mutation results in an extension of the reading frame by 64

nucleotides This deletion occurs in the codon before the translation stop codon (TGA)

resulting in a gene product with an extra 21 amino acid at its C-terminus The

glycosyltransferases encoded by the A2 allele have lower efficiency leading to a weaker

A phenotype The enzyme activity is decreased by 30-50 times compared to A1

(22)Other variants of A2 alleles have subsequently been elucidated (A202-206) Three

A2-like alleles were shown to have three different single mutations near the 3acute end of

exon 7 by Ogasawara A2-2 (A202) contains 1054CrarrT (Arg352Trp) A2-3 (A203) with

1054CrarrG (Arg352Gly) (42) and A2-4 (A205) with both 467CrarrT and an additional

new mutation 1009ArarrG (Arg337Gly) (44) A204 with four common B-related base

substitutions (297ArarrG 526CrarrG 657CrarrT and 703GrarrA) seems to be a hybrid with

two extra substitutions 771CrarrT (silent mutation) and 829GrarrA (Val277Met) One

other rare A2 subgroup A2-5 (A206) carried only the single deletion (1061delC)

(4546)

31

2412 B Subgroups

Subtypes of blood type B are classified by the quantity of B antigen and the

amount of B antigen decreases in the order B B3 Bx Bm Bel B1 allele (B101)

showed seven single nucleotide substitutions 297 526 657 703 796 803 and 930

throughout exon 6 and 7 and later one extra mutation outside the coding region at the 3acute

end at nt 1096 (33) The four amino acids substitutions governed by nt 526CrarrG

(Arg176Gly) 703GrarrA (Gly235Ser) 796CrarrA (Leu266Met) and 803GrarrC

(Gly268Ala) discriminate GTA from GTB (4147) while the substitutions 297 657 and

930 are silent

ABO glycosyltransferases can accordingly be described by using the letters A

and B to illustrate the derivation of the amino acid at these four residues GTA would be

represented by AAAA indicating the presence of ArgGlyLeuGly and similarly BBBB

would describe the GTB with GlySerMetAla at residues 176235266 and 268

respectively The substitutions at positions 266 and 268 were shown to be responsible

for the nucleotidedonor specificity of the transferases (47) and the other residues may

have a role in acceptor binding and turnover (48)Some other variants have been

reported afterwards that differ from B1 (B101) by lacking the 930 substitution for B2

(B102) the 657 substitution for B3 (B103) (47) the 526 substitution for B4 (B107) (44)

and 297 for B108 (43)

242 O Subgroups

The blood group O demonstrates the absence of A and B antigens on the RBC

surface in forward blood typing Reverse blood typing indicates the presence of both

anti-A and -B in the plasma The first O allele (O1-1 [O01]) was shown to be identical

to the consensus A allele (A101) except for a nucleotide deletion 261delG in exon 6

This results in a shift in the reading frame giving rise to a truncated protein that alters

the protein sequence after amino acid 88 A stop codon halts translation after amino acid

117 and the resulting protein is enzymatically inactive (41) Some other O1-1-like

alleles that are characterized by the presence of the 261delG and at least one additional

point mutation A second kind of O allele has the same inactivating deletion (261delG)

as the original O allele (O1-1 [O01]) but in addition has nine point mutations spread

34

throughout exons 3 to 7 (4145) and a further 13 mutations have been found amongst

the intron 6 (50)

Additional polymorphisms associated with blood group O have been found up to

approximately 4300 bp from ABO exon 7 (51) some of which correlated with O1 and

O1v alleles (52) Other O alleles not due to 261delG also exist that are caused by other

inactivating mutations along the reading frame The ( O2-1 [O03]) was the first allele

described of this type (5354) which has a critical mutation (802G rarr A) causing an

amino acid change (Gly268Arg) that prevents the enzyme from utilizing the nucleotide

sugar donor (55) This O allele comprises approximately 2 to 5 percent of O alleles in

Caucasian persons but seems to be absent or at least very rare in other populations

(5456) Other rare O alleles such as O3 [O08] that does not have 261delG but instead

contains both the common A2 allele polymorphisms 467CrarrT and C-deletion at nt

1059-1061 and an insertion of an extra guanosine in the 7-guanosine sequence at nt

798-804 (45) Two other rare O alleles lacking 261delG were reported in the Japanese

population by Ogasawara O301 [O14] has the missense mutation 893CrarrT (Ala268Val)

on an A102 background whereas O302 [O15] has the nonsense mutation 927CrarrA

(Tyr309Stop) on an A101 background (43)

243 Weak subgroups

In addition to the major phenotypes characterized by either strong or absent

haemagglutination with anti-A-A1 and -B reagents the ABO blood group system also

includes phenotypes in which erythrocytes react weakly with the anti-A and ndashB

reagents for example A3 Ax Afinn Ael B3 Bx Bv Bel cis-AB (57)

It is difficult to determine clearly their specific ABO subgroup by conventional

serological methods The weak subgroups are important in more than one way First

they risk complicating patient and donor blood group determination At worst the

wrong group can be assigned if eg a weak antigen is missed Second they allow us to

characterize and understand glycosyltransferase mechanisms by studying the results of

mutations in the underlying alleles

35

2431 Weak A alleles

The RBCs from individuals with the A3 phenotype agglutinate strongly with

anti-A and anti-AB in vitro but show a large number of free cells One A3B individual

had a novel point mutation 871GrarrA (Asp291Asn) on the A1 [A101] (58) and this

allele was named A301 Ax alleles are responsible for the rare subgroup Ax and the

RBCs typically show a weak positive reaction with anti-AB and anti-H There are four

base substitutions involved in these alleles 646TrarrA (Phe216Ile) 681GrarrA (silent)

771CrarrT (silent) 829GrarrA (Val277Met) The encoded transferase is expected to be 37

aa longer than the normal consensus allele and 16 aa longer than A2-encoded

transferase The serological characteristics of some other minor phenotypes included

among A subgroups eg Aend Am Ay Afinn Abantu and the collective description Aw are

more unclear Table 22 shows the serological reaction patterns of weak A alleles

Table 22 Serological reaction patterns

A negative reaction is noted by 0 and positive reactions are denoted from + (very weak

agglutination) to 4+ (maximal agglutination)mf mixed field agglutination

2432 Weak B alleles

B variants are much more uncommon than A variants this may reflect the

relatively low frequency of the B blood group in many populations These phenotypes

Subgroup

of A

RBC reactions with Anti-A1

in serum

Anti-A Anti-AB Anti-A1 Anti-H

A1 4+ 4+ 4+ 0 No

A2 4+ 4+ 0 4+ Sometimes

Aint 4+ 4+ 2+3+ 2+3+ No

A3 2++mf 2++ mf 0 4+ No

Ax 0+ 2++ 0 4+ Often

Ael 0 0 0 4+ Sometimes

Aend + + 0 4+ Sometimes

Afinn + + 0 4+ Yes

Abantu +(+) +(+) 0 4+ Yes

Am 0+ 0+ 0 4+ No

Ay 0 0 0 4+ No

36

often appear to result from missense mutations at the ABO locus causing single aa

changes in the GTB Characteristics of some B variants are summarized in Table 23

(57)

Table 23 Characteristics of some more frequent B weak phenotypes

mf mixed field w very weak agglutination

The B3 phenotype have missense mutation 1054CrarrT (Arg352Trp) on a B1

(B101) background and named B301 (58) A Bx allele responsible for the Bx phenotype

had a point mutation at nt 871GrarrA (Asp291Asn) (59) which was also found in an A3

sample (58) The Bel phenotype was divided into two suballeles Bel-1 and Bel-2 (Bel01

and Bel02) which had substitutions at 641TrarrG (Met214Arg) and 669GrarrT

(Glu223Asp) respectively (59) these missense mutations reduce the enzymatic

activities of the GTB Ogasawara et al (1996) also found another B3 allele B302 which

differs from B consensus by two nucleotide substitutions 646TrarrA (Phe216Ile) and

657TrarrC

CisAB alleles rare phenotype was described by Seyfried in 1964 It represents a

very interesting phenomenon that proved that it is possible for a child with blood group

O to have a parent with blood group AB Seyfried hypothesized that both A and B

determinants of the AB blood group could be located on the same chromosome Later

on this hypothesis confirmed in that the existence of an exceptional ABO allele

encoding a glycosyltransferase is indeed able to produce both A and B enzymes at the

same time (57) Cis-ABO1 was sequenced and showed the substitution 803GrarrC

(Gly268Ala) on the A1-2 (A102) background and thus can be described as AAAB (60)

Another allele named cis-ABO2 was discovered when a Vietnamese man who was to

undergo organ transplantation showed irregular blood grouping results The sequencing

Subgroup

of B

RBC reactions with Anti-B

in serum

Anti-A Anti-AB Anti-B Anti-H

B 0 ++++ ++++ ++ None

B3 1 mf mf +++ None

Bx 1 w w +++ Yes

Bel 0 w 0w +++ None

Bm 0 0 0 +++ Sometimes

37

showed that his ABO genes were nearly identical to the normal B allele except for a

796ArarrC (Met266Leu) substitution (61)

25 ABO antibodies

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after

stimulation by a specific antigen The antibody formed binds to the specific antigen in

order to mark the antigen for destruction The type of antigenic exposure occurring in

the body determines if the antibody is a naturally occurring or immune antibody The

term lsquonaturally occurringrsquo is used for blood group antibodies produced in individuals

who have never been transfused with red cells carrying the relevant antigen or been

pregnant with a fetus carrying the relevant antigen Naturally occurring antibodies can

be formed after exposure to environmental agents that are similar to red cell antigens

such as bacteria dust or pollen (62)

Most of these antibodies are not clinically significant with the exception of ABO

antibodies It is possible that food and environmental antigens (bacterial viral or plant

antigens) have epitopes similar enough to A and B glycoprotein antigens The

antibodies created against these environmental antigens in the first years of life can

cross-react with ABO-incompatible red blood cells (RBCs) that it comes in contact with

during blood transfusion later in life Anti-A antibodies are hypothesized to originate

from immune response towards influenza virus whose epitopes are similar enough to

the α-D-N-galactosamine on the A glycoprotein to be able to elicit a cross-reaction

Anti-B antibodies are hypothesized to originate from antibodies produced against

Gram-negative bacteria such as E coli cross-reacting with the α-D-galactose on the B

glycoprotein (49) Anti-A and anti-B antibodies (called isohaemagglutinins) which are

not present in the newborn appear in the first years of life They are isoantibodies that

are produced by an individual against antigens produced by members of the same

species (isoantigens) Anti-A and anti-B antibodies are usually IgM type which are not

able to pass through the placenta to the fetal blood circulation and react best at room

temperature or lower The ABO antibodies may also be found in various body fluids

including saliva milk cervical secretions tears and cysts These antibodies are

detected at about 3 months and increase their titer until the 5th

to 10th

year of life

6

CHAPTER 2

LITIRATURE REVIEW

21 Background

The International Society of Blood Transfusion (ISBT) Working Committee on

Terminology for Red Cell Surface Antigens was set up in 1980 to establish and define a

meaningful nomenclature for different blood groups (12) Every valid blood group

antigen is given a six digit identification number There are 29 different systems to date

and the first three digits represent the systems (001-029)

The symbol for a gene or cluster of genes controlling a blood group system is

often the italicized symbol for the system The ABO genotypes should consequently be

written in italicized capital letters (Table 21)

Table 21 Some of Human blood group systems recognized by the ISBT (12)

No Name Symbol No of

antigens

Gene name(s) Chromosome

001 ABO ABO 4 ABO 9

002 MNS MNS 43 GYPA GYPB GYPE 4

003 P P1 1 P1 22

004 Rh RH 49 RHD RHCE 1

005 Lutheran LU 19 LU 19

006 Kell KEL 25 KEL 7

007 Lewis LE 6 FUT3 19

008 Duffy FY 6 DARC 1

009 Kidd JK 3 SLC14A1 18

22 Biosynthesis of ABH antigens

The biochemical basis of the ABO and H antigens is well understood due to

intensive studies during the 1950s and 1960s by the pioneering work on ovarian cyst

fluids (which contain large amounts of water-soluble blood-group-active glycoproteins)

by Morgan amp Watkins and Kabat The ABO antigens are not limited to erythroid

tissues but are also found in different tissues and on some epithelial cells (13)The

antigens are also present in the secretory fluids in the majority of humans therefore they

7

can sometimes be noted as histo-blood group antigens (14) ABH antigens are

carbohydrate structures These oligosaccharide chains are generally conjugated with

polypeptides to form glycoproteins Oligosaccharides are synthesized in a stepwise

fashion the addition of each monosaccharide being catalyzed by a specific

glycosyltransferase enzyme (15)

The glycoproteins contain a peptide backbone to which multiple oligosaccharide

chains are attached through an alkali-labile glycosidic bond (1617) to the hydroxyl

group of serine or threonine (18) Most of the oligosaccharide chains are linked to the

backbone through an N-acetylgalactosamine residue The carbohydrate moiety of the

ABH glycoproteins consists primarily of four sugars D-galactose L-fucose N-acetyl-

D-galactosamine and N-acetyl-D-glucosamine (19) The amino acid compositions of the

different blood group glycoproteins are similar to each other and unrelated to blood-

group specificity

Expression of H A and B antigens is dependent on the presence of specific

monosaccharides attached to various precursor disaccharides at the non-reducing end of

a carbohydrate chain (14) A transferase product of A allele transfers the

monosaccharide N acetylgalactosamine from the donor substrate uridine diphosphate

(UDP)-N-acetylgalactosamine to the fucosylated galactosyl residue of the H antigen to

produce an active structure The B transferase product of B allele transfers galactose

from UDP-galactose to the fucosylated galactose of H to produce a B-active structure

(20)Individuals with the gene for Glycosyltransferases A have blood group A those

with the gene for Glycosyltransferases B have blood group B those with genes for both

enzymes or a cis-acting form of GTA or GTB have blood type AB and those with a

mutated inactive form of enzyme have blood group O (Figure 21 )

8

Figure 21 Biosynthesis of ABO Antigens (2)

The major alleles at the ABO locus are A B and O and to-date a number of ABO

blood groups variants have been reported with approximately 250 different alleles

registered in the Blood Group antigen gene Mutation database BGMUT (21)

221 H antigen

In nature there exists at least four H antigens on glycolipids and glycoproteins

that are recognized by GTA and GTB (22)The most common are the type I and the type

II H antigens (23)H antigen is produced when an α12-Lfucosyltransferase catalyses the

transfer of L-fucose from a guanosine diphosphate (GDP)-L-fucose donor to the C-2

position of the terminal galactose of one of the precursor structures (Figure 21) Two

α12-L-fucosyltransferases produced by two genes FUT1 (H) and FUT2 (Se) catalyze

9

the biosynthesis of H-active structures in different tissues (24) mainly in epithelial cells

and body fluids such as saliva

The H antigen (Figure 22) is the natural precursor of A and B antigens and its

fucose residue is required for A and B glycosyltransferases to recognize it as the

acceptor and transfer GalNAc or Gal to its terminal Gal Depending on the disaccharide

precursor core chain on which ABH determinants are synthesized they can be further

divided into different types

Figure 22 H antigen structure

Everybody expresses H antigen on his red cells but only about 80 of

Europeans have H antigen in their body secretions These people are called ABH

secretors because if they have an A andor B gene they also secrete A andor B

antigens The remaining 20 are called ABH non-secretors as they do not secrete H A

or B regardless of ABO genotype (2526)

222 A and B antigens

A and B antigens can be produced by the presence of the appropriate A- or B

transferase (Figure 21) The A gene product is an α13-N-acetyl galactosaminyl-

transferase which transfers N-acetyl-galactosamine from a uridine diphosphate (UDP)-

N acetylgalactosamine donor to the fucosylated galactosyl residue of H antigen The B

gene product an α 13-D-galactosyltransferase transfers D-galactose from UDP-

galactose to the fucosylated galactose of H A and B are alleles at the ABO locus on

chromosome 9 A third allele O does not produce an active enzyme and in persons

homozygous for O the H antigen remains unmodified (Figure 23)

31

Figure 23 Summary of A B and O(H) blood group structures and their synthesis(28)

The A and B antigens are carbohydrate molecules built stepwise from

saccharides such as galactosamine (GalNAc) glucosamine (GlcNAc) fucose (Fuc)

galactose (Gal) and glucose (Glc) H-antigen is the requisite precursor and

galactosylamine (GalNAcα3) or galactosyl (Galα3) with α1-3 linkage onto H antigen

become the A and B antigens respectively (514) (Figure 24)

Figure 24 A and B antigen structure

The majority of ABO antigens on red cells are linked to glycoproteins

(approximately 70) thus they very much influence the blood group activity on the red

cells At each step in the biosynthesis of ABO antigens and carbohydrate chains

synthesis is facilitated by glycosyltransferases which are competing for available

precursors and substrates This represents approximately 80 of the total complement

of red cell ABH determinants Another 5 times 105 ABH determinants localize to the red

33

cell glucose transport protein (Band 45) Small numbers of ABH antigens are also

expressed by other red cell glycoproteins (28)

23 ABO Glycosyltransferases

Glycosyltransferases (GTs) constitute a large family of enzymes that are

involved in the biosynthesis of oligosaccharides polysaccharides and glycoconjugates

(29)Particularly abundant are the GTs that transfer a sugar residue from an activated

nucleotide sugar donor to specific acceptor molecules forming glycosidic bonds

Glycosyltransferases are classified into 87 different families based on substrateproduct

stereochemistry according to the CAZy database (30)

Knowledge of the sequences of the ABO genes (9) have established that

mammalian Glycosyltransferases A and Glycosyltransferases B are type II integral

membrane proteins containing 354 amino acids and are localized in the lumen of the

Golgi apparatus These enzymes typically have a short amino-terminal cytoplasmic tail

a hydrophobic membrane domain a short protease-sensitive stem region and a large

catalytic domain that includes the carboxy terminus(31) GTA and GTB are very similar

in the coding regions and the soluble enzyme can be found in serum (32)urine (33)and

milk (34)The enzyme contains an acceptor recognition domain that binds H antigen and

a donor recognition domain that binds UDP-GalNAc or UDP-Gal GTA and GTB

require the metal ion Mn++ (manganese) for activity (35)

Glycosyltransferases are antigenic structures Human antibodies to blood group

transferases are often produced following organ transplantation (36-39)

24 ABO Subgroups

241 A and B Subgroups

An ABO blood group subtype is called a subgroup andor a variant Subgroups

of ABO are distinguished by decreased amounts of A B or O (H) antigens on red blood

cells The most common are subgroups of A and B Blood type A appears to have the

most variation in subgroups The two most common subgroups of blood group A are A1

and A2 expressing on average 1 million and 250 000 A determinants respectively (40)

32

2411 A Subgroups

The A1 (A101) allele is the reference allele often denoted as the consensus

sequence in an ABO genotype context (9)but has additionally eight variant alleles

There are variants of A1 alleles one of which is very common in Asian populations with

a 467CrarrT (A102) polymorphism resulting in the substitution Pro156Leu (41)Two

minor A1 alleles (A103 and A104) have been described and differ as follows The first

one also has the 467CrarrT point mutation but an additional silent mutation 567CrarrT

the second allele contains a silent polymorphism in nucleotide 297ArarrG (42) A105 is

like A102 with the same mutation in exon 7 467CrarrT but analysis of intron 6 showed

additional single-nucleotide polymorphism (SNP) compared to A102 Another A1 allele

(A106) contains both 297ArarrG and 467CrarrT (43)

The A2 subgroup is the most common A phenotype after the A1 subgroup The

main genetic difference between A1 and A2 alleles is one point mutation in exon 7

467CrarrT (Pro156Leu) and a deletion of one of the three cytosines at nt 1059-1061

(CCC to CC) The latter mutation results in an extension of the reading frame by 64

nucleotides This deletion occurs in the codon before the translation stop codon (TGA)

resulting in a gene product with an extra 21 amino acid at its C-terminus The

glycosyltransferases encoded by the A2 allele have lower efficiency leading to a weaker

A phenotype The enzyme activity is decreased by 30-50 times compared to A1

(22)Other variants of A2 alleles have subsequently been elucidated (A202-206) Three

A2-like alleles were shown to have three different single mutations near the 3acute end of

exon 7 by Ogasawara A2-2 (A202) contains 1054CrarrT (Arg352Trp) A2-3 (A203) with

1054CrarrG (Arg352Gly) (42) and A2-4 (A205) with both 467CrarrT and an additional

new mutation 1009ArarrG (Arg337Gly) (44) A204 with four common B-related base

substitutions (297ArarrG 526CrarrG 657CrarrT and 703GrarrA) seems to be a hybrid with

two extra substitutions 771CrarrT (silent mutation) and 829GrarrA (Val277Met) One

other rare A2 subgroup A2-5 (A206) carried only the single deletion (1061delC)

(4546)

31

2412 B Subgroups

Subtypes of blood type B are classified by the quantity of B antigen and the

amount of B antigen decreases in the order B B3 Bx Bm Bel B1 allele (B101)

showed seven single nucleotide substitutions 297 526 657 703 796 803 and 930

throughout exon 6 and 7 and later one extra mutation outside the coding region at the 3acute

end at nt 1096 (33) The four amino acids substitutions governed by nt 526CrarrG

(Arg176Gly) 703GrarrA (Gly235Ser) 796CrarrA (Leu266Met) and 803GrarrC

(Gly268Ala) discriminate GTA from GTB (4147) while the substitutions 297 657 and

930 are silent

ABO glycosyltransferases can accordingly be described by using the letters A

and B to illustrate the derivation of the amino acid at these four residues GTA would be

represented by AAAA indicating the presence of ArgGlyLeuGly and similarly BBBB

would describe the GTB with GlySerMetAla at residues 176235266 and 268

respectively The substitutions at positions 266 and 268 were shown to be responsible

for the nucleotidedonor specificity of the transferases (47) and the other residues may

have a role in acceptor binding and turnover (48)Some other variants have been

reported afterwards that differ from B1 (B101) by lacking the 930 substitution for B2

(B102) the 657 substitution for B3 (B103) (47) the 526 substitution for B4 (B107) (44)

and 297 for B108 (43)

242 O Subgroups

The blood group O demonstrates the absence of A and B antigens on the RBC

surface in forward blood typing Reverse blood typing indicates the presence of both

anti-A and -B in the plasma The first O allele (O1-1 [O01]) was shown to be identical

to the consensus A allele (A101) except for a nucleotide deletion 261delG in exon 6

This results in a shift in the reading frame giving rise to a truncated protein that alters

the protein sequence after amino acid 88 A stop codon halts translation after amino acid

117 and the resulting protein is enzymatically inactive (41) Some other O1-1-like

alleles that are characterized by the presence of the 261delG and at least one additional

point mutation A second kind of O allele has the same inactivating deletion (261delG)

as the original O allele (O1-1 [O01]) but in addition has nine point mutations spread

34

throughout exons 3 to 7 (4145) and a further 13 mutations have been found amongst

the intron 6 (50)

Additional polymorphisms associated with blood group O have been found up to

approximately 4300 bp from ABO exon 7 (51) some of which correlated with O1 and

O1v alleles (52) Other O alleles not due to 261delG also exist that are caused by other

inactivating mutations along the reading frame The ( O2-1 [O03]) was the first allele

described of this type (5354) which has a critical mutation (802G rarr A) causing an

amino acid change (Gly268Arg) that prevents the enzyme from utilizing the nucleotide

sugar donor (55) This O allele comprises approximately 2 to 5 percent of O alleles in

Caucasian persons but seems to be absent or at least very rare in other populations

(5456) Other rare O alleles such as O3 [O08] that does not have 261delG but instead

contains both the common A2 allele polymorphisms 467CrarrT and C-deletion at nt

1059-1061 and an insertion of an extra guanosine in the 7-guanosine sequence at nt

798-804 (45) Two other rare O alleles lacking 261delG were reported in the Japanese

population by Ogasawara O301 [O14] has the missense mutation 893CrarrT (Ala268Val)

on an A102 background whereas O302 [O15] has the nonsense mutation 927CrarrA

(Tyr309Stop) on an A101 background (43)

243 Weak subgroups

In addition to the major phenotypes characterized by either strong or absent

haemagglutination with anti-A-A1 and -B reagents the ABO blood group system also

includes phenotypes in which erythrocytes react weakly with the anti-A and ndashB

reagents for example A3 Ax Afinn Ael B3 Bx Bv Bel cis-AB (57)

It is difficult to determine clearly their specific ABO subgroup by conventional

serological methods The weak subgroups are important in more than one way First

they risk complicating patient and donor blood group determination At worst the

wrong group can be assigned if eg a weak antigen is missed Second they allow us to

characterize and understand glycosyltransferase mechanisms by studying the results of

mutations in the underlying alleles

35

2431 Weak A alleles

The RBCs from individuals with the A3 phenotype agglutinate strongly with

anti-A and anti-AB in vitro but show a large number of free cells One A3B individual

had a novel point mutation 871GrarrA (Asp291Asn) on the A1 [A101] (58) and this

allele was named A301 Ax alleles are responsible for the rare subgroup Ax and the

RBCs typically show a weak positive reaction with anti-AB and anti-H There are four

base substitutions involved in these alleles 646TrarrA (Phe216Ile) 681GrarrA (silent)

771CrarrT (silent) 829GrarrA (Val277Met) The encoded transferase is expected to be 37

aa longer than the normal consensus allele and 16 aa longer than A2-encoded

transferase The serological characteristics of some other minor phenotypes included

among A subgroups eg Aend Am Ay Afinn Abantu and the collective description Aw are

more unclear Table 22 shows the serological reaction patterns of weak A alleles

Table 22 Serological reaction patterns

A negative reaction is noted by 0 and positive reactions are denoted from + (very weak

agglutination) to 4+ (maximal agglutination)mf mixed field agglutination

2432 Weak B alleles

B variants are much more uncommon than A variants this may reflect the

relatively low frequency of the B blood group in many populations These phenotypes

Subgroup

of A

RBC reactions with Anti-A1

in serum

Anti-A Anti-AB Anti-A1 Anti-H

A1 4+ 4+ 4+ 0 No

A2 4+ 4+ 0 4+ Sometimes

Aint 4+ 4+ 2+3+ 2+3+ No

A3 2++mf 2++ mf 0 4+ No

Ax 0+ 2++ 0 4+ Often

Ael 0 0 0 4+ Sometimes

Aend + + 0 4+ Sometimes

Afinn + + 0 4+ Yes

Abantu +(+) +(+) 0 4+ Yes

Am 0+ 0+ 0 4+ No

Ay 0 0 0 4+ No

36

often appear to result from missense mutations at the ABO locus causing single aa

changes in the GTB Characteristics of some B variants are summarized in Table 23

(57)

Table 23 Characteristics of some more frequent B weak phenotypes

mf mixed field w very weak agglutination

The B3 phenotype have missense mutation 1054CrarrT (Arg352Trp) on a B1

(B101) background and named B301 (58) A Bx allele responsible for the Bx phenotype

had a point mutation at nt 871GrarrA (Asp291Asn) (59) which was also found in an A3

sample (58) The Bel phenotype was divided into two suballeles Bel-1 and Bel-2 (Bel01

and Bel02) which had substitutions at 641TrarrG (Met214Arg) and 669GrarrT

(Glu223Asp) respectively (59) these missense mutations reduce the enzymatic

activities of the GTB Ogasawara et al (1996) also found another B3 allele B302 which

differs from B consensus by two nucleotide substitutions 646TrarrA (Phe216Ile) and

657TrarrC

CisAB alleles rare phenotype was described by Seyfried in 1964 It represents a

very interesting phenomenon that proved that it is possible for a child with blood group

O to have a parent with blood group AB Seyfried hypothesized that both A and B

determinants of the AB blood group could be located on the same chromosome Later

on this hypothesis confirmed in that the existence of an exceptional ABO allele

encoding a glycosyltransferase is indeed able to produce both A and B enzymes at the

same time (57) Cis-ABO1 was sequenced and showed the substitution 803GrarrC

(Gly268Ala) on the A1-2 (A102) background and thus can be described as AAAB (60)

Another allele named cis-ABO2 was discovered when a Vietnamese man who was to

undergo organ transplantation showed irregular blood grouping results The sequencing

Subgroup

of B

RBC reactions with Anti-B

in serum

Anti-A Anti-AB Anti-B Anti-H

B 0 ++++ ++++ ++ None

B3 1 mf mf +++ None

Bx 1 w w +++ Yes

Bel 0 w 0w +++ None

Bm 0 0 0 +++ Sometimes

37

showed that his ABO genes were nearly identical to the normal B allele except for a

796ArarrC (Met266Leu) substitution (61)

25 ABO antibodies

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after

stimulation by a specific antigen The antibody formed binds to the specific antigen in

order to mark the antigen for destruction The type of antigenic exposure occurring in

the body determines if the antibody is a naturally occurring or immune antibody The

term lsquonaturally occurringrsquo is used for blood group antibodies produced in individuals

who have never been transfused with red cells carrying the relevant antigen or been

pregnant with a fetus carrying the relevant antigen Naturally occurring antibodies can

be formed after exposure to environmental agents that are similar to red cell antigens

such as bacteria dust or pollen (62)

Most of these antibodies are not clinically significant with the exception of ABO

antibodies It is possible that food and environmental antigens (bacterial viral or plant

antigens) have epitopes similar enough to A and B glycoprotein antigens The

antibodies created against these environmental antigens in the first years of life can

cross-react with ABO-incompatible red blood cells (RBCs) that it comes in contact with

during blood transfusion later in life Anti-A antibodies are hypothesized to originate

from immune response towards influenza virus whose epitopes are similar enough to

the α-D-N-galactosamine on the A glycoprotein to be able to elicit a cross-reaction

Anti-B antibodies are hypothesized to originate from antibodies produced against

Gram-negative bacteria such as E coli cross-reacting with the α-D-galactose on the B

glycoprotein (49) Anti-A and anti-B antibodies (called isohaemagglutinins) which are

not present in the newborn appear in the first years of life They are isoantibodies that

are produced by an individual against antigens produced by members of the same

species (isoantigens) Anti-A and anti-B antibodies are usually IgM type which are not

able to pass through the placenta to the fetal blood circulation and react best at room

temperature or lower The ABO antibodies may also be found in various body fluids

including saliva milk cervical secretions tears and cysts These antibodies are

detected at about 3 months and increase their titer until the 5th

to 10th

year of life

7

can sometimes be noted as histo-blood group antigens (14) ABH antigens are

carbohydrate structures These oligosaccharide chains are generally conjugated with

polypeptides to form glycoproteins Oligosaccharides are synthesized in a stepwise

fashion the addition of each monosaccharide being catalyzed by a specific

glycosyltransferase enzyme (15)

The glycoproteins contain a peptide backbone to which multiple oligosaccharide

chains are attached through an alkali-labile glycosidic bond (1617) to the hydroxyl

group of serine or threonine (18) Most of the oligosaccharide chains are linked to the

backbone through an N-acetylgalactosamine residue The carbohydrate moiety of the

ABH glycoproteins consists primarily of four sugars D-galactose L-fucose N-acetyl-

D-galactosamine and N-acetyl-D-glucosamine (19) The amino acid compositions of the

different blood group glycoproteins are similar to each other and unrelated to blood-

group specificity

Expression of H A and B antigens is dependent on the presence of specific

monosaccharides attached to various precursor disaccharides at the non-reducing end of

a carbohydrate chain (14) A transferase product of A allele transfers the

monosaccharide N acetylgalactosamine from the donor substrate uridine diphosphate

(UDP)-N-acetylgalactosamine to the fucosylated galactosyl residue of the H antigen to

produce an active structure The B transferase product of B allele transfers galactose

from UDP-galactose to the fucosylated galactose of H to produce a B-active structure

(20)Individuals with the gene for Glycosyltransferases A have blood group A those

with the gene for Glycosyltransferases B have blood group B those with genes for both

enzymes or a cis-acting form of GTA or GTB have blood type AB and those with a

mutated inactive form of enzyme have blood group O (Figure 21 )

8

Figure 21 Biosynthesis of ABO Antigens (2)

The major alleles at the ABO locus are A B and O and to-date a number of ABO

blood groups variants have been reported with approximately 250 different alleles

registered in the Blood Group antigen gene Mutation database BGMUT (21)

221 H antigen

In nature there exists at least four H antigens on glycolipids and glycoproteins

that are recognized by GTA and GTB (22)The most common are the type I and the type

II H antigens (23)H antigen is produced when an α12-Lfucosyltransferase catalyses the

transfer of L-fucose from a guanosine diphosphate (GDP)-L-fucose donor to the C-2

position of the terminal galactose of one of the precursor structures (Figure 21) Two

α12-L-fucosyltransferases produced by two genes FUT1 (H) and FUT2 (Se) catalyze

9

the biosynthesis of H-active structures in different tissues (24) mainly in epithelial cells

and body fluids such as saliva

The H antigen (Figure 22) is the natural precursor of A and B antigens and its

fucose residue is required for A and B glycosyltransferases to recognize it as the

acceptor and transfer GalNAc or Gal to its terminal Gal Depending on the disaccharide

precursor core chain on which ABH determinants are synthesized they can be further

divided into different types

Figure 22 H antigen structure

Everybody expresses H antigen on his red cells but only about 80 of

Europeans have H antigen in their body secretions These people are called ABH

secretors because if they have an A andor B gene they also secrete A andor B

antigens The remaining 20 are called ABH non-secretors as they do not secrete H A

or B regardless of ABO genotype (2526)

222 A and B antigens

A and B antigens can be produced by the presence of the appropriate A- or B

transferase (Figure 21) The A gene product is an α13-N-acetyl galactosaminyl-

transferase which transfers N-acetyl-galactosamine from a uridine diphosphate (UDP)-

N acetylgalactosamine donor to the fucosylated galactosyl residue of H antigen The B

gene product an α 13-D-galactosyltransferase transfers D-galactose from UDP-

galactose to the fucosylated galactose of H A and B are alleles at the ABO locus on

chromosome 9 A third allele O does not produce an active enzyme and in persons

homozygous for O the H antigen remains unmodified (Figure 23)

31

Figure 23 Summary of A B and O(H) blood group structures and their synthesis(28)

The A and B antigens are carbohydrate molecules built stepwise from

saccharides such as galactosamine (GalNAc) glucosamine (GlcNAc) fucose (Fuc)

galactose (Gal) and glucose (Glc) H-antigen is the requisite precursor and

galactosylamine (GalNAcα3) or galactosyl (Galα3) with α1-3 linkage onto H antigen

become the A and B antigens respectively (514) (Figure 24)

Figure 24 A and B antigen structure

The majority of ABO antigens on red cells are linked to glycoproteins

(approximately 70) thus they very much influence the blood group activity on the red

cells At each step in the biosynthesis of ABO antigens and carbohydrate chains

synthesis is facilitated by glycosyltransferases which are competing for available

precursors and substrates This represents approximately 80 of the total complement

of red cell ABH determinants Another 5 times 105 ABH determinants localize to the red

33

cell glucose transport protein (Band 45) Small numbers of ABH antigens are also

expressed by other red cell glycoproteins (28)

23 ABO Glycosyltransferases

Glycosyltransferases (GTs) constitute a large family of enzymes that are

involved in the biosynthesis of oligosaccharides polysaccharides and glycoconjugates

(29)Particularly abundant are the GTs that transfer a sugar residue from an activated

nucleotide sugar donor to specific acceptor molecules forming glycosidic bonds

Glycosyltransferases are classified into 87 different families based on substrateproduct

stereochemistry according to the CAZy database (30)

Knowledge of the sequences of the ABO genes (9) have established that

mammalian Glycosyltransferases A and Glycosyltransferases B are type II integral

membrane proteins containing 354 amino acids and are localized in the lumen of the

Golgi apparatus These enzymes typically have a short amino-terminal cytoplasmic tail

a hydrophobic membrane domain a short protease-sensitive stem region and a large

catalytic domain that includes the carboxy terminus(31) GTA and GTB are very similar

in the coding regions and the soluble enzyme can be found in serum (32)urine (33)and

milk (34)The enzyme contains an acceptor recognition domain that binds H antigen and

a donor recognition domain that binds UDP-GalNAc or UDP-Gal GTA and GTB

require the metal ion Mn++ (manganese) for activity (35)

Glycosyltransferases are antigenic structures Human antibodies to blood group

transferases are often produced following organ transplantation (36-39)

24 ABO Subgroups

241 A and B Subgroups

An ABO blood group subtype is called a subgroup andor a variant Subgroups

of ABO are distinguished by decreased amounts of A B or O (H) antigens on red blood

cells The most common are subgroups of A and B Blood type A appears to have the

most variation in subgroups The two most common subgroups of blood group A are A1

and A2 expressing on average 1 million and 250 000 A determinants respectively (40)

32

2411 A Subgroups

The A1 (A101) allele is the reference allele often denoted as the consensus

sequence in an ABO genotype context (9)but has additionally eight variant alleles

There are variants of A1 alleles one of which is very common in Asian populations with

a 467CrarrT (A102) polymorphism resulting in the substitution Pro156Leu (41)Two

minor A1 alleles (A103 and A104) have been described and differ as follows The first

one also has the 467CrarrT point mutation but an additional silent mutation 567CrarrT

the second allele contains a silent polymorphism in nucleotide 297ArarrG (42) A105 is

like A102 with the same mutation in exon 7 467CrarrT but analysis of intron 6 showed

additional single-nucleotide polymorphism (SNP) compared to A102 Another A1 allele

(A106) contains both 297ArarrG and 467CrarrT (43)

The A2 subgroup is the most common A phenotype after the A1 subgroup The

main genetic difference between A1 and A2 alleles is one point mutation in exon 7

467CrarrT (Pro156Leu) and a deletion of one of the three cytosines at nt 1059-1061

(CCC to CC) The latter mutation results in an extension of the reading frame by 64

nucleotides This deletion occurs in the codon before the translation stop codon (TGA)

resulting in a gene product with an extra 21 amino acid at its C-terminus The

glycosyltransferases encoded by the A2 allele have lower efficiency leading to a weaker

A phenotype The enzyme activity is decreased by 30-50 times compared to A1

(22)Other variants of A2 alleles have subsequently been elucidated (A202-206) Three

A2-like alleles were shown to have three different single mutations near the 3acute end of

exon 7 by Ogasawara A2-2 (A202) contains 1054CrarrT (Arg352Trp) A2-3 (A203) with

1054CrarrG (Arg352Gly) (42) and A2-4 (A205) with both 467CrarrT and an additional

new mutation 1009ArarrG (Arg337Gly) (44) A204 with four common B-related base

substitutions (297ArarrG 526CrarrG 657CrarrT and 703GrarrA) seems to be a hybrid with

two extra substitutions 771CrarrT (silent mutation) and 829GrarrA (Val277Met) One

other rare A2 subgroup A2-5 (A206) carried only the single deletion (1061delC)

(4546)

31

2412 B Subgroups

Subtypes of blood type B are classified by the quantity of B antigen and the

amount of B antigen decreases in the order B B3 Bx Bm Bel B1 allele (B101)

showed seven single nucleotide substitutions 297 526 657 703 796 803 and 930

throughout exon 6 and 7 and later one extra mutation outside the coding region at the 3acute

end at nt 1096 (33) The four amino acids substitutions governed by nt 526CrarrG

(Arg176Gly) 703GrarrA (Gly235Ser) 796CrarrA (Leu266Met) and 803GrarrC

(Gly268Ala) discriminate GTA from GTB (4147) while the substitutions 297 657 and

930 are silent

ABO glycosyltransferases can accordingly be described by using the letters A

and B to illustrate the derivation of the amino acid at these four residues GTA would be

represented by AAAA indicating the presence of ArgGlyLeuGly and similarly BBBB

would describe the GTB with GlySerMetAla at residues 176235266 and 268

respectively The substitutions at positions 266 and 268 were shown to be responsible

for the nucleotidedonor specificity of the transferases (47) and the other residues may

have a role in acceptor binding and turnover (48)Some other variants have been

reported afterwards that differ from B1 (B101) by lacking the 930 substitution for B2

(B102) the 657 substitution for B3 (B103) (47) the 526 substitution for B4 (B107) (44)

and 297 for B108 (43)

242 O Subgroups

The blood group O demonstrates the absence of A and B antigens on the RBC

surface in forward blood typing Reverse blood typing indicates the presence of both

anti-A and -B in the plasma The first O allele (O1-1 [O01]) was shown to be identical

to the consensus A allele (A101) except for a nucleotide deletion 261delG in exon 6

This results in a shift in the reading frame giving rise to a truncated protein that alters

the protein sequence after amino acid 88 A stop codon halts translation after amino acid

117 and the resulting protein is enzymatically inactive (41) Some other O1-1-like

alleles that are characterized by the presence of the 261delG and at least one additional

point mutation A second kind of O allele has the same inactivating deletion (261delG)

as the original O allele (O1-1 [O01]) but in addition has nine point mutations spread

34

throughout exons 3 to 7 (4145) and a further 13 mutations have been found amongst

the intron 6 (50)

Additional polymorphisms associated with blood group O have been found up to

approximately 4300 bp from ABO exon 7 (51) some of which correlated with O1 and

O1v alleles (52) Other O alleles not due to 261delG also exist that are caused by other

inactivating mutations along the reading frame The ( O2-1 [O03]) was the first allele

described of this type (5354) which has a critical mutation (802G rarr A) causing an

amino acid change (Gly268Arg) that prevents the enzyme from utilizing the nucleotide

sugar donor (55) This O allele comprises approximately 2 to 5 percent of O alleles in

Caucasian persons but seems to be absent or at least very rare in other populations

(5456) Other rare O alleles such as O3 [O08] that does not have 261delG but instead

contains both the common A2 allele polymorphisms 467CrarrT and C-deletion at nt

1059-1061 and an insertion of an extra guanosine in the 7-guanosine sequence at nt

798-804 (45) Two other rare O alleles lacking 261delG were reported in the Japanese

population by Ogasawara O301 [O14] has the missense mutation 893CrarrT (Ala268Val)

on an A102 background whereas O302 [O15] has the nonsense mutation 927CrarrA

(Tyr309Stop) on an A101 background (43)

243 Weak subgroups

In addition to the major phenotypes characterized by either strong or absent

haemagglutination with anti-A-A1 and -B reagents the ABO blood group system also

includes phenotypes in which erythrocytes react weakly with the anti-A and ndashB

reagents for example A3 Ax Afinn Ael B3 Bx Bv Bel cis-AB (57)

It is difficult to determine clearly their specific ABO subgroup by conventional

serological methods The weak subgroups are important in more than one way First

they risk complicating patient and donor blood group determination At worst the

wrong group can be assigned if eg a weak antigen is missed Second they allow us to

characterize and understand glycosyltransferase mechanisms by studying the results of

mutations in the underlying alleles

35

2431 Weak A alleles

The RBCs from individuals with the A3 phenotype agglutinate strongly with

anti-A and anti-AB in vitro but show a large number of free cells One A3B individual

had a novel point mutation 871GrarrA (Asp291Asn) on the A1 [A101] (58) and this

allele was named A301 Ax alleles are responsible for the rare subgroup Ax and the

RBCs typically show a weak positive reaction with anti-AB and anti-H There are four

base substitutions involved in these alleles 646TrarrA (Phe216Ile) 681GrarrA (silent)

771CrarrT (silent) 829GrarrA (Val277Met) The encoded transferase is expected to be 37

aa longer than the normal consensus allele and 16 aa longer than A2-encoded

transferase The serological characteristics of some other minor phenotypes included

among A subgroups eg Aend Am Ay Afinn Abantu and the collective description Aw are

more unclear Table 22 shows the serological reaction patterns of weak A alleles

Table 22 Serological reaction patterns

A negative reaction is noted by 0 and positive reactions are denoted from + (very weak

agglutination) to 4+ (maximal agglutination)mf mixed field agglutination

2432 Weak B alleles

B variants are much more uncommon than A variants this may reflect the

relatively low frequency of the B blood group in many populations These phenotypes

Subgroup

of A

RBC reactions with Anti-A1

in serum

Anti-A Anti-AB Anti-A1 Anti-H

A1 4+ 4+ 4+ 0 No

A2 4+ 4+ 0 4+ Sometimes

Aint 4+ 4+ 2+3+ 2+3+ No

A3 2++mf 2++ mf 0 4+ No

Ax 0+ 2++ 0 4+ Often

Ael 0 0 0 4+ Sometimes

Aend + + 0 4+ Sometimes

Afinn + + 0 4+ Yes

Abantu +(+) +(+) 0 4+ Yes

Am 0+ 0+ 0 4+ No

Ay 0 0 0 4+ No

36

often appear to result from missense mutations at the ABO locus causing single aa

changes in the GTB Characteristics of some B variants are summarized in Table 23

(57)

Table 23 Characteristics of some more frequent B weak phenotypes

mf mixed field w very weak agglutination

The B3 phenotype have missense mutation 1054CrarrT (Arg352Trp) on a B1

(B101) background and named B301 (58) A Bx allele responsible for the Bx phenotype

had a point mutation at nt 871GrarrA (Asp291Asn) (59) which was also found in an A3

sample (58) The Bel phenotype was divided into two suballeles Bel-1 and Bel-2 (Bel01

and Bel02) which had substitutions at 641TrarrG (Met214Arg) and 669GrarrT

(Glu223Asp) respectively (59) these missense mutations reduce the enzymatic

activities of the GTB Ogasawara et al (1996) also found another B3 allele B302 which

differs from B consensus by two nucleotide substitutions 646TrarrA (Phe216Ile) and

657TrarrC

CisAB alleles rare phenotype was described by Seyfried in 1964 It represents a

very interesting phenomenon that proved that it is possible for a child with blood group

O to have a parent with blood group AB Seyfried hypothesized that both A and B

determinants of the AB blood group could be located on the same chromosome Later

on this hypothesis confirmed in that the existence of an exceptional ABO allele

encoding a glycosyltransferase is indeed able to produce both A and B enzymes at the

same time (57) Cis-ABO1 was sequenced and showed the substitution 803GrarrC

(Gly268Ala) on the A1-2 (A102) background and thus can be described as AAAB (60)

Another allele named cis-ABO2 was discovered when a Vietnamese man who was to

undergo organ transplantation showed irregular blood grouping results The sequencing

Subgroup

of B

RBC reactions with Anti-B

in serum

Anti-A Anti-AB Anti-B Anti-H

B 0 ++++ ++++ ++ None

B3 1 mf mf +++ None

Bx 1 w w +++ Yes

Bel 0 w 0w +++ None

Bm 0 0 0 +++ Sometimes

37

showed that his ABO genes were nearly identical to the normal B allele except for a

796ArarrC (Met266Leu) substitution (61)

25 ABO antibodies

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after

stimulation by a specific antigen The antibody formed binds to the specific antigen in

order to mark the antigen for destruction The type of antigenic exposure occurring in

the body determines if the antibody is a naturally occurring or immune antibody The

term lsquonaturally occurringrsquo is used for blood group antibodies produced in individuals

who have never been transfused with red cells carrying the relevant antigen or been

pregnant with a fetus carrying the relevant antigen Naturally occurring antibodies can

be formed after exposure to environmental agents that are similar to red cell antigens

such as bacteria dust or pollen (62)

Most of these antibodies are not clinically significant with the exception of ABO

antibodies It is possible that food and environmental antigens (bacterial viral or plant

antigens) have epitopes similar enough to A and B glycoprotein antigens The

antibodies created against these environmental antigens in the first years of life can

cross-react with ABO-incompatible red blood cells (RBCs) that it comes in contact with

during blood transfusion later in life Anti-A antibodies are hypothesized to originate

from immune response towards influenza virus whose epitopes are similar enough to

the α-D-N-galactosamine on the A glycoprotein to be able to elicit a cross-reaction

Anti-B antibodies are hypothesized to originate from antibodies produced against

Gram-negative bacteria such as E coli cross-reacting with the α-D-galactose on the B

glycoprotein (49) Anti-A and anti-B antibodies (called isohaemagglutinins) which are

not present in the newborn appear in the first years of life They are isoantibodies that

are produced by an individual against antigens produced by members of the same

species (isoantigens) Anti-A and anti-B antibodies are usually IgM type which are not

able to pass through the placenta to the fetal blood circulation and react best at room

temperature or lower The ABO antibodies may also be found in various body fluids

including saliva milk cervical secretions tears and cysts These antibodies are

detected at about 3 months and increase their titer until the 5th

to 10th

year of life

8

Figure 21 Biosynthesis of ABO Antigens (2)

The major alleles at the ABO locus are A B and O and to-date a number of ABO

blood groups variants have been reported with approximately 250 different alleles

registered in the Blood Group antigen gene Mutation database BGMUT (21)

221 H antigen

In nature there exists at least four H antigens on glycolipids and glycoproteins

that are recognized by GTA and GTB (22)The most common are the type I and the type

II H antigens (23)H antigen is produced when an α12-Lfucosyltransferase catalyses the

transfer of L-fucose from a guanosine diphosphate (GDP)-L-fucose donor to the C-2

position of the terminal galactose of one of the precursor structures (Figure 21) Two

α12-L-fucosyltransferases produced by two genes FUT1 (H) and FUT2 (Se) catalyze

9

the biosynthesis of H-active structures in different tissues (24) mainly in epithelial cells

and body fluids such as saliva

The H antigen (Figure 22) is the natural precursor of A and B antigens and its

fucose residue is required for A and B glycosyltransferases to recognize it as the

acceptor and transfer GalNAc or Gal to its terminal Gal Depending on the disaccharide

precursor core chain on which ABH determinants are synthesized they can be further

divided into different types

Figure 22 H antigen structure

Everybody expresses H antigen on his red cells but only about 80 of

Europeans have H antigen in their body secretions These people are called ABH

secretors because if they have an A andor B gene they also secrete A andor B

antigens The remaining 20 are called ABH non-secretors as they do not secrete H A

or B regardless of ABO genotype (2526)

222 A and B antigens

A and B antigens can be produced by the presence of the appropriate A- or B

transferase (Figure 21) The A gene product is an α13-N-acetyl galactosaminyl-

transferase which transfers N-acetyl-galactosamine from a uridine diphosphate (UDP)-

N acetylgalactosamine donor to the fucosylated galactosyl residue of H antigen The B

gene product an α 13-D-galactosyltransferase transfers D-galactose from UDP-

galactose to the fucosylated galactose of H A and B are alleles at the ABO locus on

chromosome 9 A third allele O does not produce an active enzyme and in persons

homozygous for O the H antigen remains unmodified (Figure 23)

31

Figure 23 Summary of A B and O(H) blood group structures and their synthesis(28)

The A and B antigens are carbohydrate molecules built stepwise from

saccharides such as galactosamine (GalNAc) glucosamine (GlcNAc) fucose (Fuc)

galactose (Gal) and glucose (Glc) H-antigen is the requisite precursor and

galactosylamine (GalNAcα3) or galactosyl (Galα3) with α1-3 linkage onto H antigen

become the A and B antigens respectively (514) (Figure 24)

Figure 24 A and B antigen structure

The majority of ABO antigens on red cells are linked to glycoproteins

(approximately 70) thus they very much influence the blood group activity on the red

cells At each step in the biosynthesis of ABO antigens and carbohydrate chains

synthesis is facilitated by glycosyltransferases which are competing for available

precursors and substrates This represents approximately 80 of the total complement

of red cell ABH determinants Another 5 times 105 ABH determinants localize to the red

33

cell glucose transport protein (Band 45) Small numbers of ABH antigens are also

expressed by other red cell glycoproteins (28)

23 ABO Glycosyltransferases

Glycosyltransferases (GTs) constitute a large family of enzymes that are

involved in the biosynthesis of oligosaccharides polysaccharides and glycoconjugates

(29)Particularly abundant are the GTs that transfer a sugar residue from an activated

nucleotide sugar donor to specific acceptor molecules forming glycosidic bonds

Glycosyltransferases are classified into 87 different families based on substrateproduct

stereochemistry according to the CAZy database (30)

Knowledge of the sequences of the ABO genes (9) have established that

mammalian Glycosyltransferases A and Glycosyltransferases B are type II integral

membrane proteins containing 354 amino acids and are localized in the lumen of the

Golgi apparatus These enzymes typically have a short amino-terminal cytoplasmic tail

a hydrophobic membrane domain a short protease-sensitive stem region and a large

catalytic domain that includes the carboxy terminus(31) GTA and GTB are very similar

in the coding regions and the soluble enzyme can be found in serum (32)urine (33)and

milk (34)The enzyme contains an acceptor recognition domain that binds H antigen and

a donor recognition domain that binds UDP-GalNAc or UDP-Gal GTA and GTB

require the metal ion Mn++ (manganese) for activity (35)

Glycosyltransferases are antigenic structures Human antibodies to blood group

transferases are often produced following organ transplantation (36-39)

24 ABO Subgroups

241 A and B Subgroups

An ABO blood group subtype is called a subgroup andor a variant Subgroups

of ABO are distinguished by decreased amounts of A B or O (H) antigens on red blood

cells The most common are subgroups of A and B Blood type A appears to have the

most variation in subgroups The two most common subgroups of blood group A are A1

and A2 expressing on average 1 million and 250 000 A determinants respectively (40)

32

2411 A Subgroups

The A1 (A101) allele is the reference allele often denoted as the consensus

sequence in an ABO genotype context (9)but has additionally eight variant alleles

There are variants of A1 alleles one of which is very common in Asian populations with

a 467CrarrT (A102) polymorphism resulting in the substitution Pro156Leu (41)Two

minor A1 alleles (A103 and A104) have been described and differ as follows The first

one also has the 467CrarrT point mutation but an additional silent mutation 567CrarrT

the second allele contains a silent polymorphism in nucleotide 297ArarrG (42) A105 is

like A102 with the same mutation in exon 7 467CrarrT but analysis of intron 6 showed

additional single-nucleotide polymorphism (SNP) compared to A102 Another A1 allele

(A106) contains both 297ArarrG and 467CrarrT (43)

The A2 subgroup is the most common A phenotype after the A1 subgroup The

main genetic difference between A1 and A2 alleles is one point mutation in exon 7

467CrarrT (Pro156Leu) and a deletion of one of the three cytosines at nt 1059-1061

(CCC to CC) The latter mutation results in an extension of the reading frame by 64

nucleotides This deletion occurs in the codon before the translation stop codon (TGA)

resulting in a gene product with an extra 21 amino acid at its C-terminus The

glycosyltransferases encoded by the A2 allele have lower efficiency leading to a weaker

A phenotype The enzyme activity is decreased by 30-50 times compared to A1

(22)Other variants of A2 alleles have subsequently been elucidated (A202-206) Three

A2-like alleles were shown to have three different single mutations near the 3acute end of

exon 7 by Ogasawara A2-2 (A202) contains 1054CrarrT (Arg352Trp) A2-3 (A203) with

1054CrarrG (Arg352Gly) (42) and A2-4 (A205) with both 467CrarrT and an additional

new mutation 1009ArarrG (Arg337Gly) (44) A204 with four common B-related base

substitutions (297ArarrG 526CrarrG 657CrarrT and 703GrarrA) seems to be a hybrid with

two extra substitutions 771CrarrT (silent mutation) and 829GrarrA (Val277Met) One

other rare A2 subgroup A2-5 (A206) carried only the single deletion (1061delC)

(4546)

31

2412 B Subgroups

Subtypes of blood type B are classified by the quantity of B antigen and the

amount of B antigen decreases in the order B B3 Bx Bm Bel B1 allele (B101)

showed seven single nucleotide substitutions 297 526 657 703 796 803 and 930

throughout exon 6 and 7 and later one extra mutation outside the coding region at the 3acute

end at nt 1096 (33) The four amino acids substitutions governed by nt 526CrarrG

(Arg176Gly) 703GrarrA (Gly235Ser) 796CrarrA (Leu266Met) and 803GrarrC

(Gly268Ala) discriminate GTA from GTB (4147) while the substitutions 297 657 and

930 are silent

ABO glycosyltransferases can accordingly be described by using the letters A

and B to illustrate the derivation of the amino acid at these four residues GTA would be

represented by AAAA indicating the presence of ArgGlyLeuGly and similarly BBBB

would describe the GTB with GlySerMetAla at residues 176235266 and 268

respectively The substitutions at positions 266 and 268 were shown to be responsible

for the nucleotidedonor specificity of the transferases (47) and the other residues may

have a role in acceptor binding and turnover (48)Some other variants have been

reported afterwards that differ from B1 (B101) by lacking the 930 substitution for B2

(B102) the 657 substitution for B3 (B103) (47) the 526 substitution for B4 (B107) (44)

and 297 for B108 (43)

242 O Subgroups

The blood group O demonstrates the absence of A and B antigens on the RBC

surface in forward blood typing Reverse blood typing indicates the presence of both

anti-A and -B in the plasma The first O allele (O1-1 [O01]) was shown to be identical

to the consensus A allele (A101) except for a nucleotide deletion 261delG in exon 6

This results in a shift in the reading frame giving rise to a truncated protein that alters

the protein sequence after amino acid 88 A stop codon halts translation after amino acid

117 and the resulting protein is enzymatically inactive (41) Some other O1-1-like

alleles that are characterized by the presence of the 261delG and at least one additional

point mutation A second kind of O allele has the same inactivating deletion (261delG)

as the original O allele (O1-1 [O01]) but in addition has nine point mutations spread

34

throughout exons 3 to 7 (4145) and a further 13 mutations have been found amongst

the intron 6 (50)

Additional polymorphisms associated with blood group O have been found up to

approximately 4300 bp from ABO exon 7 (51) some of which correlated with O1 and

O1v alleles (52) Other O alleles not due to 261delG also exist that are caused by other

inactivating mutations along the reading frame The ( O2-1 [O03]) was the first allele

described of this type (5354) which has a critical mutation (802G rarr A) causing an

amino acid change (Gly268Arg) that prevents the enzyme from utilizing the nucleotide

sugar donor (55) This O allele comprises approximately 2 to 5 percent of O alleles in

Caucasian persons but seems to be absent or at least very rare in other populations

(5456) Other rare O alleles such as O3 [O08] that does not have 261delG but instead

contains both the common A2 allele polymorphisms 467CrarrT and C-deletion at nt

1059-1061 and an insertion of an extra guanosine in the 7-guanosine sequence at nt

798-804 (45) Two other rare O alleles lacking 261delG were reported in the Japanese

population by Ogasawara O301 [O14] has the missense mutation 893CrarrT (Ala268Val)

on an A102 background whereas O302 [O15] has the nonsense mutation 927CrarrA

(Tyr309Stop) on an A101 background (43)

243 Weak subgroups

In addition to the major phenotypes characterized by either strong or absent

haemagglutination with anti-A-A1 and -B reagents the ABO blood group system also

includes phenotypes in which erythrocytes react weakly with the anti-A and ndashB

reagents for example A3 Ax Afinn Ael B3 Bx Bv Bel cis-AB (57)

It is difficult to determine clearly their specific ABO subgroup by conventional

serological methods The weak subgroups are important in more than one way First

they risk complicating patient and donor blood group determination At worst the

wrong group can be assigned if eg a weak antigen is missed Second they allow us to

characterize and understand glycosyltransferase mechanisms by studying the results of

mutations in the underlying alleles

35

2431 Weak A alleles

The RBCs from individuals with the A3 phenotype agglutinate strongly with

anti-A and anti-AB in vitro but show a large number of free cells One A3B individual

had a novel point mutation 871GrarrA (Asp291Asn) on the A1 [A101] (58) and this

allele was named A301 Ax alleles are responsible for the rare subgroup Ax and the

RBCs typically show a weak positive reaction with anti-AB and anti-H There are four

base substitutions involved in these alleles 646TrarrA (Phe216Ile) 681GrarrA (silent)

771CrarrT (silent) 829GrarrA (Val277Met) The encoded transferase is expected to be 37

aa longer than the normal consensus allele and 16 aa longer than A2-encoded

transferase The serological characteristics of some other minor phenotypes included

among A subgroups eg Aend Am Ay Afinn Abantu and the collective description Aw are

more unclear Table 22 shows the serological reaction patterns of weak A alleles

Table 22 Serological reaction patterns

A negative reaction is noted by 0 and positive reactions are denoted from + (very weak

agglutination) to 4+ (maximal agglutination)mf mixed field agglutination

2432 Weak B alleles

B variants are much more uncommon than A variants this may reflect the

relatively low frequency of the B blood group in many populations These phenotypes

Subgroup

of A

RBC reactions with Anti-A1

in serum

Anti-A Anti-AB Anti-A1 Anti-H

A1 4+ 4+ 4+ 0 No

A2 4+ 4+ 0 4+ Sometimes

Aint 4+ 4+ 2+3+ 2+3+ No

A3 2++mf 2++ mf 0 4+ No

Ax 0+ 2++ 0 4+ Often

Ael 0 0 0 4+ Sometimes

Aend + + 0 4+ Sometimes

Afinn + + 0 4+ Yes

Abantu +(+) +(+) 0 4+ Yes

Am 0+ 0+ 0 4+ No

Ay 0 0 0 4+ No

36

often appear to result from missense mutations at the ABO locus causing single aa

changes in the GTB Characteristics of some B variants are summarized in Table 23

(57)

Table 23 Characteristics of some more frequent B weak phenotypes

mf mixed field w very weak agglutination

The B3 phenotype have missense mutation 1054CrarrT (Arg352Trp) on a B1

(B101) background and named B301 (58) A Bx allele responsible for the Bx phenotype

had a point mutation at nt 871GrarrA (Asp291Asn) (59) which was also found in an A3

sample (58) The Bel phenotype was divided into two suballeles Bel-1 and Bel-2 (Bel01

and Bel02) which had substitutions at 641TrarrG (Met214Arg) and 669GrarrT

(Glu223Asp) respectively (59) these missense mutations reduce the enzymatic

activities of the GTB Ogasawara et al (1996) also found another B3 allele B302 which

differs from B consensus by two nucleotide substitutions 646TrarrA (Phe216Ile) and

657TrarrC

CisAB alleles rare phenotype was described by Seyfried in 1964 It represents a

very interesting phenomenon that proved that it is possible for a child with blood group

O to have a parent with blood group AB Seyfried hypothesized that both A and B

determinants of the AB blood group could be located on the same chromosome Later

on this hypothesis confirmed in that the existence of an exceptional ABO allele

encoding a glycosyltransferase is indeed able to produce both A and B enzymes at the

same time (57) Cis-ABO1 was sequenced and showed the substitution 803GrarrC

(Gly268Ala) on the A1-2 (A102) background and thus can be described as AAAB (60)

Another allele named cis-ABO2 was discovered when a Vietnamese man who was to

undergo organ transplantation showed irregular blood grouping results The sequencing

Subgroup

of B

RBC reactions with Anti-B

in serum

Anti-A Anti-AB Anti-B Anti-H

B 0 ++++ ++++ ++ None

B3 1 mf mf +++ None

Bx 1 w w +++ Yes

Bel 0 w 0w +++ None

Bm 0 0 0 +++ Sometimes

37

showed that his ABO genes were nearly identical to the normal B allele except for a

796ArarrC (Met266Leu) substitution (61)

25 ABO antibodies

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after

stimulation by a specific antigen The antibody formed binds to the specific antigen in

order to mark the antigen for destruction The type of antigenic exposure occurring in

the body determines if the antibody is a naturally occurring or immune antibody The

term lsquonaturally occurringrsquo is used for blood group antibodies produced in individuals

who have never been transfused with red cells carrying the relevant antigen or been

pregnant with a fetus carrying the relevant antigen Naturally occurring antibodies can

be formed after exposure to environmental agents that are similar to red cell antigens

such as bacteria dust or pollen (62)

Most of these antibodies are not clinically significant with the exception of ABO

antibodies It is possible that food and environmental antigens (bacterial viral or plant

antigens) have epitopes similar enough to A and B glycoprotein antigens The

antibodies created against these environmental antigens in the first years of life can

cross-react with ABO-incompatible red blood cells (RBCs) that it comes in contact with

during blood transfusion later in life Anti-A antibodies are hypothesized to originate

from immune response towards influenza virus whose epitopes are similar enough to

the α-D-N-galactosamine on the A glycoprotein to be able to elicit a cross-reaction

Anti-B antibodies are hypothesized to originate from antibodies produced against

Gram-negative bacteria such as E coli cross-reacting with the α-D-galactose on the B

glycoprotein (49) Anti-A and anti-B antibodies (called isohaemagglutinins) which are

not present in the newborn appear in the first years of life They are isoantibodies that

are produced by an individual against antigens produced by members of the same

species (isoantigens) Anti-A and anti-B antibodies are usually IgM type which are not

able to pass through the placenta to the fetal blood circulation and react best at room

temperature or lower The ABO antibodies may also be found in various body fluids

including saliva milk cervical secretions tears and cysts These antibodies are

detected at about 3 months and increase their titer until the 5th

to 10th

year of life

9

the biosynthesis of H-active structures in different tissues (24) mainly in epithelial cells

and body fluids such as saliva

The H antigen (Figure 22) is the natural precursor of A and B antigens and its

fucose residue is required for A and B glycosyltransferases to recognize it as the

acceptor and transfer GalNAc or Gal to its terminal Gal Depending on the disaccharide

precursor core chain on which ABH determinants are synthesized they can be further

divided into different types

Figure 22 H antigen structure

Everybody expresses H antigen on his red cells but only about 80 of

Europeans have H antigen in their body secretions These people are called ABH

secretors because if they have an A andor B gene they also secrete A andor B

antigens The remaining 20 are called ABH non-secretors as they do not secrete H A

or B regardless of ABO genotype (2526)

222 A and B antigens

A and B antigens can be produced by the presence of the appropriate A- or B

transferase (Figure 21) The A gene product is an α13-N-acetyl galactosaminyl-

transferase which transfers N-acetyl-galactosamine from a uridine diphosphate (UDP)-

N acetylgalactosamine donor to the fucosylated galactosyl residue of H antigen The B

gene product an α 13-D-galactosyltransferase transfers D-galactose from UDP-

galactose to the fucosylated galactose of H A and B are alleles at the ABO locus on

chromosome 9 A third allele O does not produce an active enzyme and in persons

homozygous for O the H antigen remains unmodified (Figure 23)

31

Figure 23 Summary of A B and O(H) blood group structures and their synthesis(28)

The A and B antigens are carbohydrate molecules built stepwise from

saccharides such as galactosamine (GalNAc) glucosamine (GlcNAc) fucose (Fuc)

galactose (Gal) and glucose (Glc) H-antigen is the requisite precursor and

galactosylamine (GalNAcα3) or galactosyl (Galα3) with α1-3 linkage onto H antigen

become the A and B antigens respectively (514) (Figure 24)

Figure 24 A and B antigen structure

The majority of ABO antigens on red cells are linked to glycoproteins

(approximately 70) thus they very much influence the blood group activity on the red

cells At each step in the biosynthesis of ABO antigens and carbohydrate chains

synthesis is facilitated by glycosyltransferases which are competing for available

precursors and substrates This represents approximately 80 of the total complement

of red cell ABH determinants Another 5 times 105 ABH determinants localize to the red

33

cell glucose transport protein (Band 45) Small numbers of ABH antigens are also

expressed by other red cell glycoproteins (28)

23 ABO Glycosyltransferases

Glycosyltransferases (GTs) constitute a large family of enzymes that are

involved in the biosynthesis of oligosaccharides polysaccharides and glycoconjugates

(29)Particularly abundant are the GTs that transfer a sugar residue from an activated

nucleotide sugar donor to specific acceptor molecules forming glycosidic bonds

Glycosyltransferases are classified into 87 different families based on substrateproduct

stereochemistry according to the CAZy database (30)

Knowledge of the sequences of the ABO genes (9) have established that

mammalian Glycosyltransferases A and Glycosyltransferases B are type II integral

membrane proteins containing 354 amino acids and are localized in the lumen of the

Golgi apparatus These enzymes typically have a short amino-terminal cytoplasmic tail

a hydrophobic membrane domain a short protease-sensitive stem region and a large

catalytic domain that includes the carboxy terminus(31) GTA and GTB are very similar

in the coding regions and the soluble enzyme can be found in serum (32)urine (33)and

milk (34)The enzyme contains an acceptor recognition domain that binds H antigen and

a donor recognition domain that binds UDP-GalNAc or UDP-Gal GTA and GTB

require the metal ion Mn++ (manganese) for activity (35)

Glycosyltransferases are antigenic structures Human antibodies to blood group

transferases are often produced following organ transplantation (36-39)

24 ABO Subgroups

241 A and B Subgroups

An ABO blood group subtype is called a subgroup andor a variant Subgroups

of ABO are distinguished by decreased amounts of A B or O (H) antigens on red blood

cells The most common are subgroups of A and B Blood type A appears to have the

most variation in subgroups The two most common subgroups of blood group A are A1

and A2 expressing on average 1 million and 250 000 A determinants respectively (40)

32

2411 A Subgroups

The A1 (A101) allele is the reference allele often denoted as the consensus

sequence in an ABO genotype context (9)but has additionally eight variant alleles

There are variants of A1 alleles one of which is very common in Asian populations with

a 467CrarrT (A102) polymorphism resulting in the substitution Pro156Leu (41)Two

minor A1 alleles (A103 and A104) have been described and differ as follows The first

one also has the 467CrarrT point mutation but an additional silent mutation 567CrarrT

the second allele contains a silent polymorphism in nucleotide 297ArarrG (42) A105 is

like A102 with the same mutation in exon 7 467CrarrT but analysis of intron 6 showed

additional single-nucleotide polymorphism (SNP) compared to A102 Another A1 allele

(A106) contains both 297ArarrG and 467CrarrT (43)

The A2 subgroup is the most common A phenotype after the A1 subgroup The

main genetic difference between A1 and A2 alleles is one point mutation in exon 7

467CrarrT (Pro156Leu) and a deletion of one of the three cytosines at nt 1059-1061

(CCC to CC) The latter mutation results in an extension of the reading frame by 64

nucleotides This deletion occurs in the codon before the translation stop codon (TGA)

resulting in a gene product with an extra 21 amino acid at its C-terminus The

glycosyltransferases encoded by the A2 allele have lower efficiency leading to a weaker

A phenotype The enzyme activity is decreased by 30-50 times compared to A1

(22)Other variants of A2 alleles have subsequently been elucidated (A202-206) Three

A2-like alleles were shown to have three different single mutations near the 3acute end of

exon 7 by Ogasawara A2-2 (A202) contains 1054CrarrT (Arg352Trp) A2-3 (A203) with

1054CrarrG (Arg352Gly) (42) and A2-4 (A205) with both 467CrarrT and an additional

new mutation 1009ArarrG (Arg337Gly) (44) A204 with four common B-related base

substitutions (297ArarrG 526CrarrG 657CrarrT and 703GrarrA) seems to be a hybrid with

two extra substitutions 771CrarrT (silent mutation) and 829GrarrA (Val277Met) One

other rare A2 subgroup A2-5 (A206) carried only the single deletion (1061delC)

(4546)

31

2412 B Subgroups

Subtypes of blood type B are classified by the quantity of B antigen and the

amount of B antigen decreases in the order B B3 Bx Bm Bel B1 allele (B101)

showed seven single nucleotide substitutions 297 526 657 703 796 803 and 930

throughout exon 6 and 7 and later one extra mutation outside the coding region at the 3acute

end at nt 1096 (33) The four amino acids substitutions governed by nt 526CrarrG

(Arg176Gly) 703GrarrA (Gly235Ser) 796CrarrA (Leu266Met) and 803GrarrC

(Gly268Ala) discriminate GTA from GTB (4147) while the substitutions 297 657 and

930 are silent

ABO glycosyltransferases can accordingly be described by using the letters A

and B to illustrate the derivation of the amino acid at these four residues GTA would be

represented by AAAA indicating the presence of ArgGlyLeuGly and similarly BBBB

would describe the GTB with GlySerMetAla at residues 176235266 and 268

respectively The substitutions at positions 266 and 268 were shown to be responsible

for the nucleotidedonor specificity of the transferases (47) and the other residues may

have a role in acceptor binding and turnover (48)Some other variants have been

reported afterwards that differ from B1 (B101) by lacking the 930 substitution for B2

(B102) the 657 substitution for B3 (B103) (47) the 526 substitution for B4 (B107) (44)

and 297 for B108 (43)

242 O Subgroups

The blood group O demonstrates the absence of A and B antigens on the RBC

surface in forward blood typing Reverse blood typing indicates the presence of both

anti-A and -B in the plasma The first O allele (O1-1 [O01]) was shown to be identical

to the consensus A allele (A101) except for a nucleotide deletion 261delG in exon 6

This results in a shift in the reading frame giving rise to a truncated protein that alters

the protein sequence after amino acid 88 A stop codon halts translation after amino acid

117 and the resulting protein is enzymatically inactive (41) Some other O1-1-like

alleles that are characterized by the presence of the 261delG and at least one additional

point mutation A second kind of O allele has the same inactivating deletion (261delG)

as the original O allele (O1-1 [O01]) but in addition has nine point mutations spread

34

throughout exons 3 to 7 (4145) and a further 13 mutations have been found amongst

the intron 6 (50)

Additional polymorphisms associated with blood group O have been found up to

approximately 4300 bp from ABO exon 7 (51) some of which correlated with O1 and

O1v alleles (52) Other O alleles not due to 261delG also exist that are caused by other

inactivating mutations along the reading frame The ( O2-1 [O03]) was the first allele

described of this type (5354) which has a critical mutation (802G rarr A) causing an

amino acid change (Gly268Arg) that prevents the enzyme from utilizing the nucleotide

sugar donor (55) This O allele comprises approximately 2 to 5 percent of O alleles in

Caucasian persons but seems to be absent or at least very rare in other populations

(5456) Other rare O alleles such as O3 [O08] that does not have 261delG but instead

contains both the common A2 allele polymorphisms 467CrarrT and C-deletion at nt

1059-1061 and an insertion of an extra guanosine in the 7-guanosine sequence at nt

798-804 (45) Two other rare O alleles lacking 261delG were reported in the Japanese

population by Ogasawara O301 [O14] has the missense mutation 893CrarrT (Ala268Val)

on an A102 background whereas O302 [O15] has the nonsense mutation 927CrarrA

(Tyr309Stop) on an A101 background (43)

243 Weak subgroups

In addition to the major phenotypes characterized by either strong or absent

haemagglutination with anti-A-A1 and -B reagents the ABO blood group system also

includes phenotypes in which erythrocytes react weakly with the anti-A and ndashB

reagents for example A3 Ax Afinn Ael B3 Bx Bv Bel cis-AB (57)

It is difficult to determine clearly their specific ABO subgroup by conventional

serological methods The weak subgroups are important in more than one way First

they risk complicating patient and donor blood group determination At worst the

wrong group can be assigned if eg a weak antigen is missed Second they allow us to

characterize and understand glycosyltransferase mechanisms by studying the results of

mutations in the underlying alleles

35

2431 Weak A alleles

The RBCs from individuals with the A3 phenotype agglutinate strongly with

anti-A and anti-AB in vitro but show a large number of free cells One A3B individual

had a novel point mutation 871GrarrA (Asp291Asn) on the A1 [A101] (58) and this

allele was named A301 Ax alleles are responsible for the rare subgroup Ax and the

RBCs typically show a weak positive reaction with anti-AB and anti-H There are four

base substitutions involved in these alleles 646TrarrA (Phe216Ile) 681GrarrA (silent)

771CrarrT (silent) 829GrarrA (Val277Met) The encoded transferase is expected to be 37

aa longer than the normal consensus allele and 16 aa longer than A2-encoded

transferase The serological characteristics of some other minor phenotypes included

among A subgroups eg Aend Am Ay Afinn Abantu and the collective description Aw are

more unclear Table 22 shows the serological reaction patterns of weak A alleles

Table 22 Serological reaction patterns

A negative reaction is noted by 0 and positive reactions are denoted from + (very weak

agglutination) to 4+ (maximal agglutination)mf mixed field agglutination

2432 Weak B alleles

B variants are much more uncommon than A variants this may reflect the

relatively low frequency of the B blood group in many populations These phenotypes

Subgroup

of A

RBC reactions with Anti-A1

in serum

Anti-A Anti-AB Anti-A1 Anti-H

A1 4+ 4+ 4+ 0 No

A2 4+ 4+ 0 4+ Sometimes

Aint 4+ 4+ 2+3+ 2+3+ No

A3 2++mf 2++ mf 0 4+ No

Ax 0+ 2++ 0 4+ Often

Ael 0 0 0 4+ Sometimes

Aend + + 0 4+ Sometimes

Afinn + + 0 4+ Yes

Abantu +(+) +(+) 0 4+ Yes

Am 0+ 0+ 0 4+ No

Ay 0 0 0 4+ No

36

often appear to result from missense mutations at the ABO locus causing single aa

changes in the GTB Characteristics of some B variants are summarized in Table 23

(57)

Table 23 Characteristics of some more frequent B weak phenotypes

mf mixed field w very weak agglutination

The B3 phenotype have missense mutation 1054CrarrT (Arg352Trp) on a B1

(B101) background and named B301 (58) A Bx allele responsible for the Bx phenotype

had a point mutation at nt 871GrarrA (Asp291Asn) (59) which was also found in an A3

sample (58) The Bel phenotype was divided into two suballeles Bel-1 and Bel-2 (Bel01

and Bel02) which had substitutions at 641TrarrG (Met214Arg) and 669GrarrT

(Glu223Asp) respectively (59) these missense mutations reduce the enzymatic

activities of the GTB Ogasawara et al (1996) also found another B3 allele B302 which

differs from B consensus by two nucleotide substitutions 646TrarrA (Phe216Ile) and

657TrarrC

CisAB alleles rare phenotype was described by Seyfried in 1964 It represents a

very interesting phenomenon that proved that it is possible for a child with blood group

O to have a parent with blood group AB Seyfried hypothesized that both A and B

determinants of the AB blood group could be located on the same chromosome Later

on this hypothesis confirmed in that the existence of an exceptional ABO allele

encoding a glycosyltransferase is indeed able to produce both A and B enzymes at the

same time (57) Cis-ABO1 was sequenced and showed the substitution 803GrarrC

(Gly268Ala) on the A1-2 (A102) background and thus can be described as AAAB (60)

Another allele named cis-ABO2 was discovered when a Vietnamese man who was to

undergo organ transplantation showed irregular blood grouping results The sequencing

Subgroup

of B

RBC reactions with Anti-B

in serum

Anti-A Anti-AB Anti-B Anti-H

B 0 ++++ ++++ ++ None

B3 1 mf mf +++ None

Bx 1 w w +++ Yes

Bel 0 w 0w +++ None

Bm 0 0 0 +++ Sometimes

37

showed that his ABO genes were nearly identical to the normal B allele except for a

796ArarrC (Met266Leu) substitution (61)

25 ABO antibodies

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after

stimulation by a specific antigen The antibody formed binds to the specific antigen in

order to mark the antigen for destruction The type of antigenic exposure occurring in

the body determines if the antibody is a naturally occurring or immune antibody The

term lsquonaturally occurringrsquo is used for blood group antibodies produced in individuals

who have never been transfused with red cells carrying the relevant antigen or been

pregnant with a fetus carrying the relevant antigen Naturally occurring antibodies can

be formed after exposure to environmental agents that are similar to red cell antigens

such as bacteria dust or pollen (62)

Most of these antibodies are not clinically significant with the exception of ABO

antibodies It is possible that food and environmental antigens (bacterial viral or plant

antigens) have epitopes similar enough to A and B glycoprotein antigens The

antibodies created against these environmental antigens in the first years of life can

cross-react with ABO-incompatible red blood cells (RBCs) that it comes in contact with

during blood transfusion later in life Anti-A antibodies are hypothesized to originate

from immune response towards influenza virus whose epitopes are similar enough to

the α-D-N-galactosamine on the A glycoprotein to be able to elicit a cross-reaction

Anti-B antibodies are hypothesized to originate from antibodies produced against

Gram-negative bacteria such as E coli cross-reacting with the α-D-galactose on the B

glycoprotein (49) Anti-A and anti-B antibodies (called isohaemagglutinins) which are

not present in the newborn appear in the first years of life They are isoantibodies that

are produced by an individual against antigens produced by members of the same

species (isoantigens) Anti-A and anti-B antibodies are usually IgM type which are not

able to pass through the placenta to the fetal blood circulation and react best at room

temperature or lower The ABO antibodies may also be found in various body fluids

including saliva milk cervical secretions tears and cysts These antibodies are

detected at about 3 months and increase their titer until the 5th

to 10th

year of life

31

Figure 23 Summary of A B and O(H) blood group structures and their synthesis(28)

The A and B antigens are carbohydrate molecules built stepwise from

saccharides such as galactosamine (GalNAc) glucosamine (GlcNAc) fucose (Fuc)

galactose (Gal) and glucose (Glc) H-antigen is the requisite precursor and

galactosylamine (GalNAcα3) or galactosyl (Galα3) with α1-3 linkage onto H antigen

become the A and B antigens respectively (514) (Figure 24)

Figure 24 A and B antigen structure

The majority of ABO antigens on red cells are linked to glycoproteins

(approximately 70) thus they very much influence the blood group activity on the red

cells At each step in the biosynthesis of ABO antigens and carbohydrate chains

synthesis is facilitated by glycosyltransferases which are competing for available

precursors and substrates This represents approximately 80 of the total complement

of red cell ABH determinants Another 5 times 105 ABH determinants localize to the red

33

cell glucose transport protein (Band 45) Small numbers of ABH antigens are also

expressed by other red cell glycoproteins (28)

23 ABO Glycosyltransferases

Glycosyltransferases (GTs) constitute a large family of enzymes that are

involved in the biosynthesis of oligosaccharides polysaccharides and glycoconjugates

(29)Particularly abundant are the GTs that transfer a sugar residue from an activated

nucleotide sugar donor to specific acceptor molecules forming glycosidic bonds

Glycosyltransferases are classified into 87 different families based on substrateproduct

stereochemistry according to the CAZy database (30)

Knowledge of the sequences of the ABO genes (9) have established that

mammalian Glycosyltransferases A and Glycosyltransferases B are type II integral

membrane proteins containing 354 amino acids and are localized in the lumen of the

Golgi apparatus These enzymes typically have a short amino-terminal cytoplasmic tail

a hydrophobic membrane domain a short protease-sensitive stem region and a large

catalytic domain that includes the carboxy terminus(31) GTA and GTB are very similar

in the coding regions and the soluble enzyme can be found in serum (32)urine (33)and

milk (34)The enzyme contains an acceptor recognition domain that binds H antigen and

a donor recognition domain that binds UDP-GalNAc or UDP-Gal GTA and GTB

require the metal ion Mn++ (manganese) for activity (35)

Glycosyltransferases are antigenic structures Human antibodies to blood group

transferases are often produced following organ transplantation (36-39)

24 ABO Subgroups

241 A and B Subgroups

An ABO blood group subtype is called a subgroup andor a variant Subgroups

of ABO are distinguished by decreased amounts of A B or O (H) antigens on red blood

cells The most common are subgroups of A and B Blood type A appears to have the

most variation in subgroups The two most common subgroups of blood group A are A1

and A2 expressing on average 1 million and 250 000 A determinants respectively (40)

32

2411 A Subgroups

The A1 (A101) allele is the reference allele often denoted as the consensus

sequence in an ABO genotype context (9)but has additionally eight variant alleles

There are variants of A1 alleles one of which is very common in Asian populations with

a 467CrarrT (A102) polymorphism resulting in the substitution Pro156Leu (41)Two

minor A1 alleles (A103 and A104) have been described and differ as follows The first

one also has the 467CrarrT point mutation but an additional silent mutation 567CrarrT

the second allele contains a silent polymorphism in nucleotide 297ArarrG (42) A105 is

like A102 with the same mutation in exon 7 467CrarrT but analysis of intron 6 showed

additional single-nucleotide polymorphism (SNP) compared to A102 Another A1 allele

(A106) contains both 297ArarrG and 467CrarrT (43)

The A2 subgroup is the most common A phenotype after the A1 subgroup The

main genetic difference between A1 and A2 alleles is one point mutation in exon 7

467CrarrT (Pro156Leu) and a deletion of one of the three cytosines at nt 1059-1061

(CCC to CC) The latter mutation results in an extension of the reading frame by 64

nucleotides This deletion occurs in the codon before the translation stop codon (TGA)

resulting in a gene product with an extra 21 amino acid at its C-terminus The

glycosyltransferases encoded by the A2 allele have lower efficiency leading to a weaker

A phenotype The enzyme activity is decreased by 30-50 times compared to A1

(22)Other variants of A2 alleles have subsequently been elucidated (A202-206) Three

A2-like alleles were shown to have three different single mutations near the 3acute end of

exon 7 by Ogasawara A2-2 (A202) contains 1054CrarrT (Arg352Trp) A2-3 (A203) with

1054CrarrG (Arg352Gly) (42) and A2-4 (A205) with both 467CrarrT and an additional

new mutation 1009ArarrG (Arg337Gly) (44) A204 with four common B-related base

substitutions (297ArarrG 526CrarrG 657CrarrT and 703GrarrA) seems to be a hybrid with

two extra substitutions 771CrarrT (silent mutation) and 829GrarrA (Val277Met) One

other rare A2 subgroup A2-5 (A206) carried only the single deletion (1061delC)

(4546)

31

2412 B Subgroups

Subtypes of blood type B are classified by the quantity of B antigen and the

amount of B antigen decreases in the order B B3 Bx Bm Bel B1 allele (B101)

showed seven single nucleotide substitutions 297 526 657 703 796 803 and 930

throughout exon 6 and 7 and later one extra mutation outside the coding region at the 3acute

end at nt 1096 (33) The four amino acids substitutions governed by nt 526CrarrG

(Arg176Gly) 703GrarrA (Gly235Ser) 796CrarrA (Leu266Met) and 803GrarrC

(Gly268Ala) discriminate GTA from GTB (4147) while the substitutions 297 657 and

930 are silent

ABO glycosyltransferases can accordingly be described by using the letters A

and B to illustrate the derivation of the amino acid at these four residues GTA would be

represented by AAAA indicating the presence of ArgGlyLeuGly and similarly BBBB

would describe the GTB with GlySerMetAla at residues 176235266 and 268

respectively The substitutions at positions 266 and 268 were shown to be responsible

for the nucleotidedonor specificity of the transferases (47) and the other residues may

have a role in acceptor binding and turnover (48)Some other variants have been

reported afterwards that differ from B1 (B101) by lacking the 930 substitution for B2

(B102) the 657 substitution for B3 (B103) (47) the 526 substitution for B4 (B107) (44)

and 297 for B108 (43)

242 O Subgroups

The blood group O demonstrates the absence of A and B antigens on the RBC

surface in forward blood typing Reverse blood typing indicates the presence of both

anti-A and -B in the plasma The first O allele (O1-1 [O01]) was shown to be identical

to the consensus A allele (A101) except for a nucleotide deletion 261delG in exon 6

This results in a shift in the reading frame giving rise to a truncated protein that alters

the protein sequence after amino acid 88 A stop codon halts translation after amino acid

117 and the resulting protein is enzymatically inactive (41) Some other O1-1-like

alleles that are characterized by the presence of the 261delG and at least one additional

point mutation A second kind of O allele has the same inactivating deletion (261delG)

as the original O allele (O1-1 [O01]) but in addition has nine point mutations spread

34

throughout exons 3 to 7 (4145) and a further 13 mutations have been found amongst

the intron 6 (50)

Additional polymorphisms associated with blood group O have been found up to

approximately 4300 bp from ABO exon 7 (51) some of which correlated with O1 and

O1v alleles (52) Other O alleles not due to 261delG also exist that are caused by other

inactivating mutations along the reading frame The ( O2-1 [O03]) was the first allele

described of this type (5354) which has a critical mutation (802G rarr A) causing an

amino acid change (Gly268Arg) that prevents the enzyme from utilizing the nucleotide

sugar donor (55) This O allele comprises approximately 2 to 5 percent of O alleles in

Caucasian persons but seems to be absent or at least very rare in other populations

(5456) Other rare O alleles such as O3 [O08] that does not have 261delG but instead

contains both the common A2 allele polymorphisms 467CrarrT and C-deletion at nt

1059-1061 and an insertion of an extra guanosine in the 7-guanosine sequence at nt

798-804 (45) Two other rare O alleles lacking 261delG were reported in the Japanese

population by Ogasawara O301 [O14] has the missense mutation 893CrarrT (Ala268Val)

on an A102 background whereas O302 [O15] has the nonsense mutation 927CrarrA

(Tyr309Stop) on an A101 background (43)

243 Weak subgroups

In addition to the major phenotypes characterized by either strong or absent

haemagglutination with anti-A-A1 and -B reagents the ABO blood group system also

includes phenotypes in which erythrocytes react weakly with the anti-A and ndashB

reagents for example A3 Ax Afinn Ael B3 Bx Bv Bel cis-AB (57)

It is difficult to determine clearly their specific ABO subgroup by conventional

serological methods The weak subgroups are important in more than one way First

they risk complicating patient and donor blood group determination At worst the

wrong group can be assigned if eg a weak antigen is missed Second they allow us to

characterize and understand glycosyltransferase mechanisms by studying the results of

mutations in the underlying alleles

35

2431 Weak A alleles

The RBCs from individuals with the A3 phenotype agglutinate strongly with

anti-A and anti-AB in vitro but show a large number of free cells One A3B individual

had a novel point mutation 871GrarrA (Asp291Asn) on the A1 [A101] (58) and this

allele was named A301 Ax alleles are responsible for the rare subgroup Ax and the

RBCs typically show a weak positive reaction with anti-AB and anti-H There are four

base substitutions involved in these alleles 646TrarrA (Phe216Ile) 681GrarrA (silent)

771CrarrT (silent) 829GrarrA (Val277Met) The encoded transferase is expected to be 37

aa longer than the normal consensus allele and 16 aa longer than A2-encoded

transferase The serological characteristics of some other minor phenotypes included

among A subgroups eg Aend Am Ay Afinn Abantu and the collective description Aw are

more unclear Table 22 shows the serological reaction patterns of weak A alleles

Table 22 Serological reaction patterns

A negative reaction is noted by 0 and positive reactions are denoted from + (very weak

agglutination) to 4+ (maximal agglutination)mf mixed field agglutination

2432 Weak B alleles

B variants are much more uncommon than A variants this may reflect the

relatively low frequency of the B blood group in many populations These phenotypes

Subgroup

of A

RBC reactions with Anti-A1

in serum

Anti-A Anti-AB Anti-A1 Anti-H

A1 4+ 4+ 4+ 0 No

A2 4+ 4+ 0 4+ Sometimes

Aint 4+ 4+ 2+3+ 2+3+ No

A3 2++mf 2++ mf 0 4+ No

Ax 0+ 2++ 0 4+ Often

Ael 0 0 0 4+ Sometimes

Aend + + 0 4+ Sometimes

Afinn + + 0 4+ Yes

Abantu +(+) +(+) 0 4+ Yes

Am 0+ 0+ 0 4+ No

Ay 0 0 0 4+ No

36

often appear to result from missense mutations at the ABO locus causing single aa

changes in the GTB Characteristics of some B variants are summarized in Table 23

(57)

Table 23 Characteristics of some more frequent B weak phenotypes

mf mixed field w very weak agglutination

The B3 phenotype have missense mutation 1054CrarrT (Arg352Trp) on a B1

(B101) background and named B301 (58) A Bx allele responsible for the Bx phenotype

had a point mutation at nt 871GrarrA (Asp291Asn) (59) which was also found in an A3

sample (58) The Bel phenotype was divided into two suballeles Bel-1 and Bel-2 (Bel01

and Bel02) which had substitutions at 641TrarrG (Met214Arg) and 669GrarrT

(Glu223Asp) respectively (59) these missense mutations reduce the enzymatic

activities of the GTB Ogasawara et al (1996) also found another B3 allele B302 which

differs from B consensus by two nucleotide substitutions 646TrarrA (Phe216Ile) and

657TrarrC

CisAB alleles rare phenotype was described by Seyfried in 1964 It represents a

very interesting phenomenon that proved that it is possible for a child with blood group

O to have a parent with blood group AB Seyfried hypothesized that both A and B

determinants of the AB blood group could be located on the same chromosome Later

on this hypothesis confirmed in that the existence of an exceptional ABO allele

encoding a glycosyltransferase is indeed able to produce both A and B enzymes at the

same time (57) Cis-ABO1 was sequenced and showed the substitution 803GrarrC

(Gly268Ala) on the A1-2 (A102) background and thus can be described as AAAB (60)

Another allele named cis-ABO2 was discovered when a Vietnamese man who was to

undergo organ transplantation showed irregular blood grouping results The sequencing

Subgroup

of B

RBC reactions with Anti-B

in serum

Anti-A Anti-AB Anti-B Anti-H

B 0 ++++ ++++ ++ None

B3 1 mf mf +++ None

Bx 1 w w +++ Yes

Bel 0 w 0w +++ None

Bm 0 0 0 +++ Sometimes

37

showed that his ABO genes were nearly identical to the normal B allele except for a

796ArarrC (Met266Leu) substitution (61)

25 ABO antibodies

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after

stimulation by a specific antigen The antibody formed binds to the specific antigen in

order to mark the antigen for destruction The type of antigenic exposure occurring in

the body determines if the antibody is a naturally occurring or immune antibody The

term lsquonaturally occurringrsquo is used for blood group antibodies produced in individuals

who have never been transfused with red cells carrying the relevant antigen or been

pregnant with a fetus carrying the relevant antigen Naturally occurring antibodies can

be formed after exposure to environmental agents that are similar to red cell antigens

such as bacteria dust or pollen (62)

Most of these antibodies are not clinically significant with the exception of ABO

antibodies It is possible that food and environmental antigens (bacterial viral or plant

antigens) have epitopes similar enough to A and B glycoprotein antigens The

antibodies created against these environmental antigens in the first years of life can

cross-react with ABO-incompatible red blood cells (RBCs) that it comes in contact with

during blood transfusion later in life Anti-A antibodies are hypothesized to originate

from immune response towards influenza virus whose epitopes are similar enough to

the α-D-N-galactosamine on the A glycoprotein to be able to elicit a cross-reaction

Anti-B antibodies are hypothesized to originate from antibodies produced against

Gram-negative bacteria such as E coli cross-reacting with the α-D-galactose on the B

glycoprotein (49) Anti-A and anti-B antibodies (called isohaemagglutinins) which are

not present in the newborn appear in the first years of life They are isoantibodies that

are produced by an individual against antigens produced by members of the same

species (isoantigens) Anti-A and anti-B antibodies are usually IgM type which are not

able to pass through the placenta to the fetal blood circulation and react best at room

temperature or lower The ABO antibodies may also be found in various body fluids

including saliva milk cervical secretions tears and cysts These antibodies are

detected at about 3 months and increase their titer until the 5th

to 10th

year of life

33

cell glucose transport protein (Band 45) Small numbers of ABH antigens are also

expressed by other red cell glycoproteins (28)

23 ABO Glycosyltransferases

Glycosyltransferases (GTs) constitute a large family of enzymes that are

involved in the biosynthesis of oligosaccharides polysaccharides and glycoconjugates

(29)Particularly abundant are the GTs that transfer a sugar residue from an activated

nucleotide sugar donor to specific acceptor molecules forming glycosidic bonds

Glycosyltransferases are classified into 87 different families based on substrateproduct

stereochemistry according to the CAZy database (30)

Knowledge of the sequences of the ABO genes (9) have established that

mammalian Glycosyltransferases A and Glycosyltransferases B are type II integral

membrane proteins containing 354 amino acids and are localized in the lumen of the

Golgi apparatus These enzymes typically have a short amino-terminal cytoplasmic tail

a hydrophobic membrane domain a short protease-sensitive stem region and a large

catalytic domain that includes the carboxy terminus(31) GTA and GTB are very similar

in the coding regions and the soluble enzyme can be found in serum (32)urine (33)and

milk (34)The enzyme contains an acceptor recognition domain that binds H antigen and

a donor recognition domain that binds UDP-GalNAc or UDP-Gal GTA and GTB

require the metal ion Mn++ (manganese) for activity (35)

Glycosyltransferases are antigenic structures Human antibodies to blood group

transferases are often produced following organ transplantation (36-39)

24 ABO Subgroups

241 A and B Subgroups

An ABO blood group subtype is called a subgroup andor a variant Subgroups

of ABO are distinguished by decreased amounts of A B or O (H) antigens on red blood

cells The most common are subgroups of A and B Blood type A appears to have the

most variation in subgroups The two most common subgroups of blood group A are A1

and A2 expressing on average 1 million and 250 000 A determinants respectively (40)

32

2411 A Subgroups

The A1 (A101) allele is the reference allele often denoted as the consensus

sequence in an ABO genotype context (9)but has additionally eight variant alleles

There are variants of A1 alleles one of which is very common in Asian populations with

a 467CrarrT (A102) polymorphism resulting in the substitution Pro156Leu (41)Two

minor A1 alleles (A103 and A104) have been described and differ as follows The first

one also has the 467CrarrT point mutation but an additional silent mutation 567CrarrT

the second allele contains a silent polymorphism in nucleotide 297ArarrG (42) A105 is

like A102 with the same mutation in exon 7 467CrarrT but analysis of intron 6 showed

additional single-nucleotide polymorphism (SNP) compared to A102 Another A1 allele

(A106) contains both 297ArarrG and 467CrarrT (43)

The A2 subgroup is the most common A phenotype after the A1 subgroup The

main genetic difference between A1 and A2 alleles is one point mutation in exon 7

467CrarrT (Pro156Leu) and a deletion of one of the three cytosines at nt 1059-1061

(CCC to CC) The latter mutation results in an extension of the reading frame by 64

nucleotides This deletion occurs in the codon before the translation stop codon (TGA)

resulting in a gene product with an extra 21 amino acid at its C-terminus The

glycosyltransferases encoded by the A2 allele have lower efficiency leading to a weaker

A phenotype The enzyme activity is decreased by 30-50 times compared to A1

(22)Other variants of A2 alleles have subsequently been elucidated (A202-206) Three

A2-like alleles were shown to have three different single mutations near the 3acute end of

exon 7 by Ogasawara A2-2 (A202) contains 1054CrarrT (Arg352Trp) A2-3 (A203) with

1054CrarrG (Arg352Gly) (42) and A2-4 (A205) with both 467CrarrT and an additional

new mutation 1009ArarrG (Arg337Gly) (44) A204 with four common B-related base

substitutions (297ArarrG 526CrarrG 657CrarrT and 703GrarrA) seems to be a hybrid with

two extra substitutions 771CrarrT (silent mutation) and 829GrarrA (Val277Met) One

other rare A2 subgroup A2-5 (A206) carried only the single deletion (1061delC)

(4546)

31

2412 B Subgroups

Subtypes of blood type B are classified by the quantity of B antigen and the

amount of B antigen decreases in the order B B3 Bx Bm Bel B1 allele (B101)

showed seven single nucleotide substitutions 297 526 657 703 796 803 and 930

throughout exon 6 and 7 and later one extra mutation outside the coding region at the 3acute

end at nt 1096 (33) The four amino acids substitutions governed by nt 526CrarrG

(Arg176Gly) 703GrarrA (Gly235Ser) 796CrarrA (Leu266Met) and 803GrarrC

(Gly268Ala) discriminate GTA from GTB (4147) while the substitutions 297 657 and

930 are silent

ABO glycosyltransferases can accordingly be described by using the letters A

and B to illustrate the derivation of the amino acid at these four residues GTA would be

represented by AAAA indicating the presence of ArgGlyLeuGly and similarly BBBB

would describe the GTB with GlySerMetAla at residues 176235266 and 268

respectively The substitutions at positions 266 and 268 were shown to be responsible

for the nucleotidedonor specificity of the transferases (47) and the other residues may

have a role in acceptor binding and turnover (48)Some other variants have been

reported afterwards that differ from B1 (B101) by lacking the 930 substitution for B2

(B102) the 657 substitution for B3 (B103) (47) the 526 substitution for B4 (B107) (44)

and 297 for B108 (43)

242 O Subgroups

The blood group O demonstrates the absence of A and B antigens on the RBC

surface in forward blood typing Reverse blood typing indicates the presence of both

anti-A and -B in the plasma The first O allele (O1-1 [O01]) was shown to be identical

to the consensus A allele (A101) except for a nucleotide deletion 261delG in exon 6

This results in a shift in the reading frame giving rise to a truncated protein that alters

the protein sequence after amino acid 88 A stop codon halts translation after amino acid

117 and the resulting protein is enzymatically inactive (41) Some other O1-1-like

alleles that are characterized by the presence of the 261delG and at least one additional

point mutation A second kind of O allele has the same inactivating deletion (261delG)

as the original O allele (O1-1 [O01]) but in addition has nine point mutations spread

34

throughout exons 3 to 7 (4145) and a further 13 mutations have been found amongst

the intron 6 (50)

Additional polymorphisms associated with blood group O have been found up to

approximately 4300 bp from ABO exon 7 (51) some of which correlated with O1 and

O1v alleles (52) Other O alleles not due to 261delG also exist that are caused by other

inactivating mutations along the reading frame The ( O2-1 [O03]) was the first allele

described of this type (5354) which has a critical mutation (802G rarr A) causing an

amino acid change (Gly268Arg) that prevents the enzyme from utilizing the nucleotide

sugar donor (55) This O allele comprises approximately 2 to 5 percent of O alleles in

Caucasian persons but seems to be absent or at least very rare in other populations

(5456) Other rare O alleles such as O3 [O08] that does not have 261delG but instead

contains both the common A2 allele polymorphisms 467CrarrT and C-deletion at nt

1059-1061 and an insertion of an extra guanosine in the 7-guanosine sequence at nt

798-804 (45) Two other rare O alleles lacking 261delG were reported in the Japanese

population by Ogasawara O301 [O14] has the missense mutation 893CrarrT (Ala268Val)

on an A102 background whereas O302 [O15] has the nonsense mutation 927CrarrA

(Tyr309Stop) on an A101 background (43)

243 Weak subgroups

In addition to the major phenotypes characterized by either strong or absent

haemagglutination with anti-A-A1 and -B reagents the ABO blood group system also

includes phenotypes in which erythrocytes react weakly with the anti-A and ndashB

reagents for example A3 Ax Afinn Ael B3 Bx Bv Bel cis-AB (57)

It is difficult to determine clearly their specific ABO subgroup by conventional

serological methods The weak subgroups are important in more than one way First

they risk complicating patient and donor blood group determination At worst the

wrong group can be assigned if eg a weak antigen is missed Second they allow us to

characterize and understand glycosyltransferase mechanisms by studying the results of

mutations in the underlying alleles

35

2431 Weak A alleles

The RBCs from individuals with the A3 phenotype agglutinate strongly with

anti-A and anti-AB in vitro but show a large number of free cells One A3B individual

had a novel point mutation 871GrarrA (Asp291Asn) on the A1 [A101] (58) and this

allele was named A301 Ax alleles are responsible for the rare subgroup Ax and the

RBCs typically show a weak positive reaction with anti-AB and anti-H There are four

base substitutions involved in these alleles 646TrarrA (Phe216Ile) 681GrarrA (silent)

771CrarrT (silent) 829GrarrA (Val277Met) The encoded transferase is expected to be 37

aa longer than the normal consensus allele and 16 aa longer than A2-encoded

transferase The serological characteristics of some other minor phenotypes included

among A subgroups eg Aend Am Ay Afinn Abantu and the collective description Aw are

more unclear Table 22 shows the serological reaction patterns of weak A alleles

Table 22 Serological reaction patterns

A negative reaction is noted by 0 and positive reactions are denoted from + (very weak

agglutination) to 4+ (maximal agglutination)mf mixed field agglutination

2432 Weak B alleles

B variants are much more uncommon than A variants this may reflect the

relatively low frequency of the B blood group in many populations These phenotypes

Subgroup

of A

RBC reactions with Anti-A1

in serum

Anti-A Anti-AB Anti-A1 Anti-H

A1 4+ 4+ 4+ 0 No

A2 4+ 4+ 0 4+ Sometimes

Aint 4+ 4+ 2+3+ 2+3+ No

A3 2++mf 2++ mf 0 4+ No

Ax 0+ 2++ 0 4+ Often

Ael 0 0 0 4+ Sometimes

Aend + + 0 4+ Sometimes

Afinn + + 0 4+ Yes

Abantu +(+) +(+) 0 4+ Yes

Am 0+ 0+ 0 4+ No

Ay 0 0 0 4+ No

36

often appear to result from missense mutations at the ABO locus causing single aa

changes in the GTB Characteristics of some B variants are summarized in Table 23

(57)

Table 23 Characteristics of some more frequent B weak phenotypes

mf mixed field w very weak agglutination

The B3 phenotype have missense mutation 1054CrarrT (Arg352Trp) on a B1

(B101) background and named B301 (58) A Bx allele responsible for the Bx phenotype

had a point mutation at nt 871GrarrA (Asp291Asn) (59) which was also found in an A3

sample (58) The Bel phenotype was divided into two suballeles Bel-1 and Bel-2 (Bel01

and Bel02) which had substitutions at 641TrarrG (Met214Arg) and 669GrarrT

(Glu223Asp) respectively (59) these missense mutations reduce the enzymatic

activities of the GTB Ogasawara et al (1996) also found another B3 allele B302 which

differs from B consensus by two nucleotide substitutions 646TrarrA (Phe216Ile) and

657TrarrC

CisAB alleles rare phenotype was described by Seyfried in 1964 It represents a

very interesting phenomenon that proved that it is possible for a child with blood group

O to have a parent with blood group AB Seyfried hypothesized that both A and B

determinants of the AB blood group could be located on the same chromosome Later

on this hypothesis confirmed in that the existence of an exceptional ABO allele

encoding a glycosyltransferase is indeed able to produce both A and B enzymes at the

same time (57) Cis-ABO1 was sequenced and showed the substitution 803GrarrC

(Gly268Ala) on the A1-2 (A102) background and thus can be described as AAAB (60)

Another allele named cis-ABO2 was discovered when a Vietnamese man who was to

undergo organ transplantation showed irregular blood grouping results The sequencing

Subgroup

of B

RBC reactions with Anti-B

in serum

Anti-A Anti-AB Anti-B Anti-H

B 0 ++++ ++++ ++ None

B3 1 mf mf +++ None

Bx 1 w w +++ Yes

Bel 0 w 0w +++ None

Bm 0 0 0 +++ Sometimes

37

showed that his ABO genes were nearly identical to the normal B allele except for a

796ArarrC (Met266Leu) substitution (61)

25 ABO antibodies

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after

stimulation by a specific antigen The antibody formed binds to the specific antigen in

order to mark the antigen for destruction The type of antigenic exposure occurring in

the body determines if the antibody is a naturally occurring or immune antibody The

term lsquonaturally occurringrsquo is used for blood group antibodies produced in individuals

who have never been transfused with red cells carrying the relevant antigen or been

pregnant with a fetus carrying the relevant antigen Naturally occurring antibodies can

be formed after exposure to environmental agents that are similar to red cell antigens

such as bacteria dust or pollen (62)

Most of these antibodies are not clinically significant with the exception of ABO

antibodies It is possible that food and environmental antigens (bacterial viral or plant

antigens) have epitopes similar enough to A and B glycoprotein antigens The

antibodies created against these environmental antigens in the first years of life can

cross-react with ABO-incompatible red blood cells (RBCs) that it comes in contact with

during blood transfusion later in life Anti-A antibodies are hypothesized to originate

from immune response towards influenza virus whose epitopes are similar enough to

the α-D-N-galactosamine on the A glycoprotein to be able to elicit a cross-reaction

Anti-B antibodies are hypothesized to originate from antibodies produced against

Gram-negative bacteria such as E coli cross-reacting with the α-D-galactose on the B

glycoprotein (49) Anti-A and anti-B antibodies (called isohaemagglutinins) which are

not present in the newborn appear in the first years of life They are isoantibodies that

are produced by an individual against antigens produced by members of the same

species (isoantigens) Anti-A and anti-B antibodies are usually IgM type which are not

able to pass through the placenta to the fetal blood circulation and react best at room

temperature or lower The ABO antibodies may also be found in various body fluids

including saliva milk cervical secretions tears and cysts These antibodies are

detected at about 3 months and increase their titer until the 5th

to 10th

year of life

32

2411 A Subgroups

The A1 (A101) allele is the reference allele often denoted as the consensus

sequence in an ABO genotype context (9)but has additionally eight variant alleles

There are variants of A1 alleles one of which is very common in Asian populations with

a 467CrarrT (A102) polymorphism resulting in the substitution Pro156Leu (41)Two

minor A1 alleles (A103 and A104) have been described and differ as follows The first

one also has the 467CrarrT point mutation but an additional silent mutation 567CrarrT

the second allele contains a silent polymorphism in nucleotide 297ArarrG (42) A105 is

like A102 with the same mutation in exon 7 467CrarrT but analysis of intron 6 showed

additional single-nucleotide polymorphism (SNP) compared to A102 Another A1 allele

(A106) contains both 297ArarrG and 467CrarrT (43)

The A2 subgroup is the most common A phenotype after the A1 subgroup The

main genetic difference between A1 and A2 alleles is one point mutation in exon 7

467CrarrT (Pro156Leu) and a deletion of one of the three cytosines at nt 1059-1061

(CCC to CC) The latter mutation results in an extension of the reading frame by 64

nucleotides This deletion occurs in the codon before the translation stop codon (TGA)

resulting in a gene product with an extra 21 amino acid at its C-terminus The

glycosyltransferases encoded by the A2 allele have lower efficiency leading to a weaker

A phenotype The enzyme activity is decreased by 30-50 times compared to A1

(22)Other variants of A2 alleles have subsequently been elucidated (A202-206) Three

A2-like alleles were shown to have three different single mutations near the 3acute end of

exon 7 by Ogasawara A2-2 (A202) contains 1054CrarrT (Arg352Trp) A2-3 (A203) with

1054CrarrG (Arg352Gly) (42) and A2-4 (A205) with both 467CrarrT and an additional

new mutation 1009ArarrG (Arg337Gly) (44) A204 with four common B-related base

substitutions (297ArarrG 526CrarrG 657CrarrT and 703GrarrA) seems to be a hybrid with

two extra substitutions 771CrarrT (silent mutation) and 829GrarrA (Val277Met) One

other rare A2 subgroup A2-5 (A206) carried only the single deletion (1061delC)

(4546)

31

2412 B Subgroups

Subtypes of blood type B are classified by the quantity of B antigen and the

amount of B antigen decreases in the order B B3 Bx Bm Bel B1 allele (B101)

showed seven single nucleotide substitutions 297 526 657 703 796 803 and 930

throughout exon 6 and 7 and later one extra mutation outside the coding region at the 3acute

end at nt 1096 (33) The four amino acids substitutions governed by nt 526CrarrG

(Arg176Gly) 703GrarrA (Gly235Ser) 796CrarrA (Leu266Met) and 803GrarrC

(Gly268Ala) discriminate GTA from GTB (4147) while the substitutions 297 657 and

930 are silent

ABO glycosyltransferases can accordingly be described by using the letters A

and B to illustrate the derivation of the amino acid at these four residues GTA would be

represented by AAAA indicating the presence of ArgGlyLeuGly and similarly BBBB

would describe the GTB with GlySerMetAla at residues 176235266 and 268

respectively The substitutions at positions 266 and 268 were shown to be responsible

for the nucleotidedonor specificity of the transferases (47) and the other residues may

have a role in acceptor binding and turnover (48)Some other variants have been

reported afterwards that differ from B1 (B101) by lacking the 930 substitution for B2

(B102) the 657 substitution for B3 (B103) (47) the 526 substitution for B4 (B107) (44)

and 297 for B108 (43)

242 O Subgroups

The blood group O demonstrates the absence of A and B antigens on the RBC

surface in forward blood typing Reverse blood typing indicates the presence of both

anti-A and -B in the plasma The first O allele (O1-1 [O01]) was shown to be identical

to the consensus A allele (A101) except for a nucleotide deletion 261delG in exon 6

This results in a shift in the reading frame giving rise to a truncated protein that alters

the protein sequence after amino acid 88 A stop codon halts translation after amino acid

117 and the resulting protein is enzymatically inactive (41) Some other O1-1-like

alleles that are characterized by the presence of the 261delG and at least one additional

point mutation A second kind of O allele has the same inactivating deletion (261delG)

as the original O allele (O1-1 [O01]) but in addition has nine point mutations spread

34

throughout exons 3 to 7 (4145) and a further 13 mutations have been found amongst

the intron 6 (50)

Additional polymorphisms associated with blood group O have been found up to

approximately 4300 bp from ABO exon 7 (51) some of which correlated with O1 and

O1v alleles (52) Other O alleles not due to 261delG also exist that are caused by other

inactivating mutations along the reading frame The ( O2-1 [O03]) was the first allele

described of this type (5354) which has a critical mutation (802G rarr A) causing an

amino acid change (Gly268Arg) that prevents the enzyme from utilizing the nucleotide

sugar donor (55) This O allele comprises approximately 2 to 5 percent of O alleles in

Caucasian persons but seems to be absent or at least very rare in other populations

(5456) Other rare O alleles such as O3 [O08] that does not have 261delG but instead

contains both the common A2 allele polymorphisms 467CrarrT and C-deletion at nt

1059-1061 and an insertion of an extra guanosine in the 7-guanosine sequence at nt

798-804 (45) Two other rare O alleles lacking 261delG were reported in the Japanese

population by Ogasawara O301 [O14] has the missense mutation 893CrarrT (Ala268Val)

on an A102 background whereas O302 [O15] has the nonsense mutation 927CrarrA

(Tyr309Stop) on an A101 background (43)

243 Weak subgroups

In addition to the major phenotypes characterized by either strong or absent

haemagglutination with anti-A-A1 and -B reagents the ABO blood group system also

includes phenotypes in which erythrocytes react weakly with the anti-A and ndashB

reagents for example A3 Ax Afinn Ael B3 Bx Bv Bel cis-AB (57)

It is difficult to determine clearly their specific ABO subgroup by conventional

serological methods The weak subgroups are important in more than one way First

they risk complicating patient and donor blood group determination At worst the

wrong group can be assigned if eg a weak antigen is missed Second they allow us to

characterize and understand glycosyltransferase mechanisms by studying the results of

mutations in the underlying alleles

35

2431 Weak A alleles

The RBCs from individuals with the A3 phenotype agglutinate strongly with

anti-A and anti-AB in vitro but show a large number of free cells One A3B individual

had a novel point mutation 871GrarrA (Asp291Asn) on the A1 [A101] (58) and this

allele was named A301 Ax alleles are responsible for the rare subgroup Ax and the

RBCs typically show a weak positive reaction with anti-AB and anti-H There are four

base substitutions involved in these alleles 646TrarrA (Phe216Ile) 681GrarrA (silent)

771CrarrT (silent) 829GrarrA (Val277Met) The encoded transferase is expected to be 37

aa longer than the normal consensus allele and 16 aa longer than A2-encoded

transferase The serological characteristics of some other minor phenotypes included

among A subgroups eg Aend Am Ay Afinn Abantu and the collective description Aw are

more unclear Table 22 shows the serological reaction patterns of weak A alleles

Table 22 Serological reaction patterns

A negative reaction is noted by 0 and positive reactions are denoted from + (very weak

agglutination) to 4+ (maximal agglutination)mf mixed field agglutination

2432 Weak B alleles

B variants are much more uncommon than A variants this may reflect the

relatively low frequency of the B blood group in many populations These phenotypes

Subgroup

of A

RBC reactions with Anti-A1

in serum

Anti-A Anti-AB Anti-A1 Anti-H

A1 4+ 4+ 4+ 0 No

A2 4+ 4+ 0 4+ Sometimes

Aint 4+ 4+ 2+3+ 2+3+ No

A3 2++mf 2++ mf 0 4+ No

Ax 0+ 2++ 0 4+ Often

Ael 0 0 0 4+ Sometimes

Aend + + 0 4+ Sometimes

Afinn + + 0 4+ Yes

Abantu +(+) +(+) 0 4+ Yes

Am 0+ 0+ 0 4+ No

Ay 0 0 0 4+ No

36

often appear to result from missense mutations at the ABO locus causing single aa

changes in the GTB Characteristics of some B variants are summarized in Table 23

(57)

Table 23 Characteristics of some more frequent B weak phenotypes

mf mixed field w very weak agglutination

The B3 phenotype have missense mutation 1054CrarrT (Arg352Trp) on a B1

(B101) background and named B301 (58) A Bx allele responsible for the Bx phenotype

had a point mutation at nt 871GrarrA (Asp291Asn) (59) which was also found in an A3

sample (58) The Bel phenotype was divided into two suballeles Bel-1 and Bel-2 (Bel01

and Bel02) which had substitutions at 641TrarrG (Met214Arg) and 669GrarrT

(Glu223Asp) respectively (59) these missense mutations reduce the enzymatic

activities of the GTB Ogasawara et al (1996) also found another B3 allele B302 which

differs from B consensus by two nucleotide substitutions 646TrarrA (Phe216Ile) and

657TrarrC

CisAB alleles rare phenotype was described by Seyfried in 1964 It represents a

very interesting phenomenon that proved that it is possible for a child with blood group

O to have a parent with blood group AB Seyfried hypothesized that both A and B

determinants of the AB blood group could be located on the same chromosome Later

on this hypothesis confirmed in that the existence of an exceptional ABO allele

encoding a glycosyltransferase is indeed able to produce both A and B enzymes at the

same time (57) Cis-ABO1 was sequenced and showed the substitution 803GrarrC

(Gly268Ala) on the A1-2 (A102) background and thus can be described as AAAB (60)

Another allele named cis-ABO2 was discovered when a Vietnamese man who was to

undergo organ transplantation showed irregular blood grouping results The sequencing

Subgroup

of B

RBC reactions with Anti-B

in serum

Anti-A Anti-AB Anti-B Anti-H

B 0 ++++ ++++ ++ None

B3 1 mf mf +++ None

Bx 1 w w +++ Yes

Bel 0 w 0w +++ None

Bm 0 0 0 +++ Sometimes

37

showed that his ABO genes were nearly identical to the normal B allele except for a

796ArarrC (Met266Leu) substitution (61)

25 ABO antibodies

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after

stimulation by a specific antigen The antibody formed binds to the specific antigen in

order to mark the antigen for destruction The type of antigenic exposure occurring in

the body determines if the antibody is a naturally occurring or immune antibody The

term lsquonaturally occurringrsquo is used for blood group antibodies produced in individuals

who have never been transfused with red cells carrying the relevant antigen or been

pregnant with a fetus carrying the relevant antigen Naturally occurring antibodies can

be formed after exposure to environmental agents that are similar to red cell antigens

such as bacteria dust or pollen (62)

Most of these antibodies are not clinically significant with the exception of ABO

antibodies It is possible that food and environmental antigens (bacterial viral or plant

antigens) have epitopes similar enough to A and B glycoprotein antigens The

antibodies created against these environmental antigens in the first years of life can

cross-react with ABO-incompatible red blood cells (RBCs) that it comes in contact with

during blood transfusion later in life Anti-A antibodies are hypothesized to originate

from immune response towards influenza virus whose epitopes are similar enough to

the α-D-N-galactosamine on the A glycoprotein to be able to elicit a cross-reaction

Anti-B antibodies are hypothesized to originate from antibodies produced against

Gram-negative bacteria such as E coli cross-reacting with the α-D-galactose on the B

glycoprotein (49) Anti-A and anti-B antibodies (called isohaemagglutinins) which are

not present in the newborn appear in the first years of life They are isoantibodies that

are produced by an individual against antigens produced by members of the same

species (isoantigens) Anti-A and anti-B antibodies are usually IgM type which are not

able to pass through the placenta to the fetal blood circulation and react best at room

temperature or lower The ABO antibodies may also be found in various body fluids

including saliva milk cervical secretions tears and cysts These antibodies are

detected at about 3 months and increase their titer until the 5th

to 10th

year of life

31

2412 B Subgroups

Subtypes of blood type B are classified by the quantity of B antigen and the

amount of B antigen decreases in the order B B3 Bx Bm Bel B1 allele (B101)

showed seven single nucleotide substitutions 297 526 657 703 796 803 and 930

throughout exon 6 and 7 and later one extra mutation outside the coding region at the 3acute

end at nt 1096 (33) The four amino acids substitutions governed by nt 526CrarrG

(Arg176Gly) 703GrarrA (Gly235Ser) 796CrarrA (Leu266Met) and 803GrarrC

(Gly268Ala) discriminate GTA from GTB (4147) while the substitutions 297 657 and

930 are silent

ABO glycosyltransferases can accordingly be described by using the letters A

and B to illustrate the derivation of the amino acid at these four residues GTA would be

represented by AAAA indicating the presence of ArgGlyLeuGly and similarly BBBB

would describe the GTB with GlySerMetAla at residues 176235266 and 268

respectively The substitutions at positions 266 and 268 were shown to be responsible

for the nucleotidedonor specificity of the transferases (47) and the other residues may

have a role in acceptor binding and turnover (48)Some other variants have been

reported afterwards that differ from B1 (B101) by lacking the 930 substitution for B2

(B102) the 657 substitution for B3 (B103) (47) the 526 substitution for B4 (B107) (44)

and 297 for B108 (43)

242 O Subgroups

The blood group O demonstrates the absence of A and B antigens on the RBC

surface in forward blood typing Reverse blood typing indicates the presence of both

anti-A and -B in the plasma The first O allele (O1-1 [O01]) was shown to be identical

to the consensus A allele (A101) except for a nucleotide deletion 261delG in exon 6

This results in a shift in the reading frame giving rise to a truncated protein that alters

the protein sequence after amino acid 88 A stop codon halts translation after amino acid

117 and the resulting protein is enzymatically inactive (41) Some other O1-1-like

alleles that are characterized by the presence of the 261delG and at least one additional

point mutation A second kind of O allele has the same inactivating deletion (261delG)

as the original O allele (O1-1 [O01]) but in addition has nine point mutations spread

34

throughout exons 3 to 7 (4145) and a further 13 mutations have been found amongst

the intron 6 (50)

Additional polymorphisms associated with blood group O have been found up to

approximately 4300 bp from ABO exon 7 (51) some of which correlated with O1 and

O1v alleles (52) Other O alleles not due to 261delG also exist that are caused by other

inactivating mutations along the reading frame The ( O2-1 [O03]) was the first allele

described of this type (5354) which has a critical mutation (802G rarr A) causing an

amino acid change (Gly268Arg) that prevents the enzyme from utilizing the nucleotide

sugar donor (55) This O allele comprises approximately 2 to 5 percent of O alleles in

Caucasian persons but seems to be absent or at least very rare in other populations

(5456) Other rare O alleles such as O3 [O08] that does not have 261delG but instead

contains both the common A2 allele polymorphisms 467CrarrT and C-deletion at nt

1059-1061 and an insertion of an extra guanosine in the 7-guanosine sequence at nt

798-804 (45) Two other rare O alleles lacking 261delG were reported in the Japanese

population by Ogasawara O301 [O14] has the missense mutation 893CrarrT (Ala268Val)

on an A102 background whereas O302 [O15] has the nonsense mutation 927CrarrA

(Tyr309Stop) on an A101 background (43)

243 Weak subgroups

In addition to the major phenotypes characterized by either strong or absent

haemagglutination with anti-A-A1 and -B reagents the ABO blood group system also

includes phenotypes in which erythrocytes react weakly with the anti-A and ndashB

reagents for example A3 Ax Afinn Ael B3 Bx Bv Bel cis-AB (57)

It is difficult to determine clearly their specific ABO subgroup by conventional

serological methods The weak subgroups are important in more than one way First

they risk complicating patient and donor blood group determination At worst the

wrong group can be assigned if eg a weak antigen is missed Second they allow us to

characterize and understand glycosyltransferase mechanisms by studying the results of

mutations in the underlying alleles

35

2431 Weak A alleles

The RBCs from individuals with the A3 phenotype agglutinate strongly with

anti-A and anti-AB in vitro but show a large number of free cells One A3B individual

had a novel point mutation 871GrarrA (Asp291Asn) on the A1 [A101] (58) and this

allele was named A301 Ax alleles are responsible for the rare subgroup Ax and the

RBCs typically show a weak positive reaction with anti-AB and anti-H There are four

base substitutions involved in these alleles 646TrarrA (Phe216Ile) 681GrarrA (silent)

771CrarrT (silent) 829GrarrA (Val277Met) The encoded transferase is expected to be 37

aa longer than the normal consensus allele and 16 aa longer than A2-encoded

transferase The serological characteristics of some other minor phenotypes included

among A subgroups eg Aend Am Ay Afinn Abantu and the collective description Aw are

more unclear Table 22 shows the serological reaction patterns of weak A alleles

Table 22 Serological reaction patterns

A negative reaction is noted by 0 and positive reactions are denoted from + (very weak

agglutination) to 4+ (maximal agglutination)mf mixed field agglutination

2432 Weak B alleles

B variants are much more uncommon than A variants this may reflect the

relatively low frequency of the B blood group in many populations These phenotypes

Subgroup

of A

RBC reactions with Anti-A1

in serum

Anti-A Anti-AB Anti-A1 Anti-H

A1 4+ 4+ 4+ 0 No

A2 4+ 4+ 0 4+ Sometimes

Aint 4+ 4+ 2+3+ 2+3+ No

A3 2++mf 2++ mf 0 4+ No

Ax 0+ 2++ 0 4+ Often

Ael 0 0 0 4+ Sometimes

Aend + + 0 4+ Sometimes

Afinn + + 0 4+ Yes

Abantu +(+) +(+) 0 4+ Yes

Am 0+ 0+ 0 4+ No

Ay 0 0 0 4+ No

36

often appear to result from missense mutations at the ABO locus causing single aa

changes in the GTB Characteristics of some B variants are summarized in Table 23

(57)

Table 23 Characteristics of some more frequent B weak phenotypes

mf mixed field w very weak agglutination

The B3 phenotype have missense mutation 1054CrarrT (Arg352Trp) on a B1

(B101) background and named B301 (58) A Bx allele responsible for the Bx phenotype

had a point mutation at nt 871GrarrA (Asp291Asn) (59) which was also found in an A3

sample (58) The Bel phenotype was divided into two suballeles Bel-1 and Bel-2 (Bel01

and Bel02) which had substitutions at 641TrarrG (Met214Arg) and 669GrarrT

(Glu223Asp) respectively (59) these missense mutations reduce the enzymatic

activities of the GTB Ogasawara et al (1996) also found another B3 allele B302 which

differs from B consensus by two nucleotide substitutions 646TrarrA (Phe216Ile) and

657TrarrC

CisAB alleles rare phenotype was described by Seyfried in 1964 It represents a

very interesting phenomenon that proved that it is possible for a child with blood group

O to have a parent with blood group AB Seyfried hypothesized that both A and B

determinants of the AB blood group could be located on the same chromosome Later

on this hypothesis confirmed in that the existence of an exceptional ABO allele

encoding a glycosyltransferase is indeed able to produce both A and B enzymes at the

same time (57) Cis-ABO1 was sequenced and showed the substitution 803GrarrC

(Gly268Ala) on the A1-2 (A102) background and thus can be described as AAAB (60)

Another allele named cis-ABO2 was discovered when a Vietnamese man who was to

undergo organ transplantation showed irregular blood grouping results The sequencing

Subgroup

of B

RBC reactions with Anti-B

in serum

Anti-A Anti-AB Anti-B Anti-H

B 0 ++++ ++++ ++ None

B3 1 mf mf +++ None

Bx 1 w w +++ Yes

Bel 0 w 0w +++ None

Bm 0 0 0 +++ Sometimes

37

showed that his ABO genes were nearly identical to the normal B allele except for a

796ArarrC (Met266Leu) substitution (61)

25 ABO antibodies

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after

stimulation by a specific antigen The antibody formed binds to the specific antigen in

order to mark the antigen for destruction The type of antigenic exposure occurring in

the body determines if the antibody is a naturally occurring or immune antibody The

term lsquonaturally occurringrsquo is used for blood group antibodies produced in individuals

who have never been transfused with red cells carrying the relevant antigen or been

pregnant with a fetus carrying the relevant antigen Naturally occurring antibodies can

be formed after exposure to environmental agents that are similar to red cell antigens

such as bacteria dust or pollen (62)

Most of these antibodies are not clinically significant with the exception of ABO

antibodies It is possible that food and environmental antigens (bacterial viral or plant

antigens) have epitopes similar enough to A and B glycoprotein antigens The

antibodies created against these environmental antigens in the first years of life can

cross-react with ABO-incompatible red blood cells (RBCs) that it comes in contact with

during blood transfusion later in life Anti-A antibodies are hypothesized to originate

from immune response towards influenza virus whose epitopes are similar enough to

the α-D-N-galactosamine on the A glycoprotein to be able to elicit a cross-reaction

Anti-B antibodies are hypothesized to originate from antibodies produced against

Gram-negative bacteria such as E coli cross-reacting with the α-D-galactose on the B

glycoprotein (49) Anti-A and anti-B antibodies (called isohaemagglutinins) which are

not present in the newborn appear in the first years of life They are isoantibodies that

are produced by an individual against antigens produced by members of the same

species (isoantigens) Anti-A and anti-B antibodies are usually IgM type which are not

able to pass through the placenta to the fetal blood circulation and react best at room

temperature or lower The ABO antibodies may also be found in various body fluids

including saliva milk cervical secretions tears and cysts These antibodies are

detected at about 3 months and increase their titer until the 5th

to 10th

year of life

34

throughout exons 3 to 7 (4145) and a further 13 mutations have been found amongst

the intron 6 (50)

Additional polymorphisms associated with blood group O have been found up to

approximately 4300 bp from ABO exon 7 (51) some of which correlated with O1 and

O1v alleles (52) Other O alleles not due to 261delG also exist that are caused by other

inactivating mutations along the reading frame The ( O2-1 [O03]) was the first allele

described of this type (5354) which has a critical mutation (802G rarr A) causing an

amino acid change (Gly268Arg) that prevents the enzyme from utilizing the nucleotide

sugar donor (55) This O allele comprises approximately 2 to 5 percent of O alleles in

Caucasian persons but seems to be absent or at least very rare in other populations

(5456) Other rare O alleles such as O3 [O08] that does not have 261delG but instead

contains both the common A2 allele polymorphisms 467CrarrT and C-deletion at nt

1059-1061 and an insertion of an extra guanosine in the 7-guanosine sequence at nt

798-804 (45) Two other rare O alleles lacking 261delG were reported in the Japanese

population by Ogasawara O301 [O14] has the missense mutation 893CrarrT (Ala268Val)

on an A102 background whereas O302 [O15] has the nonsense mutation 927CrarrA

(Tyr309Stop) on an A101 background (43)

243 Weak subgroups

In addition to the major phenotypes characterized by either strong or absent

haemagglutination with anti-A-A1 and -B reagents the ABO blood group system also

includes phenotypes in which erythrocytes react weakly with the anti-A and ndashB

reagents for example A3 Ax Afinn Ael B3 Bx Bv Bel cis-AB (57)

It is difficult to determine clearly their specific ABO subgroup by conventional

serological methods The weak subgroups are important in more than one way First

they risk complicating patient and donor blood group determination At worst the

wrong group can be assigned if eg a weak antigen is missed Second they allow us to

characterize and understand glycosyltransferase mechanisms by studying the results of

mutations in the underlying alleles

35

2431 Weak A alleles

The RBCs from individuals with the A3 phenotype agglutinate strongly with

anti-A and anti-AB in vitro but show a large number of free cells One A3B individual

had a novel point mutation 871GrarrA (Asp291Asn) on the A1 [A101] (58) and this

allele was named A301 Ax alleles are responsible for the rare subgroup Ax and the

RBCs typically show a weak positive reaction with anti-AB and anti-H There are four

base substitutions involved in these alleles 646TrarrA (Phe216Ile) 681GrarrA (silent)

771CrarrT (silent) 829GrarrA (Val277Met) The encoded transferase is expected to be 37

aa longer than the normal consensus allele and 16 aa longer than A2-encoded

transferase The serological characteristics of some other minor phenotypes included

among A subgroups eg Aend Am Ay Afinn Abantu and the collective description Aw are

more unclear Table 22 shows the serological reaction patterns of weak A alleles

Table 22 Serological reaction patterns

A negative reaction is noted by 0 and positive reactions are denoted from + (very weak

agglutination) to 4+ (maximal agglutination)mf mixed field agglutination

2432 Weak B alleles

B variants are much more uncommon than A variants this may reflect the

relatively low frequency of the B blood group in many populations These phenotypes

Subgroup

of A

RBC reactions with Anti-A1

in serum

Anti-A Anti-AB Anti-A1 Anti-H

A1 4+ 4+ 4+ 0 No

A2 4+ 4+ 0 4+ Sometimes

Aint 4+ 4+ 2+3+ 2+3+ No

A3 2++mf 2++ mf 0 4+ No

Ax 0+ 2++ 0 4+ Often

Ael 0 0 0 4+ Sometimes

Aend + + 0 4+ Sometimes

Afinn + + 0 4+ Yes

Abantu +(+) +(+) 0 4+ Yes

Am 0+ 0+ 0 4+ No

Ay 0 0 0 4+ No

36

often appear to result from missense mutations at the ABO locus causing single aa

changes in the GTB Characteristics of some B variants are summarized in Table 23

(57)

Table 23 Characteristics of some more frequent B weak phenotypes

mf mixed field w very weak agglutination

The B3 phenotype have missense mutation 1054CrarrT (Arg352Trp) on a B1

(B101) background and named B301 (58) A Bx allele responsible for the Bx phenotype

had a point mutation at nt 871GrarrA (Asp291Asn) (59) which was also found in an A3

sample (58) The Bel phenotype was divided into two suballeles Bel-1 and Bel-2 (Bel01

and Bel02) which had substitutions at 641TrarrG (Met214Arg) and 669GrarrT

(Glu223Asp) respectively (59) these missense mutations reduce the enzymatic

activities of the GTB Ogasawara et al (1996) also found another B3 allele B302 which

differs from B consensus by two nucleotide substitutions 646TrarrA (Phe216Ile) and

657TrarrC

CisAB alleles rare phenotype was described by Seyfried in 1964 It represents a

very interesting phenomenon that proved that it is possible for a child with blood group

O to have a parent with blood group AB Seyfried hypothesized that both A and B

determinants of the AB blood group could be located on the same chromosome Later

on this hypothesis confirmed in that the existence of an exceptional ABO allele

encoding a glycosyltransferase is indeed able to produce both A and B enzymes at the

same time (57) Cis-ABO1 was sequenced and showed the substitution 803GrarrC

(Gly268Ala) on the A1-2 (A102) background and thus can be described as AAAB (60)

Another allele named cis-ABO2 was discovered when a Vietnamese man who was to

undergo organ transplantation showed irregular blood grouping results The sequencing

Subgroup

of B

RBC reactions with Anti-B

in serum

Anti-A Anti-AB Anti-B Anti-H

B 0 ++++ ++++ ++ None

B3 1 mf mf +++ None

Bx 1 w w +++ Yes

Bel 0 w 0w +++ None

Bm 0 0 0 +++ Sometimes

37

showed that his ABO genes were nearly identical to the normal B allele except for a

796ArarrC (Met266Leu) substitution (61)

25 ABO antibodies

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after

stimulation by a specific antigen The antibody formed binds to the specific antigen in

order to mark the antigen for destruction The type of antigenic exposure occurring in

the body determines if the antibody is a naturally occurring or immune antibody The

term lsquonaturally occurringrsquo is used for blood group antibodies produced in individuals

who have never been transfused with red cells carrying the relevant antigen or been

pregnant with a fetus carrying the relevant antigen Naturally occurring antibodies can

be formed after exposure to environmental agents that are similar to red cell antigens

such as bacteria dust or pollen (62)

Most of these antibodies are not clinically significant with the exception of ABO

antibodies It is possible that food and environmental antigens (bacterial viral or plant

antigens) have epitopes similar enough to A and B glycoprotein antigens The

antibodies created against these environmental antigens in the first years of life can

cross-react with ABO-incompatible red blood cells (RBCs) that it comes in contact with

during blood transfusion later in life Anti-A antibodies are hypothesized to originate

from immune response towards influenza virus whose epitopes are similar enough to

the α-D-N-galactosamine on the A glycoprotein to be able to elicit a cross-reaction

Anti-B antibodies are hypothesized to originate from antibodies produced against

Gram-negative bacteria such as E coli cross-reacting with the α-D-galactose on the B

glycoprotein (49) Anti-A and anti-B antibodies (called isohaemagglutinins) which are

not present in the newborn appear in the first years of life They are isoantibodies that

are produced by an individual against antigens produced by members of the same

species (isoantigens) Anti-A and anti-B antibodies are usually IgM type which are not

able to pass through the placenta to the fetal blood circulation and react best at room

temperature or lower The ABO antibodies may also be found in various body fluids

including saliva milk cervical secretions tears and cysts These antibodies are

detected at about 3 months and increase their titer until the 5th

to 10th

year of life

35

2431 Weak A alleles

The RBCs from individuals with the A3 phenotype agglutinate strongly with

anti-A and anti-AB in vitro but show a large number of free cells One A3B individual

had a novel point mutation 871GrarrA (Asp291Asn) on the A1 [A101] (58) and this

allele was named A301 Ax alleles are responsible for the rare subgroup Ax and the

RBCs typically show a weak positive reaction with anti-AB and anti-H There are four

base substitutions involved in these alleles 646TrarrA (Phe216Ile) 681GrarrA (silent)

771CrarrT (silent) 829GrarrA (Val277Met) The encoded transferase is expected to be 37

aa longer than the normal consensus allele and 16 aa longer than A2-encoded

transferase The serological characteristics of some other minor phenotypes included

among A subgroups eg Aend Am Ay Afinn Abantu and the collective description Aw are

more unclear Table 22 shows the serological reaction patterns of weak A alleles

Table 22 Serological reaction patterns

A negative reaction is noted by 0 and positive reactions are denoted from + (very weak

agglutination) to 4+ (maximal agglutination)mf mixed field agglutination

2432 Weak B alleles

B variants are much more uncommon than A variants this may reflect the

relatively low frequency of the B blood group in many populations These phenotypes

Subgroup

of A

RBC reactions with Anti-A1

in serum

Anti-A Anti-AB Anti-A1 Anti-H

A1 4+ 4+ 4+ 0 No

A2 4+ 4+ 0 4+ Sometimes

Aint 4+ 4+ 2+3+ 2+3+ No

A3 2++mf 2++ mf 0 4+ No

Ax 0+ 2++ 0 4+ Often

Ael 0 0 0 4+ Sometimes

Aend + + 0 4+ Sometimes

Afinn + + 0 4+ Yes

Abantu +(+) +(+) 0 4+ Yes

Am 0+ 0+ 0 4+ No

Ay 0 0 0 4+ No

36

often appear to result from missense mutations at the ABO locus causing single aa

changes in the GTB Characteristics of some B variants are summarized in Table 23

(57)

Table 23 Characteristics of some more frequent B weak phenotypes

mf mixed field w very weak agglutination

The B3 phenotype have missense mutation 1054CrarrT (Arg352Trp) on a B1

(B101) background and named B301 (58) A Bx allele responsible for the Bx phenotype

had a point mutation at nt 871GrarrA (Asp291Asn) (59) which was also found in an A3

sample (58) The Bel phenotype was divided into two suballeles Bel-1 and Bel-2 (Bel01

and Bel02) which had substitutions at 641TrarrG (Met214Arg) and 669GrarrT

(Glu223Asp) respectively (59) these missense mutations reduce the enzymatic

activities of the GTB Ogasawara et al (1996) also found another B3 allele B302 which

differs from B consensus by two nucleotide substitutions 646TrarrA (Phe216Ile) and

657TrarrC

CisAB alleles rare phenotype was described by Seyfried in 1964 It represents a

very interesting phenomenon that proved that it is possible for a child with blood group

O to have a parent with blood group AB Seyfried hypothesized that both A and B

determinants of the AB blood group could be located on the same chromosome Later

on this hypothesis confirmed in that the existence of an exceptional ABO allele

encoding a glycosyltransferase is indeed able to produce both A and B enzymes at the

same time (57) Cis-ABO1 was sequenced and showed the substitution 803GrarrC

(Gly268Ala) on the A1-2 (A102) background and thus can be described as AAAB (60)

Another allele named cis-ABO2 was discovered when a Vietnamese man who was to

undergo organ transplantation showed irregular blood grouping results The sequencing

Subgroup

of B

RBC reactions with Anti-B

in serum

Anti-A Anti-AB Anti-B Anti-H

B 0 ++++ ++++ ++ None

B3 1 mf mf +++ None

Bx 1 w w +++ Yes

Bel 0 w 0w +++ None

Bm 0 0 0 +++ Sometimes

37

showed that his ABO genes were nearly identical to the normal B allele except for a

796ArarrC (Met266Leu) substitution (61)

25 ABO antibodies

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after

stimulation by a specific antigen The antibody formed binds to the specific antigen in

order to mark the antigen for destruction The type of antigenic exposure occurring in

the body determines if the antibody is a naturally occurring or immune antibody The

term lsquonaturally occurringrsquo is used for blood group antibodies produced in individuals

who have never been transfused with red cells carrying the relevant antigen or been

pregnant with a fetus carrying the relevant antigen Naturally occurring antibodies can

be formed after exposure to environmental agents that are similar to red cell antigens

such as bacteria dust or pollen (62)

Most of these antibodies are not clinically significant with the exception of ABO

antibodies It is possible that food and environmental antigens (bacterial viral or plant

antigens) have epitopes similar enough to A and B glycoprotein antigens The

antibodies created against these environmental antigens in the first years of life can

cross-react with ABO-incompatible red blood cells (RBCs) that it comes in contact with

during blood transfusion later in life Anti-A antibodies are hypothesized to originate

from immune response towards influenza virus whose epitopes are similar enough to

the α-D-N-galactosamine on the A glycoprotein to be able to elicit a cross-reaction

Anti-B antibodies are hypothesized to originate from antibodies produced against

Gram-negative bacteria such as E coli cross-reacting with the α-D-galactose on the B

glycoprotein (49) Anti-A and anti-B antibodies (called isohaemagglutinins) which are

not present in the newborn appear in the first years of life They are isoantibodies that

are produced by an individual against antigens produced by members of the same

species (isoantigens) Anti-A and anti-B antibodies are usually IgM type which are not

able to pass through the placenta to the fetal blood circulation and react best at room

temperature or lower The ABO antibodies may also be found in various body fluids

including saliva milk cervical secretions tears and cysts These antibodies are

detected at about 3 months and increase their titer until the 5th

to 10th

year of life

36

often appear to result from missense mutations at the ABO locus causing single aa

changes in the GTB Characteristics of some B variants are summarized in Table 23

(57)

Table 23 Characteristics of some more frequent B weak phenotypes

mf mixed field w very weak agglutination

The B3 phenotype have missense mutation 1054CrarrT (Arg352Trp) on a B1

(B101) background and named B301 (58) A Bx allele responsible for the Bx phenotype

had a point mutation at nt 871GrarrA (Asp291Asn) (59) which was also found in an A3

sample (58) The Bel phenotype was divided into two suballeles Bel-1 and Bel-2 (Bel01

and Bel02) which had substitutions at 641TrarrG (Met214Arg) and 669GrarrT

(Glu223Asp) respectively (59) these missense mutations reduce the enzymatic

activities of the GTB Ogasawara et al (1996) also found another B3 allele B302 which

differs from B consensus by two nucleotide substitutions 646TrarrA (Phe216Ile) and

657TrarrC

CisAB alleles rare phenotype was described by Seyfried in 1964 It represents a

very interesting phenomenon that proved that it is possible for a child with blood group

O to have a parent with blood group AB Seyfried hypothesized that both A and B

determinants of the AB blood group could be located on the same chromosome Later

on this hypothesis confirmed in that the existence of an exceptional ABO allele

encoding a glycosyltransferase is indeed able to produce both A and B enzymes at the

same time (57) Cis-ABO1 was sequenced and showed the substitution 803GrarrC

(Gly268Ala) on the A1-2 (A102) background and thus can be described as AAAB (60)

Another allele named cis-ABO2 was discovered when a Vietnamese man who was to

undergo organ transplantation showed irregular blood grouping results The sequencing

Subgroup

of B

RBC reactions with Anti-B

in serum

Anti-A Anti-AB Anti-B Anti-H

B 0 ++++ ++++ ++ None

B3 1 mf mf +++ None

Bx 1 w w +++ Yes

Bel 0 w 0w +++ None

Bm 0 0 0 +++ Sometimes

37

showed that his ABO genes were nearly identical to the normal B allele except for a

796ArarrC (Met266Leu) substitution (61)

25 ABO antibodies

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after

stimulation by a specific antigen The antibody formed binds to the specific antigen in

order to mark the antigen for destruction The type of antigenic exposure occurring in

the body determines if the antibody is a naturally occurring or immune antibody The

term lsquonaturally occurringrsquo is used for blood group antibodies produced in individuals

who have never been transfused with red cells carrying the relevant antigen or been

pregnant with a fetus carrying the relevant antigen Naturally occurring antibodies can

be formed after exposure to environmental agents that are similar to red cell antigens

such as bacteria dust or pollen (62)

Most of these antibodies are not clinically significant with the exception of ABO

antibodies It is possible that food and environmental antigens (bacterial viral or plant

antigens) have epitopes similar enough to A and B glycoprotein antigens The

antibodies created against these environmental antigens in the first years of life can

cross-react with ABO-incompatible red blood cells (RBCs) that it comes in contact with

during blood transfusion later in life Anti-A antibodies are hypothesized to originate

from immune response towards influenza virus whose epitopes are similar enough to

the α-D-N-galactosamine on the A glycoprotein to be able to elicit a cross-reaction

Anti-B antibodies are hypothesized to originate from antibodies produced against

Gram-negative bacteria such as E coli cross-reacting with the α-D-galactose on the B

glycoprotein (49) Anti-A and anti-B antibodies (called isohaemagglutinins) which are

not present in the newborn appear in the first years of life They are isoantibodies that

are produced by an individual against antigens produced by members of the same

species (isoantigens) Anti-A and anti-B antibodies are usually IgM type which are not

able to pass through the placenta to the fetal blood circulation and react best at room

temperature or lower The ABO antibodies may also be found in various body fluids

including saliva milk cervical secretions tears and cysts These antibodies are

detected at about 3 months and increase their titer until the 5th

to 10th

year of life

37

showed that his ABO genes were nearly identical to the normal B allele except for a

796ArarrC (Met266Leu) substitution (61)

25 ABO antibodies

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after

stimulation by a specific antigen The antibody formed binds to the specific antigen in

order to mark the antigen for destruction The type of antigenic exposure occurring in

the body determines if the antibody is a naturally occurring or immune antibody The

term lsquonaturally occurringrsquo is used for blood group antibodies produced in individuals

who have never been transfused with red cells carrying the relevant antigen or been

pregnant with a fetus carrying the relevant antigen Naturally occurring antibodies can

be formed after exposure to environmental agents that are similar to red cell antigens

such as bacteria dust or pollen (62)

Most of these antibodies are not clinically significant with the exception of ABO

antibodies It is possible that food and environmental antigens (bacterial viral or plant

antigens) have epitopes similar enough to A and B glycoprotein antigens The

antibodies created against these environmental antigens in the first years of life can

cross-react with ABO-incompatible red blood cells (RBCs) that it comes in contact with

during blood transfusion later in life Anti-A antibodies are hypothesized to originate

from immune response towards influenza virus whose epitopes are similar enough to

the α-D-N-galactosamine on the A glycoprotein to be able to elicit a cross-reaction

Anti-B antibodies are hypothesized to originate from antibodies produced against

Gram-negative bacteria such as E coli cross-reacting with the α-D-galactose on the B

glycoprotein (49) Anti-A and anti-B antibodies (called isohaemagglutinins) which are

not present in the newborn appear in the first years of life They are isoantibodies that

are produced by an individual against antigens produced by members of the same

species (isoantigens) Anti-A and anti-B antibodies are usually IgM type which are not

able to pass through the placenta to the fetal blood circulation and react best at room

temperature or lower The ABO antibodies may also be found in various body fluids

including saliva milk cervical secretions tears and cysts These antibodies are

detected at about 3 months and increase their titer until the 5th

to 10th

year of life

38

Sera from A individuals contain anti-B antibody while B individuals sera

contain two types of antibody against A antigens The first is anti-A and the second one

is specific towards A1 RBCs Anti-A reacts with both A1 and A2 cells whereas the

second only does with A1 RBCs Anti-A1 is also present in some A2 and A2B

individuals (63) Group O people produce an antibody anti-AB able to cross-react with

both A and B RBCs

26 Studies on ABO Genotype

Olsson et al (1995) studied the ABO genotype in 300 Danish blood donors with

a method using six restriction enzyme digestions following three different PCRs

detecting polymorphism at nucleotide positions (nt) 261 526 and 703 The results of the

study showed that about 3 of the O alleles were of the new type of O gene O1 The

common O allele was assigned O2 They found a restriction enzyme site for HpaIl that

is only present in non-B O2 alleles (64)

Fukumori et al (1995) performed the genotyping of ABO blood groups on a

Japanese population using the polymerase chain reaction (PCR) method The 4 DNA

fragments containing the nucleotide position 261 526 703 and 796 of cDNA from A-

transferase were amplified by PCR and the amplified DNA was subjected to restriction

fragment length polymorphism (RFLP) analysis The different nucleotide at position

803 was distinguished by electrophoresis of the PCR products amplified with allele-

specific primers By analyzing the electrophoresis patterns The frequencies of ABO

genotypes found in Japanese blood donors with A and B phenotypes were as follows in

the phenotype A group AA =198 and AO = 802 and in the phenotype B group

BB =128 and BO=872 (65)

Ogasawara et al (1996) investigated the polymorphism of the ABO blood

group gene in 262 healthy Japanese donors by a polymerase chain reaction-single-strand

conformation polymorphism (PCR-SSCP) method and 13 different alleles were

identified The number of alleles identified in each group was 4 for A1 (called ABO

A101 A102 A103 and A104) 3 for B (ABO B101 B102 and B103) and 6 for O

(ABO O101 O102 O103 O201 O202 and O203) Nucleotide sequences of the

amplified fragments with different SSCP patterns were determined by direct

sequencing These alleles were classified into three major lineages AO1 B and O2 In

39

Japanese A102 and B101 were the predominant alleles with frequencies of 83 and

97 in each group respectively whereas in group O two common alleles O101 (43)

and O201 (53) were observed (44)

Akane et al (1996) isolated DNA from peripheral blood leukocytes of 24

unrelated Japanese individuals ABO phenotypes of these samples were identified by

serological methods Using primers 200 base-pair (bp) fragment of ABO locus was

amplified by PCR which spans the site of the single nucleotide deletion associated with

O allele O allele identified by Kpn I digestion of the PCR product A and B alleles were

distinguished by Mae II digestion of the product The nucleotide substitution in the 200-

bp product between A and B alleles was also found in O allele resulting in 2 different

suballeles OA and O

G (66)

Al-Bustan et al (2002) genotyped the ABO blood group system in a Kuwaiti

population sample using polymerase chain reactionmdashrestriction fragment length

polymorphism (PCR-RFLP) analysis The positions of nucleotides 258 and 700 of

cDNA from A transferase were amplified by PCR The amplified DNA was subjected

to RFLP analysis to distinguish A B and O alleles Blood samples of known ABO

phenotype from 101 healthy unrelated Kuwaiti individuals (A 29 B 23AB 14 O 35)

were used Two DNA fragments of the ABO locus were designed to be amplified by 2

pairs of primers To identify the 258th nucleotide a 199- or 200-bp DNA fragment was

amplified by PCR and digested with KpnI For the 700th nucleotide a 128-bp DNA

fragment was amplified by PCR and digested with AluI By analyzing the

electrophoresis patterns the DNA fragments were examined The result of the study

showed that ABO genotypes of the known 101 samples were as follows AA 430

AO 2441 BB 416 BO 242 AB 846 and OO 3465 (67)

Seltsam et al (2003) analyzed the complete genomic sequences except intron

1 and 2 regulatory regions of 6 common (ABOA101 ABOA201 ABOB101

ABOO01 ABOO02 and ABOO03) and 18 rare ABO alleles by phylogenetic

analysis and correlating sequence data with the ABO phenotypes They revealed

multiple polymorphisms in noncoding regions The analysis revealed 5 main lineages

ABOA ABOB ABOO01 ABOO02 and ABOO03 Phenotype-genotype

correlation showed that sequence variations within the complete coding sequence can

21

affect A and B antigen expression All variant ABOAB alleles and one new

ABOO03- like allele were associated with weak ABO phenotypes (10)

Natsuko et al (2004) studied ABO genotypes from samples obtained from 1134

randomly selected Japanese peripheral blood samples A simple ABO genotyping

method using multiplex sequence-specific PCR and capillary electrophoresis was

developed as a supplement to serological ABO typing They found a concordance rate

of 9982 (11321134 samples) between genotypes and phenotypes defined as groups

A B AB and O Sequencing analysis revealed that one discrepant sample contained an

O allele having a point mutation at the primer binding site in exon 6 and another

discrepant sample contained an O allele lacking the guanine deletion at nt 261 (the

O301 allele) (68)

K Honda et al (2004) determined the ABO genotypes of 958 DNA samples

extracted from individuals living in Japan Mongolia and Colombia by using Single-

Strand Conformation Polymorphism (SSCP) which detects only one-base difference

between different genotypes The denatured single-stranded amplicons were

electrophoresed in sequencing gel analyzed by laser detector and visualized the peak

patterns of chromatogram As a result they were able to classify ABO genotypes into

15 groups and additional subtypes Ten kinds of fundamental genotypes (AA AOA AO

G

BB BOA BO

G O

AO

A O

AO

G O

GO

G and AB) by the combination of a base substitution

of np261 (Gdel) and np297 (A G) were detected In addition examination of 400 DNA

samples from Japan Mongolia and Columbia revealed a remarkable regional deviation

in allele frequency of A101 versus A102 and OA vs O

G (69)

Hanania et al (2007) determined the phenotypic allelic frequencies and the

genotypes of ABO blood groups in a Jordanian population Samples of 12215 randomly

healthy Jordanian voluntary blood donors during the period 1998-2003 were taken from

the National Blood Bank donor registry Amman Jordan The results of the phenotypic

distribution indicated that 4686 (3836) of the donors were type A 4473 (3662) O

2203 (1804) B and 853 (698) AB The gene frequencies were 06052 for Io allele

02607 for Ia allele and 01341 for I

b allele Using PCR-RFLP technique two separate

segments of the transferase gene containing nucleotide 261 in exon 6 and nucleotide

703 in exon 7 of the ABO gene locus were amplified and their products were analyzed

23

with two restriction enzymes (KpnI and AluI) The electrophoresis patterns of 105

samples showed that ABO genotypes were AA 6 (5714) AO 35 (33333) BB 1

(0953) BO 14 (13333) AB 10 (9524) and OO 39 (37143) (70)

EL-Zawahri and Luqmani (2008) examined the genotype of a 355 unrelated

blood donors of phenotype A1 (46) A2 (31) A1B (6) A2B (4) B (97) and O (171) by

using a multiplex PCR-RFLP technique in a Kuwaiti Arab cohort DNA fragments of

252 (251 for O1) and 843 (842 for A2) bp spanning the two major exons 6 and 7 of the

ABO gene were amplified and digested with HpaII and KpnI They identified 13

different genotypes combining the A1 A2 B O1 and O2 alleles from the digestion

patterns 1 A1A1 (028) 6 A1A2 (169) 38 A1O1 (1071) 1 A1O2 (028) 1 A2A2

(028) 30 A2O1 (845) 6 A1B (169) 4 A2B (113) 12 BB (338) 79 BO1

(2225) 6 BO2 (169) 167 O1O1 (4704) and 4 O1O2 (113) Two of the

combinations (A2O2 O2O2) were not found (71)

Sung et al (2009) evaluated ABO genotypes via multiplex allele-specific PCR

(ASPCR) amplification using whole blood samples without DNA purification of 127

randomly chosen samples The genotypes of the 127 samples were found to be A1A2

(n=1) A2A2 (n=9) A1O1 (n=3) A2O1 (n=12) A2O2 (n=14) B1B1 (n=5) B1O1 (n=18)

B1O2 (n=15) O1O1 (n=14) O2O2 (n=8) O1O2 (n=14) and A2B1 (n=14) from which

phenotypes were calculated to be A (n=39) B (n=38) O (n=36) and AB (n=14) They

found no discrepancies when the multiplex AS-PCR assay results were compared with

the serologically determined blood group phenotypes and genotypes determined by

DNA sequencing (72)

Nojavan et al (2012) examined the genotype of 744 randomly selected samples

from Azari donors of East Azerbaijan province (Iran) using multiplex allele-specific

PCR ABO genotyping technique As a result the ABO blood group genotypes were

12 A1A104 A1A2 4A1B1 24 A1O1 141 A1O2 32 A2A2 6A2B152

A2O1 69 A2O2 16 B1B1 113 B1O1 105 B1O2 93 O1O1 153 O1O2

85 O2O2 (73)

22

Bugert et al (2012) determined the major ABO alleles by PCR amplification

with sequence-specific primers (PCR-SSP) in a representative sample of 1335 blood

donors in Germany The genotypes were compared to the ABO blood groups registered

in the blood donor files Then the ABO phenotypes and genotypes were determined in

95 paternity trio cases that have been investigated They found that the prevalence of

the major ABO alleles and genotypes corresponded to the expected occurrence of ABO

blood groups in a Caucasian population In 12 of 35 exclusion cases (343) the ABO

genotype also excluded the alleged father whereas the ABO phenotype excluded the

alleged father only in 7 cases (20) (74)

27 ABO Genotyping and Susceptibility to Diseases

The presence of the A and B blood group antigens expressed on red blood cells

and other cells and molecules within the body has been associated with susceptibility to

diseases like cancer leukemia cardiovascular disease and risk of both arterial and

venous thrombosis Most studies indicated an increased risk of thrombosis associated

with the non-O blood group (7576) Nonndashgroup O patients have a greater risk of

venous thromboembolism (VTE) than patients of group O and have greater levels of

von Willebrand factor (vWF) and factor VIII (7577) The risk of VTE is probably

related to the level of vWF and factor VIII because patients of group A2 have lower

levels of these proteins than A1 B and AB and have a lower risk of VTE (76) A B

and H blood group antigens are expressed on N-glycans of vWF and influence the half-

life of the protein providing an explanation for the greater levels in non-O patients

which increase clot formation in non-O patients (78)

21

CHAPTER 3

MATERIALS amp METHODS

24

CHAPTER 3

MATERIALS amp METHODS

31 Materials

311 PCR primers

The nucleotide sequence of the PCR primers used in the current study was as

described by Yamamoto et al (79) The nucleotide substitutions in the primers are

focused on the nucleotide positions 261 297 467 and 803 so as to discriminate

between the A101 A102 B101 O01 O02 and cis-ABO1 alleles Table 31 shows the

oligonucleotide primer sequences their combinations amplification product lengths

and allele specificities The 3prime base of each primer (except int6) was designed to

correspond to the nucleotides at positions 261 297 467 and 803 which define the

polymorphisms

Table 31 PCR primers sequence used for ABO genotype

PCR

reaction

Primer pair Fragment

size (bp)

Allele specificity

1 261G 5prime-GCAGTAGGAAGGATGTCCTCGTGTTG-3prime 205 A101 A102 B101 cis-ABO1

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467C 5prime-CCACTACTATGTCTTCACCGACCATCC-3prime 381 A101 O01 O02

803G 5prime-CACCGACCCCCCGAAGATCC-3prime

2 297A 5prime-CCATTGTCTGGGAGGGCCCA-3prime 164 A101 A102 O01 cis-ABO1

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467C 5prime-CCACTACTATGTCTTCACCGACCATCC-3prime 381 B101

803C 5prime-CACCGACCCCCCGAAGATCG-3prime

3 261A 5prime-GCAGTAGGAAGGATGTCCTCGTGTTA-3prime 205 O01 O02

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467T 5prime-CCACTACTATGTCTTCACCGACCATCT-3prime 381 A102

803G 5prime-CACCGACCCCCCGAAGATCC-3prime

4 297G 5prime-CCATTGTCTGGGAGGGCCCG-3prime 164 B101 O02

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467T 5prime-CCACTACTATGTCTTCACCGACCATCT-3prime 381 cis-ABO1

803C 5prime-CACCGACCCCCCGAAGATCG-3prime

The primers are named based on the position of their 3prime end relative to the cDNA sequence of A101 allele

25

312 Kits

DNA extraction kit (Promega USA)

313 Reagents and Chemicals

Anti A (Plasmatec Monoclonal UK)

Anti B (Plasmatec Monoclonal UK)

Agarose Molecular biology grade (Promega USA)

DNA molecular weight marker 50 bp-ladder (Promega USA)

EDTA disodium salt (Promega USA)

Absolute Ethanol (Sigma USA)

Ethidium bromide (Promega USA)

Absolute Isopropanol (Sigma USA)

Tris base hydroxyl methyl amino methane (Promega USA)

DNAse RNAse free water (Promega USA)

Acetic acid (Sigma USA)

PCR Master mix (Promega USA)

314 Apparatus and Equipments

Thermal Cycler (Biometra Germany)

LG Microwave Oven

Electrophoresis Apparatus

Vortex Mixer

Digital Camera

Power Supply (Biorad)

Freezer Refrigerator

Micro-Centrifuge

Hoefer shortwave UV light table (Transilluminator)

Computer

Electrical Balance

Automatic Micropipettes

26

32 Methods

321 Study population

322 Sample collection

Blood samples were collected from 201 subjects recruited from the Islamic

University ndashGenetics Laboratory Each sample was collected into EDTA tube The

EDTA samples were kept at 4degC and were used within 24 hours for forward blood

grouping and DNA extraction and subsequent PCR analysis

323 Ethical Considerations

An authorization to carry out the study was obtained from a local ethics

committee using an agreement letter prepared by the Islamic University of Gaza All the

information that were obtained about the subjects were kept confidential

324 Data Analysis

The data were entered stored and analyzed by personal computer using the

Statistical Package for Social Sciences (SPSS) version 160 Allele frequencies were

calculated under the assumption of HardyndashWeinberg equilibrium and expressed as

percentages Chi-square test was used to compare observed allelic and genotypic

frequency distributions of the blood group antigens to that expected under the Hardyndash

Weinberg equation P values gt005 were considered statistically significant

The frequency of ABO in the studied population were compared with the

frequency of ABO in some neighbor countries

33 Blood ABO-typing

331 Forward blood group

Whole blood sample (50 microl) was mixed with Anti A and Anti B reagents by

using slide method in the proportions shown in Table 32 below

27

Table 32 Sample Anti A Anti B reaction for forward blood grouping

Sample Anti -A Anti -B

50 microl 50 microl ــــــ

50 microl 50 ــــــ microl

The contents of each slide were rotated then the agglutination (if present) was

read by naked eye after 30 seconds

34 ABO Genotyping

341 DNA Extraction DNA was isolated from fresh EDTA whole blood cells by using Promega kit for

human DNA isolation The kit contains the following components that are enough for

purifying genomic DNA from 200 samples of human blood

Cell lysis solution

Nuclei lysis solution

Protein precipitation solution

DNA rehydration solution

RNase solution

The human genomic DNA was isolated from human blood sample according to

the kit instructions and was as follows

Three hundred microl EDTA blood were transferred into a sterile 15 ml micro-

centrifuge tube containing 900 microl of cell lysis solution The tube was inverted 5-6 times

to mix the components The mixture was incubated for 10 minutes at room temperature

(with gentle mixing once during the incubation) to lyse the red blood cells The tube

was then centrifuged at 13000 rpm for 20 seconds at room temperature

Supernatant was removed and discarded as much as possible without disturbing

the visible white pellet Approximately 10-20 microl of residual liquid should be left in tube

The tube was vortexed vigorously until the white blood cells were completely

resuspended Three hundred microl of nuclei lysis solution was then added to the tube

28

containing the resuspended cells and the suspension was mixed by pipetting the solution

5-6 times to lyse the white blood cells the solution should become very viscous

RNase solution (15 microl) was added to nuclear lysate and the sample was mixed

by inverting the tube 2-5 times The mixture was incubated at 37degC for 15 minutes and

then cooled to room temperature One hundred microl of protein precipitation solution was

added to the nuclear lysate and the mixture was vortexed vigorously for 10ndash20

seconds Small protein clumps may be visible after vortexing and were removed by

centrifuge precipitation at 13000 rpm for 3 minutes at room temperature The

supernatant was transferred to a clean 15 ml micro-centrifuge tube containing 300microl

isopropanol at room temperature The mixture was gently mixed by inversion until the

white thread-like strands of DNA form a visible mass The DNA was then precipitated

by centrifugation at 13000 rpm for 1 minute at room temperature The DNA would be

visible as a small white pellet

The supernatant decanted and one volume of 70 ethanol was added to the

DNA and kept at room temperature and gently inverted several times to wash the DNA

pellet and the sides of the micro-centrifuge tube After centrifugation at 13000 rpm for

1 minute the ethanol was aspirated using a suitable pipette The DNA pellet is very

loose at this point and care must be used to avoid aspirating the pellet into the pipette

The tube was inverted on a clean absorbent paper for 10ndash15 minutes in order to air-dry

the pellet DNA rehydration solution was added to the dry pellet and the DNA was

rehydrated by incubation at 65degC for 1 hour Periodically the solution was mixed by

gently tapping the tube The DNA was then stored at 2-8degC

342 Detection of extracted DNA

The quality of the isolated DNA was determined by running 5 microl of each sample

on ethidium bromide stained 30 agarose gels and the DNA was visualized on a short

wave UV transilluminator the results were documented by photography

35 PCR reactions

PCR was performed using the primers listed in Table 31 in 4 micro-tubes as

described by Sung Ho Lee et al (70) For each PCR 5 μl master mix (Promega) 2 μl

deionized water 1 μl DNA template and 05 μl of each allele specific primer (5 pmol) in

29

one micro-tube (02 ml) were mixed The volume and concentration of a typical PCR

reaction are shown in Table 32

PCR was performed in a thermal cycler The cycling conditions were as

described below In each PCR MTHFR gene specific primers with the following

sequences (Forward 5prime-ACGATGGGGCAAGTGATGCCC -3 and reverse 5-

GAGAAGGTGTCTGCGGGATC-3) were used as a positive control that produces a 95

bp fragment Upon completion of PCR the products were analyzed by electrophoresis

on 2 ethidium bromide stained agarose gel or stored at 4degC until analysis

Table 33 Composition of PCR master mix

Reagent Composition

dNTPs 400microM each dATP dGTP dCTP dTTP

Taq DNA Polymerase 50 units ml

MgCl2 2mM

351 Temperature cycling program

The thermal cycler program was set as follows

Step 1 Denaturation for 3 minutes at 95degC

Step 2 35 cycles of

21 Melting for 40 seconds at 95degC

22 Annealing for 40 seconds at 585degC

23 Extension for 40 seconds at 72degC

Step3 Final elongation for 5 minutes at72degC

352 Expected PCR results

The PCR product size was estimated by comparing it with DNA molecular size

marker (50 bp ladder DNA ) run on the same gel

11

CHAPTER 4

RESULTS

13

CHAPTER 4

RESULTS

41 Study Population

The study population consisted of 201 subjects (50 males 151 females ) The

percentage of males was 249 while that of females was 751 ( figure 41)

0

10

20

30

40

50

60

70

80

maleFemale

Figure 41 Distribution of the study subjects according to gender

42 Phenotypic Frequency of ABO Blood Groups

Blood grouping was done by antigen antibody agglutination test by using

commercial monoclonal antisera The distribution of phenotypes in the total sample

were 363 (73) 224 (45) 75 (15) 338 (68) for groups A B AB and O

respectively Group A was the dominant one in both genders and AB was the rarest in

both males and females ( table 41)

Table 41 Phenotypic frequencies of various blood groups in the study population

Subject Sex Phenotype

Total A B AB O

Male 16 10 4 20 50

Female 57 35 11 48 151

Total 73 45 15 68 201

753

249

12

43 The Allele Frequencies of ABO Antigens

Allele frequency for the antigens was computed by the Hardy-Weinberg law on

the basis of the number of subjects with blood groups ABO The distribution of the

alleles in the total samples was 025 017 058 for IA I

B and I

O respectively

All genotyping results were compatible with the determined phenotypes by

serological method The observed genotypes as compared with the expected genotypes

are shown in Table 42 The frequencies of the five alleles in the our sample population

were O1 and O2 alleles 0376 and 0221 respectively while A1 0174 A2 0067

B 0162

Table 42 Observed and expected genotypes for the 201 samples

P-Value Expected Observed

Genotype Phenotype Percent Number Percent Number

0230 63 126 45 9 AA A

04762 29 583 318 64 AO

07205 29 58 25 5 BB B

09496 197 396 199 40 BO

05915 85 1708 75 15 AB AB

09613 336 676 338 68 OO O

100 201 100 201 Total

The statistical analysis ( using chi square test ) indicated that molecular data

were in good agreement with the ratio calculated from the estimated gene frequencies of

the ABO blood group system in the Gaza Strip population

11

44 PCR Results

441 Quality of the isolated DNA

Regarding the method of DNA isolation that was described in chapter 3 the

quality and quantity of DNA were suitable for PCR processing The quality of the

isolated DNA from human subjects is represented in the following figure (42)

Figure 42 A representative photograph of DNA isolated from human blood The samples were

run on 1 agarose gel stained with ethidium bromide

442 Blood group genotyping by Allele Specific PCR

Multiplex Allele Specific PCR (ASPCR) assay contains four independent PCR

reactions (Table 31) In the first reaction 261G-int6 primer pair was used to amplify

the 205 bp fragment to detect the A101 A102 B101 cis-ABO1 alleles and the 467C-

803G primer pair was selected to amplify the 381 bp fragment to detect the A101 O01

and O02 alleles The 297A-int6 primer pair and the 467C-803C primer pair in the

second reaction were selected to amplify the 381 bp fragment to detect the B101 allele

In the third reaction the 261A-int6 primer pair was selected to amplify the 205 bp

product to detect the O01 and O02 alleles and the 467T-803G primer pair was selected

to amplify the 381 bp product to detect the A102 allele Finally in the fourth reaction

the 297G-int6 primer pair were applied to produce the 164 bp fragment to detect the

B101 and O02 alleles and the 467T-803C primer pair to produce the 381 bp fragment

to recognize the cis-ABO1 allele Table 43 show the products of PCR reactions

according to the genotype

14

Table 43 PCR products according to the genotype

PCR Reactions Genotype

R1 R2 R3 R4

381 164 205 _ O1O1

381 _ 205 164 O2O2

381 164 205 164 O1O2

205 381 _ 164 B1B1

381205 381164 205 164 B1O1

381205 381 205 164 B1O2

381205 381164 _ 164 A1B1

205 381164 381 164 A2B1

381205 164 _ _ A1A1

381205 164 205 _ A1O1

381205 164 205 164 A1O2

381205 164 381 _ A1A2

205 164 381 _ A2A2

381205 164 381205 _ A2O1

381205 164 381205 164 A2O2

Random mating with the six different alleles at the ABO locus can result in 21

different genotype combinations Only 15 different genotype combinations were

detected from the 201 investigated samples (Figure 43)

A- 1 O1O1 2 O2O2

15

B- 1 B1B1 2 O1O2

C- 1 B1O1

2 A1B

D- 1 A1A2 2 A2O2

E- 1 A1O1 2 A1A1

16

F- 1 A1O2 2 B1O2 3 A2A2

G- 1 A2O1

2 A2B1

Figure 43 The electrophoresis pattern of recognized genotypes using the multiplex ASPCR

method This figure shows 15 different genotypes lane M indicates the 50 bp ladder The R1-

R4 show the PCR reaction number

443 Genotype Frequencies

The frequency of ABO recognized genotypes belonging to the population

residing in Gaza Strip are shown in table 44 As shown in the table the highest allele

frequencies in A B AB and O phenotypes were A1O2 (124) B1O1 (129) A1B

(55) and O1O1 (154) respectively

17

Table 44 The frequency of recognized genotypes using multiplex ASPCR method for a

population residing in Gaza Strip

Genotype Frequency Percent

A1A1 3 15

A1O1 24 119

A1O2 25 124

A1A2 4 20

A2A2 2 10

A2O1 13 65

A2O2 2 10

B1B1 5 25

B1O1 26 129

B1O2 14 70

A1B 11 55

A2B 4 20

O1O1 31 154

O1O2 26 129

O2O2 11 55

Total 201 1000

18

CHAPTER 5

DISCUSSION

19

CHAPTER 5

DISCUSSION

Since the first delineation of the molecular basis of the ABO blood group by

Yamamoto et al (11 79) it has become possible to determine the ABO genotypes using

molecular methods without the need for family investigations ABO genotyping is

commonly used in cases of an ABO discrepancy between cell typing and serum typing

as well as in forensic practices for personal identification and paternity testing (72) The

ABH antigens are ubiquitously expressed in humans and are present primarily as

glycoproteins and partly as glycolipids The ABO locus is found on the long arm

of chromosome 9 (9q341-q342)

Multiplex allele specific PCR (the technique we used here) has advantages over

PCR-RFLP in terms of cost effectiveness reaction time and simplicity of handling It is

a rapid method that is based on detecting four single nucleotide polymorphisms at

nucleotides 261 297 796 and 803 of the ABO locus which in turn discriminate the

major ABO alleles

In this study the ABO genotypes of 201 samples recruited from the Islamic

University ndashGenetics Diagnosis Laboratory were determined using multiplex AS-PCR

method The objectives of this study were to correlate ABO genotypes with phenotypes

in blood samples in a Gaza Strip population and to compare the frequency of ABO

genotypes with other populations

51 The Allele Frequencies of ABO Antigens in Gaza Strip

The frequency of the alleles in the total samples were 0250 for the I

A allele

0170 for the IB allele and 0580 for the I

O allele This distribution is in agreement with

the distribution with the samples reported in an Iranian study were they reported

023974 for IA allele 018147 for I

B allele and 057879 for I

O allele (73) The

frequencies of IA I

B and I

O alleles in our subjects are also comparable to those

reported in Iraqi and Jordanian studies where they found 0212 for IA allele 0177 for

IB allele and 06611 for I

O allele and 0270 for I

A allele 0130 for I

B allele and 060 for

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

39

Japanese A102 and B101 were the predominant alleles with frequencies of 83 and

97 in each group respectively whereas in group O two common alleles O101 (43)

and O201 (53) were observed (44)

Akane et al (1996) isolated DNA from peripheral blood leukocytes of 24

unrelated Japanese individuals ABO phenotypes of these samples were identified by

serological methods Using primers 200 base-pair (bp) fragment of ABO locus was

amplified by PCR which spans the site of the single nucleotide deletion associated with

O allele O allele identified by Kpn I digestion of the PCR product A and B alleles were

distinguished by Mae II digestion of the product The nucleotide substitution in the 200-

bp product between A and B alleles was also found in O allele resulting in 2 different

suballeles OA and O

G (66)

Al-Bustan et al (2002) genotyped the ABO blood group system in a Kuwaiti

population sample using polymerase chain reactionmdashrestriction fragment length

polymorphism (PCR-RFLP) analysis The positions of nucleotides 258 and 700 of

cDNA from A transferase were amplified by PCR The amplified DNA was subjected

to RFLP analysis to distinguish A B and O alleles Blood samples of known ABO

phenotype from 101 healthy unrelated Kuwaiti individuals (A 29 B 23AB 14 O 35)

were used Two DNA fragments of the ABO locus were designed to be amplified by 2

pairs of primers To identify the 258th nucleotide a 199- or 200-bp DNA fragment was

amplified by PCR and digested with KpnI For the 700th nucleotide a 128-bp DNA

fragment was amplified by PCR and digested with AluI By analyzing the

electrophoresis patterns the DNA fragments were examined The result of the study

showed that ABO genotypes of the known 101 samples were as follows AA 430

AO 2441 BB 416 BO 242 AB 846 and OO 3465 (67)

Seltsam et al (2003) analyzed the complete genomic sequences except intron

1 and 2 regulatory regions of 6 common (ABOA101 ABOA201 ABOB101

ABOO01 ABOO02 and ABOO03) and 18 rare ABO alleles by phylogenetic

analysis and correlating sequence data with the ABO phenotypes They revealed

multiple polymorphisms in noncoding regions The analysis revealed 5 main lineages

ABOA ABOB ABOO01 ABOO02 and ABOO03 Phenotype-genotype

correlation showed that sequence variations within the complete coding sequence can

21

affect A and B antigen expression All variant ABOAB alleles and one new

ABOO03- like allele were associated with weak ABO phenotypes (10)

Natsuko et al (2004) studied ABO genotypes from samples obtained from 1134

randomly selected Japanese peripheral blood samples A simple ABO genotyping

method using multiplex sequence-specific PCR and capillary electrophoresis was

developed as a supplement to serological ABO typing They found a concordance rate

of 9982 (11321134 samples) between genotypes and phenotypes defined as groups

A B AB and O Sequencing analysis revealed that one discrepant sample contained an

O allele having a point mutation at the primer binding site in exon 6 and another

discrepant sample contained an O allele lacking the guanine deletion at nt 261 (the

O301 allele) (68)

K Honda et al (2004) determined the ABO genotypes of 958 DNA samples

extracted from individuals living in Japan Mongolia and Colombia by using Single-

Strand Conformation Polymorphism (SSCP) which detects only one-base difference

between different genotypes The denatured single-stranded amplicons were

electrophoresed in sequencing gel analyzed by laser detector and visualized the peak

patterns of chromatogram As a result they were able to classify ABO genotypes into

15 groups and additional subtypes Ten kinds of fundamental genotypes (AA AOA AO

G

BB BOA BO

G O

AO

A O

AO

G O

GO

G and AB) by the combination of a base substitution

of np261 (Gdel) and np297 (A G) were detected In addition examination of 400 DNA

samples from Japan Mongolia and Columbia revealed a remarkable regional deviation

in allele frequency of A101 versus A102 and OA vs O

G (69)

Hanania et al (2007) determined the phenotypic allelic frequencies and the

genotypes of ABO blood groups in a Jordanian population Samples of 12215 randomly

healthy Jordanian voluntary blood donors during the period 1998-2003 were taken from

the National Blood Bank donor registry Amman Jordan The results of the phenotypic

distribution indicated that 4686 (3836) of the donors were type A 4473 (3662) O

2203 (1804) B and 853 (698) AB The gene frequencies were 06052 for Io allele

02607 for Ia allele and 01341 for I

b allele Using PCR-RFLP technique two separate

segments of the transferase gene containing nucleotide 261 in exon 6 and nucleotide

703 in exon 7 of the ABO gene locus were amplified and their products were analyzed

23

with two restriction enzymes (KpnI and AluI) The electrophoresis patterns of 105

samples showed that ABO genotypes were AA 6 (5714) AO 35 (33333) BB 1

(0953) BO 14 (13333) AB 10 (9524) and OO 39 (37143) (70)

EL-Zawahri and Luqmani (2008) examined the genotype of a 355 unrelated

blood donors of phenotype A1 (46) A2 (31) A1B (6) A2B (4) B (97) and O (171) by

using a multiplex PCR-RFLP technique in a Kuwaiti Arab cohort DNA fragments of

252 (251 for O1) and 843 (842 for A2) bp spanning the two major exons 6 and 7 of the

ABO gene were amplified and digested with HpaII and KpnI They identified 13

different genotypes combining the A1 A2 B O1 and O2 alleles from the digestion

patterns 1 A1A1 (028) 6 A1A2 (169) 38 A1O1 (1071) 1 A1O2 (028) 1 A2A2

(028) 30 A2O1 (845) 6 A1B (169) 4 A2B (113) 12 BB (338) 79 BO1

(2225) 6 BO2 (169) 167 O1O1 (4704) and 4 O1O2 (113) Two of the

combinations (A2O2 O2O2) were not found (71)

Sung et al (2009) evaluated ABO genotypes via multiplex allele-specific PCR

(ASPCR) amplification using whole blood samples without DNA purification of 127

randomly chosen samples The genotypes of the 127 samples were found to be A1A2

(n=1) A2A2 (n=9) A1O1 (n=3) A2O1 (n=12) A2O2 (n=14) B1B1 (n=5) B1O1 (n=18)

B1O2 (n=15) O1O1 (n=14) O2O2 (n=8) O1O2 (n=14) and A2B1 (n=14) from which

phenotypes were calculated to be A (n=39) B (n=38) O (n=36) and AB (n=14) They

found no discrepancies when the multiplex AS-PCR assay results were compared with

the serologically determined blood group phenotypes and genotypes determined by

DNA sequencing (72)

Nojavan et al (2012) examined the genotype of 744 randomly selected samples

from Azari donors of East Azerbaijan province (Iran) using multiplex allele-specific

PCR ABO genotyping technique As a result the ABO blood group genotypes were

12 A1A104 A1A2 4A1B1 24 A1O1 141 A1O2 32 A2A2 6A2B152

A2O1 69 A2O2 16 B1B1 113 B1O1 105 B1O2 93 O1O1 153 O1O2

85 O2O2 (73)

22

Bugert et al (2012) determined the major ABO alleles by PCR amplification

with sequence-specific primers (PCR-SSP) in a representative sample of 1335 blood

donors in Germany The genotypes were compared to the ABO blood groups registered

in the blood donor files Then the ABO phenotypes and genotypes were determined in

95 paternity trio cases that have been investigated They found that the prevalence of

the major ABO alleles and genotypes corresponded to the expected occurrence of ABO

blood groups in a Caucasian population In 12 of 35 exclusion cases (343) the ABO

genotype also excluded the alleged father whereas the ABO phenotype excluded the

alleged father only in 7 cases (20) (74)

27 ABO Genotyping and Susceptibility to Diseases

The presence of the A and B blood group antigens expressed on red blood cells

and other cells and molecules within the body has been associated with susceptibility to

diseases like cancer leukemia cardiovascular disease and risk of both arterial and

venous thrombosis Most studies indicated an increased risk of thrombosis associated

with the non-O blood group (7576) Nonndashgroup O patients have a greater risk of

venous thromboembolism (VTE) than patients of group O and have greater levels of

von Willebrand factor (vWF) and factor VIII (7577) The risk of VTE is probably

related to the level of vWF and factor VIII because patients of group A2 have lower

levels of these proteins than A1 B and AB and have a lower risk of VTE (76) A B

and H blood group antigens are expressed on N-glycans of vWF and influence the half-

life of the protein providing an explanation for the greater levels in non-O patients

which increase clot formation in non-O patients (78)

21

CHAPTER 3

MATERIALS amp METHODS

24

CHAPTER 3

MATERIALS amp METHODS

31 Materials

311 PCR primers

The nucleotide sequence of the PCR primers used in the current study was as

described by Yamamoto et al (79) The nucleotide substitutions in the primers are

focused on the nucleotide positions 261 297 467 and 803 so as to discriminate

between the A101 A102 B101 O01 O02 and cis-ABO1 alleles Table 31 shows the

oligonucleotide primer sequences their combinations amplification product lengths

and allele specificities The 3prime base of each primer (except int6) was designed to

correspond to the nucleotides at positions 261 297 467 and 803 which define the

polymorphisms

Table 31 PCR primers sequence used for ABO genotype

PCR

reaction

Primer pair Fragment

size (bp)

Allele specificity

1 261G 5prime-GCAGTAGGAAGGATGTCCTCGTGTTG-3prime 205 A101 A102 B101 cis-ABO1

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467C 5prime-CCACTACTATGTCTTCACCGACCATCC-3prime 381 A101 O01 O02

803G 5prime-CACCGACCCCCCGAAGATCC-3prime

2 297A 5prime-CCATTGTCTGGGAGGGCCCA-3prime 164 A101 A102 O01 cis-ABO1

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467C 5prime-CCACTACTATGTCTTCACCGACCATCC-3prime 381 B101

803C 5prime-CACCGACCCCCCGAAGATCG-3prime

3 261A 5prime-GCAGTAGGAAGGATGTCCTCGTGTTA-3prime 205 O01 O02

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467T 5prime-CCACTACTATGTCTTCACCGACCATCT-3prime 381 A102

803G 5prime-CACCGACCCCCCGAAGATCC-3prime

4 297G 5prime-CCATTGTCTGGGAGGGCCCG-3prime 164 B101 O02

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467T 5prime-CCACTACTATGTCTTCACCGACCATCT-3prime 381 cis-ABO1

803C 5prime-CACCGACCCCCCGAAGATCG-3prime

The primers are named based on the position of their 3prime end relative to the cDNA sequence of A101 allele

25

312 Kits

DNA extraction kit (Promega USA)

313 Reagents and Chemicals

Anti A (Plasmatec Monoclonal UK)

Anti B (Plasmatec Monoclonal UK)

Agarose Molecular biology grade (Promega USA)

DNA molecular weight marker 50 bp-ladder (Promega USA)

EDTA disodium salt (Promega USA)

Absolute Ethanol (Sigma USA)

Ethidium bromide (Promega USA)

Absolute Isopropanol (Sigma USA)

Tris base hydroxyl methyl amino methane (Promega USA)

DNAse RNAse free water (Promega USA)

Acetic acid (Sigma USA)

PCR Master mix (Promega USA)

314 Apparatus and Equipments

Thermal Cycler (Biometra Germany)

LG Microwave Oven

Electrophoresis Apparatus

Vortex Mixer

Digital Camera

Power Supply (Biorad)

Freezer Refrigerator

Micro-Centrifuge

Hoefer shortwave UV light table (Transilluminator)

Computer

Electrical Balance

Automatic Micropipettes

26

32 Methods

321 Study population

322 Sample collection

Blood samples were collected from 201 subjects recruited from the Islamic

University ndashGenetics Laboratory Each sample was collected into EDTA tube The

EDTA samples were kept at 4degC and were used within 24 hours for forward blood

grouping and DNA extraction and subsequent PCR analysis

323 Ethical Considerations

An authorization to carry out the study was obtained from a local ethics

committee using an agreement letter prepared by the Islamic University of Gaza All the

information that were obtained about the subjects were kept confidential

324 Data Analysis

The data were entered stored and analyzed by personal computer using the

Statistical Package for Social Sciences (SPSS) version 160 Allele frequencies were

calculated under the assumption of HardyndashWeinberg equilibrium and expressed as

percentages Chi-square test was used to compare observed allelic and genotypic

frequency distributions of the blood group antigens to that expected under the Hardyndash

Weinberg equation P values gt005 were considered statistically significant

The frequency of ABO in the studied population were compared with the

frequency of ABO in some neighbor countries

33 Blood ABO-typing

331 Forward blood group

Whole blood sample (50 microl) was mixed with Anti A and Anti B reagents by

using slide method in the proportions shown in Table 32 below

27

Table 32 Sample Anti A Anti B reaction for forward blood grouping

Sample Anti -A Anti -B

50 microl 50 microl ــــــ

50 microl 50 ــــــ microl

The contents of each slide were rotated then the agglutination (if present) was

read by naked eye after 30 seconds

34 ABO Genotyping

341 DNA Extraction DNA was isolated from fresh EDTA whole blood cells by using Promega kit for

human DNA isolation The kit contains the following components that are enough for

purifying genomic DNA from 200 samples of human blood

Cell lysis solution

Nuclei lysis solution

Protein precipitation solution

DNA rehydration solution

RNase solution

The human genomic DNA was isolated from human blood sample according to

the kit instructions and was as follows

Three hundred microl EDTA blood were transferred into a sterile 15 ml micro-

centrifuge tube containing 900 microl of cell lysis solution The tube was inverted 5-6 times

to mix the components The mixture was incubated for 10 minutes at room temperature

(with gentle mixing once during the incubation) to lyse the red blood cells The tube

was then centrifuged at 13000 rpm for 20 seconds at room temperature

Supernatant was removed and discarded as much as possible without disturbing

the visible white pellet Approximately 10-20 microl of residual liquid should be left in tube

The tube was vortexed vigorously until the white blood cells were completely

resuspended Three hundred microl of nuclei lysis solution was then added to the tube

28

containing the resuspended cells and the suspension was mixed by pipetting the solution

5-6 times to lyse the white blood cells the solution should become very viscous

RNase solution (15 microl) was added to nuclear lysate and the sample was mixed

by inverting the tube 2-5 times The mixture was incubated at 37degC for 15 minutes and

then cooled to room temperature One hundred microl of protein precipitation solution was

added to the nuclear lysate and the mixture was vortexed vigorously for 10ndash20

seconds Small protein clumps may be visible after vortexing and were removed by

centrifuge precipitation at 13000 rpm for 3 minutes at room temperature The

supernatant was transferred to a clean 15 ml micro-centrifuge tube containing 300microl

isopropanol at room temperature The mixture was gently mixed by inversion until the

white thread-like strands of DNA form a visible mass The DNA was then precipitated

by centrifugation at 13000 rpm for 1 minute at room temperature The DNA would be

visible as a small white pellet

The supernatant decanted and one volume of 70 ethanol was added to the

DNA and kept at room temperature and gently inverted several times to wash the DNA

pellet and the sides of the micro-centrifuge tube After centrifugation at 13000 rpm for

1 minute the ethanol was aspirated using a suitable pipette The DNA pellet is very

loose at this point and care must be used to avoid aspirating the pellet into the pipette

The tube was inverted on a clean absorbent paper for 10ndash15 minutes in order to air-dry

the pellet DNA rehydration solution was added to the dry pellet and the DNA was

rehydrated by incubation at 65degC for 1 hour Periodically the solution was mixed by

gently tapping the tube The DNA was then stored at 2-8degC

342 Detection of extracted DNA

The quality of the isolated DNA was determined by running 5 microl of each sample

on ethidium bromide stained 30 agarose gels and the DNA was visualized on a short

wave UV transilluminator the results were documented by photography

35 PCR reactions

PCR was performed using the primers listed in Table 31 in 4 micro-tubes as

described by Sung Ho Lee et al (70) For each PCR 5 μl master mix (Promega) 2 μl

deionized water 1 μl DNA template and 05 μl of each allele specific primer (5 pmol) in

29

one micro-tube (02 ml) were mixed The volume and concentration of a typical PCR

reaction are shown in Table 32

PCR was performed in a thermal cycler The cycling conditions were as

described below In each PCR MTHFR gene specific primers with the following

sequences (Forward 5prime-ACGATGGGGCAAGTGATGCCC -3 and reverse 5-

GAGAAGGTGTCTGCGGGATC-3) were used as a positive control that produces a 95

bp fragment Upon completion of PCR the products were analyzed by electrophoresis

on 2 ethidium bromide stained agarose gel or stored at 4degC until analysis

Table 33 Composition of PCR master mix

Reagent Composition

dNTPs 400microM each dATP dGTP dCTP dTTP

Taq DNA Polymerase 50 units ml

MgCl2 2mM

351 Temperature cycling program

The thermal cycler program was set as follows

Step 1 Denaturation for 3 minutes at 95degC

Step 2 35 cycles of

21 Melting for 40 seconds at 95degC

22 Annealing for 40 seconds at 585degC

23 Extension for 40 seconds at 72degC

Step3 Final elongation for 5 minutes at72degC

352 Expected PCR results

The PCR product size was estimated by comparing it with DNA molecular size

marker (50 bp ladder DNA ) run on the same gel

11

CHAPTER 4

RESULTS

13

CHAPTER 4

RESULTS

41 Study Population

The study population consisted of 201 subjects (50 males 151 females ) The

percentage of males was 249 while that of females was 751 ( figure 41)

0

10

20

30

40

50

60

70

80

maleFemale

Figure 41 Distribution of the study subjects according to gender

42 Phenotypic Frequency of ABO Blood Groups

Blood grouping was done by antigen antibody agglutination test by using

commercial monoclonal antisera The distribution of phenotypes in the total sample

were 363 (73) 224 (45) 75 (15) 338 (68) for groups A B AB and O

respectively Group A was the dominant one in both genders and AB was the rarest in

both males and females ( table 41)

Table 41 Phenotypic frequencies of various blood groups in the study population

Subject Sex Phenotype

Total A B AB O

Male 16 10 4 20 50

Female 57 35 11 48 151

Total 73 45 15 68 201

753

249

12

43 The Allele Frequencies of ABO Antigens

Allele frequency for the antigens was computed by the Hardy-Weinberg law on

the basis of the number of subjects with blood groups ABO The distribution of the

alleles in the total samples was 025 017 058 for IA I

B and I

O respectively

All genotyping results were compatible with the determined phenotypes by

serological method The observed genotypes as compared with the expected genotypes

are shown in Table 42 The frequencies of the five alleles in the our sample population

were O1 and O2 alleles 0376 and 0221 respectively while A1 0174 A2 0067

B 0162

Table 42 Observed and expected genotypes for the 201 samples

P-Value Expected Observed

Genotype Phenotype Percent Number Percent Number

0230 63 126 45 9 AA A

04762 29 583 318 64 AO

07205 29 58 25 5 BB B

09496 197 396 199 40 BO

05915 85 1708 75 15 AB AB

09613 336 676 338 68 OO O

100 201 100 201 Total

The statistical analysis ( using chi square test ) indicated that molecular data

were in good agreement with the ratio calculated from the estimated gene frequencies of

the ABO blood group system in the Gaza Strip population

11

44 PCR Results

441 Quality of the isolated DNA

Regarding the method of DNA isolation that was described in chapter 3 the

quality and quantity of DNA were suitable for PCR processing The quality of the

isolated DNA from human subjects is represented in the following figure (42)

Figure 42 A representative photograph of DNA isolated from human blood The samples were

run on 1 agarose gel stained with ethidium bromide

442 Blood group genotyping by Allele Specific PCR

Multiplex Allele Specific PCR (ASPCR) assay contains four independent PCR

reactions (Table 31) In the first reaction 261G-int6 primer pair was used to amplify

the 205 bp fragment to detect the A101 A102 B101 cis-ABO1 alleles and the 467C-

803G primer pair was selected to amplify the 381 bp fragment to detect the A101 O01

and O02 alleles The 297A-int6 primer pair and the 467C-803C primer pair in the

second reaction were selected to amplify the 381 bp fragment to detect the B101 allele

In the third reaction the 261A-int6 primer pair was selected to amplify the 205 bp

product to detect the O01 and O02 alleles and the 467T-803G primer pair was selected

to amplify the 381 bp product to detect the A102 allele Finally in the fourth reaction

the 297G-int6 primer pair were applied to produce the 164 bp fragment to detect the

B101 and O02 alleles and the 467T-803C primer pair to produce the 381 bp fragment

to recognize the cis-ABO1 allele Table 43 show the products of PCR reactions

according to the genotype

14

Table 43 PCR products according to the genotype

PCR Reactions Genotype

R1 R2 R3 R4

381 164 205 _ O1O1

381 _ 205 164 O2O2

381 164 205 164 O1O2

205 381 _ 164 B1B1

381205 381164 205 164 B1O1

381205 381 205 164 B1O2

381205 381164 _ 164 A1B1

205 381164 381 164 A2B1

381205 164 _ _ A1A1

381205 164 205 _ A1O1

381205 164 205 164 A1O2

381205 164 381 _ A1A2

205 164 381 _ A2A2

381205 164 381205 _ A2O1

381205 164 381205 164 A2O2

Random mating with the six different alleles at the ABO locus can result in 21

different genotype combinations Only 15 different genotype combinations were

detected from the 201 investigated samples (Figure 43)

A- 1 O1O1 2 O2O2

15

B- 1 B1B1 2 O1O2

C- 1 B1O1

2 A1B

D- 1 A1A2 2 A2O2

E- 1 A1O1 2 A1A1

16

F- 1 A1O2 2 B1O2 3 A2A2

G- 1 A2O1

2 A2B1

Figure 43 The electrophoresis pattern of recognized genotypes using the multiplex ASPCR

method This figure shows 15 different genotypes lane M indicates the 50 bp ladder The R1-

R4 show the PCR reaction number

443 Genotype Frequencies

The frequency of ABO recognized genotypes belonging to the population

residing in Gaza Strip are shown in table 44 As shown in the table the highest allele

frequencies in A B AB and O phenotypes were A1O2 (124) B1O1 (129) A1B

(55) and O1O1 (154) respectively

17

Table 44 The frequency of recognized genotypes using multiplex ASPCR method for a

population residing in Gaza Strip

Genotype Frequency Percent

A1A1 3 15

A1O1 24 119

A1O2 25 124

A1A2 4 20

A2A2 2 10

A2O1 13 65

A2O2 2 10

B1B1 5 25

B1O1 26 129

B1O2 14 70

A1B 11 55

A2B 4 20

O1O1 31 154

O1O2 26 129

O2O2 11 55

Total 201 1000

18

CHAPTER 5

DISCUSSION

19

CHAPTER 5

DISCUSSION

Since the first delineation of the molecular basis of the ABO blood group by

Yamamoto et al (11 79) it has become possible to determine the ABO genotypes using

molecular methods without the need for family investigations ABO genotyping is

commonly used in cases of an ABO discrepancy between cell typing and serum typing

as well as in forensic practices for personal identification and paternity testing (72) The

ABH antigens are ubiquitously expressed in humans and are present primarily as

glycoproteins and partly as glycolipids The ABO locus is found on the long arm

of chromosome 9 (9q341-q342)

Multiplex allele specific PCR (the technique we used here) has advantages over

PCR-RFLP in terms of cost effectiveness reaction time and simplicity of handling It is

a rapid method that is based on detecting four single nucleotide polymorphisms at

nucleotides 261 297 796 and 803 of the ABO locus which in turn discriminate the

major ABO alleles

In this study the ABO genotypes of 201 samples recruited from the Islamic

University ndashGenetics Diagnosis Laboratory were determined using multiplex AS-PCR

method The objectives of this study were to correlate ABO genotypes with phenotypes

in blood samples in a Gaza Strip population and to compare the frequency of ABO

genotypes with other populations

51 The Allele Frequencies of ABO Antigens in Gaza Strip

The frequency of the alleles in the total samples were 0250 for the I

A allele

0170 for the IB allele and 0580 for the I

O allele This distribution is in agreement with

the distribution with the samples reported in an Iranian study were they reported

023974 for IA allele 018147 for I

B allele and 057879 for I

O allele (73) The

frequencies of IA I

B and I

O alleles in our subjects are also comparable to those

reported in Iraqi and Jordanian studies where they found 0212 for IA allele 0177 for

IB allele and 06611 for I

O allele and 0270 for I

A allele 0130 for I

B allele and 060 for

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

21

affect A and B antigen expression All variant ABOAB alleles and one new

ABOO03- like allele were associated with weak ABO phenotypes (10)

Natsuko et al (2004) studied ABO genotypes from samples obtained from 1134

randomly selected Japanese peripheral blood samples A simple ABO genotyping

method using multiplex sequence-specific PCR and capillary electrophoresis was

developed as a supplement to serological ABO typing They found a concordance rate

of 9982 (11321134 samples) between genotypes and phenotypes defined as groups

A B AB and O Sequencing analysis revealed that one discrepant sample contained an

O allele having a point mutation at the primer binding site in exon 6 and another

discrepant sample contained an O allele lacking the guanine deletion at nt 261 (the

O301 allele) (68)

K Honda et al (2004) determined the ABO genotypes of 958 DNA samples

extracted from individuals living in Japan Mongolia and Colombia by using Single-

Strand Conformation Polymorphism (SSCP) which detects only one-base difference

between different genotypes The denatured single-stranded amplicons were

electrophoresed in sequencing gel analyzed by laser detector and visualized the peak

patterns of chromatogram As a result they were able to classify ABO genotypes into

15 groups and additional subtypes Ten kinds of fundamental genotypes (AA AOA AO

G

BB BOA BO

G O

AO

A O

AO

G O

GO

G and AB) by the combination of a base substitution

of np261 (Gdel) and np297 (A G) were detected In addition examination of 400 DNA

samples from Japan Mongolia and Columbia revealed a remarkable regional deviation

in allele frequency of A101 versus A102 and OA vs O

G (69)

Hanania et al (2007) determined the phenotypic allelic frequencies and the

genotypes of ABO blood groups in a Jordanian population Samples of 12215 randomly

healthy Jordanian voluntary blood donors during the period 1998-2003 were taken from

the National Blood Bank donor registry Amman Jordan The results of the phenotypic

distribution indicated that 4686 (3836) of the donors were type A 4473 (3662) O

2203 (1804) B and 853 (698) AB The gene frequencies were 06052 for Io allele

02607 for Ia allele and 01341 for I

b allele Using PCR-RFLP technique two separate

segments of the transferase gene containing nucleotide 261 in exon 6 and nucleotide

703 in exon 7 of the ABO gene locus were amplified and their products were analyzed

23

with two restriction enzymes (KpnI and AluI) The electrophoresis patterns of 105

samples showed that ABO genotypes were AA 6 (5714) AO 35 (33333) BB 1

(0953) BO 14 (13333) AB 10 (9524) and OO 39 (37143) (70)

EL-Zawahri and Luqmani (2008) examined the genotype of a 355 unrelated

blood donors of phenotype A1 (46) A2 (31) A1B (6) A2B (4) B (97) and O (171) by

using a multiplex PCR-RFLP technique in a Kuwaiti Arab cohort DNA fragments of

252 (251 for O1) and 843 (842 for A2) bp spanning the two major exons 6 and 7 of the

ABO gene were amplified and digested with HpaII and KpnI They identified 13

different genotypes combining the A1 A2 B O1 and O2 alleles from the digestion

patterns 1 A1A1 (028) 6 A1A2 (169) 38 A1O1 (1071) 1 A1O2 (028) 1 A2A2

(028) 30 A2O1 (845) 6 A1B (169) 4 A2B (113) 12 BB (338) 79 BO1

(2225) 6 BO2 (169) 167 O1O1 (4704) and 4 O1O2 (113) Two of the

combinations (A2O2 O2O2) were not found (71)

Sung et al (2009) evaluated ABO genotypes via multiplex allele-specific PCR

(ASPCR) amplification using whole blood samples without DNA purification of 127

randomly chosen samples The genotypes of the 127 samples were found to be A1A2

(n=1) A2A2 (n=9) A1O1 (n=3) A2O1 (n=12) A2O2 (n=14) B1B1 (n=5) B1O1 (n=18)

B1O2 (n=15) O1O1 (n=14) O2O2 (n=8) O1O2 (n=14) and A2B1 (n=14) from which

phenotypes were calculated to be A (n=39) B (n=38) O (n=36) and AB (n=14) They

found no discrepancies when the multiplex AS-PCR assay results were compared with

the serologically determined blood group phenotypes and genotypes determined by

DNA sequencing (72)

Nojavan et al (2012) examined the genotype of 744 randomly selected samples

from Azari donors of East Azerbaijan province (Iran) using multiplex allele-specific

PCR ABO genotyping technique As a result the ABO blood group genotypes were

12 A1A104 A1A2 4A1B1 24 A1O1 141 A1O2 32 A2A2 6A2B152

A2O1 69 A2O2 16 B1B1 113 B1O1 105 B1O2 93 O1O1 153 O1O2

85 O2O2 (73)

22

Bugert et al (2012) determined the major ABO alleles by PCR amplification

with sequence-specific primers (PCR-SSP) in a representative sample of 1335 blood

donors in Germany The genotypes were compared to the ABO blood groups registered

in the blood donor files Then the ABO phenotypes and genotypes were determined in

95 paternity trio cases that have been investigated They found that the prevalence of

the major ABO alleles and genotypes corresponded to the expected occurrence of ABO

blood groups in a Caucasian population In 12 of 35 exclusion cases (343) the ABO

genotype also excluded the alleged father whereas the ABO phenotype excluded the

alleged father only in 7 cases (20) (74)

27 ABO Genotyping and Susceptibility to Diseases

The presence of the A and B blood group antigens expressed on red blood cells

and other cells and molecules within the body has been associated with susceptibility to

diseases like cancer leukemia cardiovascular disease and risk of both arterial and

venous thrombosis Most studies indicated an increased risk of thrombosis associated

with the non-O blood group (7576) Nonndashgroup O patients have a greater risk of

venous thromboembolism (VTE) than patients of group O and have greater levels of

von Willebrand factor (vWF) and factor VIII (7577) The risk of VTE is probably

related to the level of vWF and factor VIII because patients of group A2 have lower

levels of these proteins than A1 B and AB and have a lower risk of VTE (76) A B

and H blood group antigens are expressed on N-glycans of vWF and influence the half-

life of the protein providing an explanation for the greater levels in non-O patients

which increase clot formation in non-O patients (78)

21

CHAPTER 3

MATERIALS amp METHODS

24

CHAPTER 3

MATERIALS amp METHODS

31 Materials

311 PCR primers

The nucleotide sequence of the PCR primers used in the current study was as

described by Yamamoto et al (79) The nucleotide substitutions in the primers are

focused on the nucleotide positions 261 297 467 and 803 so as to discriminate

between the A101 A102 B101 O01 O02 and cis-ABO1 alleles Table 31 shows the

oligonucleotide primer sequences their combinations amplification product lengths

and allele specificities The 3prime base of each primer (except int6) was designed to

correspond to the nucleotides at positions 261 297 467 and 803 which define the

polymorphisms

Table 31 PCR primers sequence used for ABO genotype

PCR

reaction

Primer pair Fragment

size (bp)

Allele specificity

1 261G 5prime-GCAGTAGGAAGGATGTCCTCGTGTTG-3prime 205 A101 A102 B101 cis-ABO1

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467C 5prime-CCACTACTATGTCTTCACCGACCATCC-3prime 381 A101 O01 O02

803G 5prime-CACCGACCCCCCGAAGATCC-3prime

2 297A 5prime-CCATTGTCTGGGAGGGCCCA-3prime 164 A101 A102 O01 cis-ABO1

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467C 5prime-CCACTACTATGTCTTCACCGACCATCC-3prime 381 B101

803C 5prime-CACCGACCCCCCGAAGATCG-3prime

3 261A 5prime-GCAGTAGGAAGGATGTCCTCGTGTTA-3prime 205 O01 O02

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467T 5prime-CCACTACTATGTCTTCACCGACCATCT-3prime 381 A102

803G 5prime-CACCGACCCCCCGAAGATCC-3prime

4 297G 5prime-CCATTGTCTGGGAGGGCCCG-3prime 164 B101 O02

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467T 5prime-CCACTACTATGTCTTCACCGACCATCT-3prime 381 cis-ABO1

803C 5prime-CACCGACCCCCCGAAGATCG-3prime

The primers are named based on the position of their 3prime end relative to the cDNA sequence of A101 allele

25

312 Kits

DNA extraction kit (Promega USA)

313 Reagents and Chemicals

Anti A (Plasmatec Monoclonal UK)

Anti B (Plasmatec Monoclonal UK)

Agarose Molecular biology grade (Promega USA)

DNA molecular weight marker 50 bp-ladder (Promega USA)

EDTA disodium salt (Promega USA)

Absolute Ethanol (Sigma USA)

Ethidium bromide (Promega USA)

Absolute Isopropanol (Sigma USA)

Tris base hydroxyl methyl amino methane (Promega USA)

DNAse RNAse free water (Promega USA)

Acetic acid (Sigma USA)

PCR Master mix (Promega USA)

314 Apparatus and Equipments

Thermal Cycler (Biometra Germany)

LG Microwave Oven

Electrophoresis Apparatus

Vortex Mixer

Digital Camera

Power Supply (Biorad)

Freezer Refrigerator

Micro-Centrifuge

Hoefer shortwave UV light table (Transilluminator)

Computer

Electrical Balance

Automatic Micropipettes

26

32 Methods

321 Study population

322 Sample collection

Blood samples were collected from 201 subjects recruited from the Islamic

University ndashGenetics Laboratory Each sample was collected into EDTA tube The

EDTA samples were kept at 4degC and were used within 24 hours for forward blood

grouping and DNA extraction and subsequent PCR analysis

323 Ethical Considerations

An authorization to carry out the study was obtained from a local ethics

committee using an agreement letter prepared by the Islamic University of Gaza All the

information that were obtained about the subjects were kept confidential

324 Data Analysis

The data were entered stored and analyzed by personal computer using the

Statistical Package for Social Sciences (SPSS) version 160 Allele frequencies were

calculated under the assumption of HardyndashWeinberg equilibrium and expressed as

percentages Chi-square test was used to compare observed allelic and genotypic

frequency distributions of the blood group antigens to that expected under the Hardyndash

Weinberg equation P values gt005 were considered statistically significant

The frequency of ABO in the studied population were compared with the

frequency of ABO in some neighbor countries

33 Blood ABO-typing

331 Forward blood group

Whole blood sample (50 microl) was mixed with Anti A and Anti B reagents by

using slide method in the proportions shown in Table 32 below

27

Table 32 Sample Anti A Anti B reaction for forward blood grouping

Sample Anti -A Anti -B

50 microl 50 microl ــــــ

50 microl 50 ــــــ microl

The contents of each slide were rotated then the agglutination (if present) was

read by naked eye after 30 seconds

34 ABO Genotyping

341 DNA Extraction DNA was isolated from fresh EDTA whole blood cells by using Promega kit for

human DNA isolation The kit contains the following components that are enough for

purifying genomic DNA from 200 samples of human blood

Cell lysis solution

Nuclei lysis solution

Protein precipitation solution

DNA rehydration solution

RNase solution

The human genomic DNA was isolated from human blood sample according to

the kit instructions and was as follows

Three hundred microl EDTA blood were transferred into a sterile 15 ml micro-

centrifuge tube containing 900 microl of cell lysis solution The tube was inverted 5-6 times

to mix the components The mixture was incubated for 10 minutes at room temperature

(with gentle mixing once during the incubation) to lyse the red blood cells The tube

was then centrifuged at 13000 rpm for 20 seconds at room temperature

Supernatant was removed and discarded as much as possible without disturbing

the visible white pellet Approximately 10-20 microl of residual liquid should be left in tube

The tube was vortexed vigorously until the white blood cells were completely

resuspended Three hundred microl of nuclei lysis solution was then added to the tube

28

containing the resuspended cells and the suspension was mixed by pipetting the solution

5-6 times to lyse the white blood cells the solution should become very viscous

RNase solution (15 microl) was added to nuclear lysate and the sample was mixed

by inverting the tube 2-5 times The mixture was incubated at 37degC for 15 minutes and

then cooled to room temperature One hundred microl of protein precipitation solution was

added to the nuclear lysate and the mixture was vortexed vigorously for 10ndash20

seconds Small protein clumps may be visible after vortexing and were removed by

centrifuge precipitation at 13000 rpm for 3 minutes at room temperature The

supernatant was transferred to a clean 15 ml micro-centrifuge tube containing 300microl

isopropanol at room temperature The mixture was gently mixed by inversion until the

white thread-like strands of DNA form a visible mass The DNA was then precipitated

by centrifugation at 13000 rpm for 1 minute at room temperature The DNA would be

visible as a small white pellet

The supernatant decanted and one volume of 70 ethanol was added to the

DNA and kept at room temperature and gently inverted several times to wash the DNA

pellet and the sides of the micro-centrifuge tube After centrifugation at 13000 rpm for

1 minute the ethanol was aspirated using a suitable pipette The DNA pellet is very

loose at this point and care must be used to avoid aspirating the pellet into the pipette

The tube was inverted on a clean absorbent paper for 10ndash15 minutes in order to air-dry

the pellet DNA rehydration solution was added to the dry pellet and the DNA was

rehydrated by incubation at 65degC for 1 hour Periodically the solution was mixed by

gently tapping the tube The DNA was then stored at 2-8degC

342 Detection of extracted DNA

The quality of the isolated DNA was determined by running 5 microl of each sample

on ethidium bromide stained 30 agarose gels and the DNA was visualized on a short

wave UV transilluminator the results were documented by photography

35 PCR reactions

PCR was performed using the primers listed in Table 31 in 4 micro-tubes as

described by Sung Ho Lee et al (70) For each PCR 5 μl master mix (Promega) 2 μl

deionized water 1 μl DNA template and 05 μl of each allele specific primer (5 pmol) in

29

one micro-tube (02 ml) were mixed The volume and concentration of a typical PCR

reaction are shown in Table 32

PCR was performed in a thermal cycler The cycling conditions were as

described below In each PCR MTHFR gene specific primers with the following

sequences (Forward 5prime-ACGATGGGGCAAGTGATGCCC -3 and reverse 5-

GAGAAGGTGTCTGCGGGATC-3) were used as a positive control that produces a 95

bp fragment Upon completion of PCR the products were analyzed by electrophoresis

on 2 ethidium bromide stained agarose gel or stored at 4degC until analysis

Table 33 Composition of PCR master mix

Reagent Composition

dNTPs 400microM each dATP dGTP dCTP dTTP

Taq DNA Polymerase 50 units ml

MgCl2 2mM

351 Temperature cycling program

The thermal cycler program was set as follows

Step 1 Denaturation for 3 minutes at 95degC

Step 2 35 cycles of

21 Melting for 40 seconds at 95degC

22 Annealing for 40 seconds at 585degC

23 Extension for 40 seconds at 72degC

Step3 Final elongation for 5 minutes at72degC

352 Expected PCR results

The PCR product size was estimated by comparing it with DNA molecular size

marker (50 bp ladder DNA ) run on the same gel

11

CHAPTER 4

RESULTS

13

CHAPTER 4

RESULTS

41 Study Population

The study population consisted of 201 subjects (50 males 151 females ) The

percentage of males was 249 while that of females was 751 ( figure 41)

0

10

20

30

40

50

60

70

80

maleFemale

Figure 41 Distribution of the study subjects according to gender

42 Phenotypic Frequency of ABO Blood Groups

Blood grouping was done by antigen antibody agglutination test by using

commercial monoclonal antisera The distribution of phenotypes in the total sample

were 363 (73) 224 (45) 75 (15) 338 (68) for groups A B AB and O

respectively Group A was the dominant one in both genders and AB was the rarest in

both males and females ( table 41)

Table 41 Phenotypic frequencies of various blood groups in the study population

Subject Sex Phenotype

Total A B AB O

Male 16 10 4 20 50

Female 57 35 11 48 151

Total 73 45 15 68 201

753

249

12

43 The Allele Frequencies of ABO Antigens

Allele frequency for the antigens was computed by the Hardy-Weinberg law on

the basis of the number of subjects with blood groups ABO The distribution of the

alleles in the total samples was 025 017 058 for IA I

B and I

O respectively

All genotyping results were compatible with the determined phenotypes by

serological method The observed genotypes as compared with the expected genotypes

are shown in Table 42 The frequencies of the five alleles in the our sample population

were O1 and O2 alleles 0376 and 0221 respectively while A1 0174 A2 0067

B 0162

Table 42 Observed and expected genotypes for the 201 samples

P-Value Expected Observed

Genotype Phenotype Percent Number Percent Number

0230 63 126 45 9 AA A

04762 29 583 318 64 AO

07205 29 58 25 5 BB B

09496 197 396 199 40 BO

05915 85 1708 75 15 AB AB

09613 336 676 338 68 OO O

100 201 100 201 Total

The statistical analysis ( using chi square test ) indicated that molecular data

were in good agreement with the ratio calculated from the estimated gene frequencies of

the ABO blood group system in the Gaza Strip population

11

44 PCR Results

441 Quality of the isolated DNA

Regarding the method of DNA isolation that was described in chapter 3 the

quality and quantity of DNA were suitable for PCR processing The quality of the

isolated DNA from human subjects is represented in the following figure (42)

Figure 42 A representative photograph of DNA isolated from human blood The samples were

run on 1 agarose gel stained with ethidium bromide

442 Blood group genotyping by Allele Specific PCR

Multiplex Allele Specific PCR (ASPCR) assay contains four independent PCR

reactions (Table 31) In the first reaction 261G-int6 primer pair was used to amplify

the 205 bp fragment to detect the A101 A102 B101 cis-ABO1 alleles and the 467C-

803G primer pair was selected to amplify the 381 bp fragment to detect the A101 O01

and O02 alleles The 297A-int6 primer pair and the 467C-803C primer pair in the

second reaction were selected to amplify the 381 bp fragment to detect the B101 allele

In the third reaction the 261A-int6 primer pair was selected to amplify the 205 bp

product to detect the O01 and O02 alleles and the 467T-803G primer pair was selected

to amplify the 381 bp product to detect the A102 allele Finally in the fourth reaction

the 297G-int6 primer pair were applied to produce the 164 bp fragment to detect the

B101 and O02 alleles and the 467T-803C primer pair to produce the 381 bp fragment

to recognize the cis-ABO1 allele Table 43 show the products of PCR reactions

according to the genotype

14

Table 43 PCR products according to the genotype

PCR Reactions Genotype

R1 R2 R3 R4

381 164 205 _ O1O1

381 _ 205 164 O2O2

381 164 205 164 O1O2

205 381 _ 164 B1B1

381205 381164 205 164 B1O1

381205 381 205 164 B1O2

381205 381164 _ 164 A1B1

205 381164 381 164 A2B1

381205 164 _ _ A1A1

381205 164 205 _ A1O1

381205 164 205 164 A1O2

381205 164 381 _ A1A2

205 164 381 _ A2A2

381205 164 381205 _ A2O1

381205 164 381205 164 A2O2

Random mating with the six different alleles at the ABO locus can result in 21

different genotype combinations Only 15 different genotype combinations were

detected from the 201 investigated samples (Figure 43)

A- 1 O1O1 2 O2O2

15

B- 1 B1B1 2 O1O2

C- 1 B1O1

2 A1B

D- 1 A1A2 2 A2O2

E- 1 A1O1 2 A1A1

16

F- 1 A1O2 2 B1O2 3 A2A2

G- 1 A2O1

2 A2B1

Figure 43 The electrophoresis pattern of recognized genotypes using the multiplex ASPCR

method This figure shows 15 different genotypes lane M indicates the 50 bp ladder The R1-

R4 show the PCR reaction number

443 Genotype Frequencies

The frequency of ABO recognized genotypes belonging to the population

residing in Gaza Strip are shown in table 44 As shown in the table the highest allele

frequencies in A B AB and O phenotypes were A1O2 (124) B1O1 (129) A1B

(55) and O1O1 (154) respectively

17

Table 44 The frequency of recognized genotypes using multiplex ASPCR method for a

population residing in Gaza Strip

Genotype Frequency Percent

A1A1 3 15

A1O1 24 119

A1O2 25 124

A1A2 4 20

A2A2 2 10

A2O1 13 65

A2O2 2 10

B1B1 5 25

B1O1 26 129

B1O2 14 70

A1B 11 55

A2B 4 20

O1O1 31 154

O1O2 26 129

O2O2 11 55

Total 201 1000

18

CHAPTER 5

DISCUSSION

19

CHAPTER 5

DISCUSSION

Since the first delineation of the molecular basis of the ABO blood group by

Yamamoto et al (11 79) it has become possible to determine the ABO genotypes using

molecular methods without the need for family investigations ABO genotyping is

commonly used in cases of an ABO discrepancy between cell typing and serum typing

as well as in forensic practices for personal identification and paternity testing (72) The

ABH antigens are ubiquitously expressed in humans and are present primarily as

glycoproteins and partly as glycolipids The ABO locus is found on the long arm

of chromosome 9 (9q341-q342)

Multiplex allele specific PCR (the technique we used here) has advantages over

PCR-RFLP in terms of cost effectiveness reaction time and simplicity of handling It is

a rapid method that is based on detecting four single nucleotide polymorphisms at

nucleotides 261 297 796 and 803 of the ABO locus which in turn discriminate the

major ABO alleles

In this study the ABO genotypes of 201 samples recruited from the Islamic

University ndashGenetics Diagnosis Laboratory were determined using multiplex AS-PCR

method The objectives of this study were to correlate ABO genotypes with phenotypes

in blood samples in a Gaza Strip population and to compare the frequency of ABO

genotypes with other populations

51 The Allele Frequencies of ABO Antigens in Gaza Strip

The frequency of the alleles in the total samples were 0250 for the I

A allele

0170 for the IB allele and 0580 for the I

O allele This distribution is in agreement with

the distribution with the samples reported in an Iranian study were they reported

023974 for IA allele 018147 for I

B allele and 057879 for I

O allele (73) The

frequencies of IA I

B and I

O alleles in our subjects are also comparable to those

reported in Iraqi and Jordanian studies where they found 0212 for IA allele 0177 for

IB allele and 06611 for I

O allele and 0270 for I

A allele 0130 for I

B allele and 060 for

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

23

with two restriction enzymes (KpnI and AluI) The electrophoresis patterns of 105

samples showed that ABO genotypes were AA 6 (5714) AO 35 (33333) BB 1

(0953) BO 14 (13333) AB 10 (9524) and OO 39 (37143) (70)

EL-Zawahri and Luqmani (2008) examined the genotype of a 355 unrelated

blood donors of phenotype A1 (46) A2 (31) A1B (6) A2B (4) B (97) and O (171) by

using a multiplex PCR-RFLP technique in a Kuwaiti Arab cohort DNA fragments of

252 (251 for O1) and 843 (842 for A2) bp spanning the two major exons 6 and 7 of the

ABO gene were amplified and digested with HpaII and KpnI They identified 13

different genotypes combining the A1 A2 B O1 and O2 alleles from the digestion

patterns 1 A1A1 (028) 6 A1A2 (169) 38 A1O1 (1071) 1 A1O2 (028) 1 A2A2

(028) 30 A2O1 (845) 6 A1B (169) 4 A2B (113) 12 BB (338) 79 BO1

(2225) 6 BO2 (169) 167 O1O1 (4704) and 4 O1O2 (113) Two of the

combinations (A2O2 O2O2) were not found (71)

Sung et al (2009) evaluated ABO genotypes via multiplex allele-specific PCR

(ASPCR) amplification using whole blood samples without DNA purification of 127

randomly chosen samples The genotypes of the 127 samples were found to be A1A2

(n=1) A2A2 (n=9) A1O1 (n=3) A2O1 (n=12) A2O2 (n=14) B1B1 (n=5) B1O1 (n=18)

B1O2 (n=15) O1O1 (n=14) O2O2 (n=8) O1O2 (n=14) and A2B1 (n=14) from which

phenotypes were calculated to be A (n=39) B (n=38) O (n=36) and AB (n=14) They

found no discrepancies when the multiplex AS-PCR assay results were compared with

the serologically determined blood group phenotypes and genotypes determined by

DNA sequencing (72)

Nojavan et al (2012) examined the genotype of 744 randomly selected samples

from Azari donors of East Azerbaijan province (Iran) using multiplex allele-specific

PCR ABO genotyping technique As a result the ABO blood group genotypes were

12 A1A104 A1A2 4A1B1 24 A1O1 141 A1O2 32 A2A2 6A2B152

A2O1 69 A2O2 16 B1B1 113 B1O1 105 B1O2 93 O1O1 153 O1O2

85 O2O2 (73)

22

Bugert et al (2012) determined the major ABO alleles by PCR amplification

with sequence-specific primers (PCR-SSP) in a representative sample of 1335 blood

donors in Germany The genotypes were compared to the ABO blood groups registered

in the blood donor files Then the ABO phenotypes and genotypes were determined in

95 paternity trio cases that have been investigated They found that the prevalence of

the major ABO alleles and genotypes corresponded to the expected occurrence of ABO

blood groups in a Caucasian population In 12 of 35 exclusion cases (343) the ABO

genotype also excluded the alleged father whereas the ABO phenotype excluded the

alleged father only in 7 cases (20) (74)

27 ABO Genotyping and Susceptibility to Diseases

The presence of the A and B blood group antigens expressed on red blood cells

and other cells and molecules within the body has been associated with susceptibility to

diseases like cancer leukemia cardiovascular disease and risk of both arterial and

venous thrombosis Most studies indicated an increased risk of thrombosis associated

with the non-O blood group (7576) Nonndashgroup O patients have a greater risk of

venous thromboembolism (VTE) than patients of group O and have greater levels of

von Willebrand factor (vWF) and factor VIII (7577) The risk of VTE is probably

related to the level of vWF and factor VIII because patients of group A2 have lower

levels of these proteins than A1 B and AB and have a lower risk of VTE (76) A B

and H blood group antigens are expressed on N-glycans of vWF and influence the half-

life of the protein providing an explanation for the greater levels in non-O patients

which increase clot formation in non-O patients (78)

21

CHAPTER 3

MATERIALS amp METHODS

24

CHAPTER 3

MATERIALS amp METHODS

31 Materials

311 PCR primers

The nucleotide sequence of the PCR primers used in the current study was as

described by Yamamoto et al (79) The nucleotide substitutions in the primers are

focused on the nucleotide positions 261 297 467 and 803 so as to discriminate

between the A101 A102 B101 O01 O02 and cis-ABO1 alleles Table 31 shows the

oligonucleotide primer sequences their combinations amplification product lengths

and allele specificities The 3prime base of each primer (except int6) was designed to

correspond to the nucleotides at positions 261 297 467 and 803 which define the

polymorphisms

Table 31 PCR primers sequence used for ABO genotype

PCR

reaction

Primer pair Fragment

size (bp)

Allele specificity

1 261G 5prime-GCAGTAGGAAGGATGTCCTCGTGTTG-3prime 205 A101 A102 B101 cis-ABO1

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467C 5prime-CCACTACTATGTCTTCACCGACCATCC-3prime 381 A101 O01 O02

803G 5prime-CACCGACCCCCCGAAGATCC-3prime

2 297A 5prime-CCATTGTCTGGGAGGGCCCA-3prime 164 A101 A102 O01 cis-ABO1

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467C 5prime-CCACTACTATGTCTTCACCGACCATCC-3prime 381 B101

803C 5prime-CACCGACCCCCCGAAGATCG-3prime

3 261A 5prime-GCAGTAGGAAGGATGTCCTCGTGTTA-3prime 205 O01 O02

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467T 5prime-CCACTACTATGTCTTCACCGACCATCT-3prime 381 A102

803G 5prime-CACCGACCCCCCGAAGATCC-3prime

4 297G 5prime-CCATTGTCTGGGAGGGCCCG-3prime 164 B101 O02

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467T 5prime-CCACTACTATGTCTTCACCGACCATCT-3prime 381 cis-ABO1

803C 5prime-CACCGACCCCCCGAAGATCG-3prime

The primers are named based on the position of their 3prime end relative to the cDNA sequence of A101 allele

25

312 Kits

DNA extraction kit (Promega USA)

313 Reagents and Chemicals

Anti A (Plasmatec Monoclonal UK)

Anti B (Plasmatec Monoclonal UK)

Agarose Molecular biology grade (Promega USA)

DNA molecular weight marker 50 bp-ladder (Promega USA)

EDTA disodium salt (Promega USA)

Absolute Ethanol (Sigma USA)

Ethidium bromide (Promega USA)

Absolute Isopropanol (Sigma USA)

Tris base hydroxyl methyl amino methane (Promega USA)

DNAse RNAse free water (Promega USA)

Acetic acid (Sigma USA)

PCR Master mix (Promega USA)

314 Apparatus and Equipments

Thermal Cycler (Biometra Germany)

LG Microwave Oven

Electrophoresis Apparatus

Vortex Mixer

Digital Camera

Power Supply (Biorad)

Freezer Refrigerator

Micro-Centrifuge

Hoefer shortwave UV light table (Transilluminator)

Computer

Electrical Balance

Automatic Micropipettes

26

32 Methods

321 Study population

322 Sample collection

Blood samples were collected from 201 subjects recruited from the Islamic

University ndashGenetics Laboratory Each sample was collected into EDTA tube The

EDTA samples were kept at 4degC and were used within 24 hours for forward blood

grouping and DNA extraction and subsequent PCR analysis

323 Ethical Considerations

An authorization to carry out the study was obtained from a local ethics

committee using an agreement letter prepared by the Islamic University of Gaza All the

information that were obtained about the subjects were kept confidential

324 Data Analysis

The data were entered stored and analyzed by personal computer using the

Statistical Package for Social Sciences (SPSS) version 160 Allele frequencies were

calculated under the assumption of HardyndashWeinberg equilibrium and expressed as

percentages Chi-square test was used to compare observed allelic and genotypic

frequency distributions of the blood group antigens to that expected under the Hardyndash

Weinberg equation P values gt005 were considered statistically significant

The frequency of ABO in the studied population were compared with the

frequency of ABO in some neighbor countries

33 Blood ABO-typing

331 Forward blood group

Whole blood sample (50 microl) was mixed with Anti A and Anti B reagents by

using slide method in the proportions shown in Table 32 below

27

Table 32 Sample Anti A Anti B reaction for forward blood grouping

Sample Anti -A Anti -B

50 microl 50 microl ــــــ

50 microl 50 ــــــ microl

The contents of each slide were rotated then the agglutination (if present) was

read by naked eye after 30 seconds

34 ABO Genotyping

341 DNA Extraction DNA was isolated from fresh EDTA whole blood cells by using Promega kit for

human DNA isolation The kit contains the following components that are enough for

purifying genomic DNA from 200 samples of human blood

Cell lysis solution

Nuclei lysis solution

Protein precipitation solution

DNA rehydration solution

RNase solution

The human genomic DNA was isolated from human blood sample according to

the kit instructions and was as follows

Three hundred microl EDTA blood were transferred into a sterile 15 ml micro-

centrifuge tube containing 900 microl of cell lysis solution The tube was inverted 5-6 times

to mix the components The mixture was incubated for 10 minutes at room temperature

(with gentle mixing once during the incubation) to lyse the red blood cells The tube

was then centrifuged at 13000 rpm for 20 seconds at room temperature

Supernatant was removed and discarded as much as possible without disturbing

the visible white pellet Approximately 10-20 microl of residual liquid should be left in tube

The tube was vortexed vigorously until the white blood cells were completely

resuspended Three hundred microl of nuclei lysis solution was then added to the tube

28

containing the resuspended cells and the suspension was mixed by pipetting the solution

5-6 times to lyse the white blood cells the solution should become very viscous

RNase solution (15 microl) was added to nuclear lysate and the sample was mixed

by inverting the tube 2-5 times The mixture was incubated at 37degC for 15 minutes and

then cooled to room temperature One hundred microl of protein precipitation solution was

added to the nuclear lysate and the mixture was vortexed vigorously for 10ndash20

seconds Small protein clumps may be visible after vortexing and were removed by

centrifuge precipitation at 13000 rpm for 3 minutes at room temperature The

supernatant was transferred to a clean 15 ml micro-centrifuge tube containing 300microl

isopropanol at room temperature The mixture was gently mixed by inversion until the

white thread-like strands of DNA form a visible mass The DNA was then precipitated

by centrifugation at 13000 rpm for 1 minute at room temperature The DNA would be

visible as a small white pellet

The supernatant decanted and one volume of 70 ethanol was added to the

DNA and kept at room temperature and gently inverted several times to wash the DNA

pellet and the sides of the micro-centrifuge tube After centrifugation at 13000 rpm for

1 minute the ethanol was aspirated using a suitable pipette The DNA pellet is very

loose at this point and care must be used to avoid aspirating the pellet into the pipette

The tube was inverted on a clean absorbent paper for 10ndash15 minutes in order to air-dry

the pellet DNA rehydration solution was added to the dry pellet and the DNA was

rehydrated by incubation at 65degC for 1 hour Periodically the solution was mixed by

gently tapping the tube The DNA was then stored at 2-8degC

342 Detection of extracted DNA

The quality of the isolated DNA was determined by running 5 microl of each sample

on ethidium bromide stained 30 agarose gels and the DNA was visualized on a short

wave UV transilluminator the results were documented by photography

35 PCR reactions

PCR was performed using the primers listed in Table 31 in 4 micro-tubes as

described by Sung Ho Lee et al (70) For each PCR 5 μl master mix (Promega) 2 μl

deionized water 1 μl DNA template and 05 μl of each allele specific primer (5 pmol) in

29

one micro-tube (02 ml) were mixed The volume and concentration of a typical PCR

reaction are shown in Table 32

PCR was performed in a thermal cycler The cycling conditions were as

described below In each PCR MTHFR gene specific primers with the following

sequences (Forward 5prime-ACGATGGGGCAAGTGATGCCC -3 and reverse 5-

GAGAAGGTGTCTGCGGGATC-3) were used as a positive control that produces a 95

bp fragment Upon completion of PCR the products were analyzed by electrophoresis

on 2 ethidium bromide stained agarose gel or stored at 4degC until analysis

Table 33 Composition of PCR master mix

Reagent Composition

dNTPs 400microM each dATP dGTP dCTP dTTP

Taq DNA Polymerase 50 units ml

MgCl2 2mM

351 Temperature cycling program

The thermal cycler program was set as follows

Step 1 Denaturation for 3 minutes at 95degC

Step 2 35 cycles of

21 Melting for 40 seconds at 95degC

22 Annealing for 40 seconds at 585degC

23 Extension for 40 seconds at 72degC

Step3 Final elongation for 5 minutes at72degC

352 Expected PCR results

The PCR product size was estimated by comparing it with DNA molecular size

marker (50 bp ladder DNA ) run on the same gel

11

CHAPTER 4

RESULTS

13

CHAPTER 4

RESULTS

41 Study Population

The study population consisted of 201 subjects (50 males 151 females ) The

percentage of males was 249 while that of females was 751 ( figure 41)

0

10

20

30

40

50

60

70

80

maleFemale

Figure 41 Distribution of the study subjects according to gender

42 Phenotypic Frequency of ABO Blood Groups

Blood grouping was done by antigen antibody agglutination test by using

commercial monoclonal antisera The distribution of phenotypes in the total sample

were 363 (73) 224 (45) 75 (15) 338 (68) for groups A B AB and O

respectively Group A was the dominant one in both genders and AB was the rarest in

both males and females ( table 41)

Table 41 Phenotypic frequencies of various blood groups in the study population

Subject Sex Phenotype

Total A B AB O

Male 16 10 4 20 50

Female 57 35 11 48 151

Total 73 45 15 68 201

753

249

12

43 The Allele Frequencies of ABO Antigens

Allele frequency for the antigens was computed by the Hardy-Weinberg law on

the basis of the number of subjects with blood groups ABO The distribution of the

alleles in the total samples was 025 017 058 for IA I

B and I

O respectively

All genotyping results were compatible with the determined phenotypes by

serological method The observed genotypes as compared with the expected genotypes

are shown in Table 42 The frequencies of the five alleles in the our sample population

were O1 and O2 alleles 0376 and 0221 respectively while A1 0174 A2 0067

B 0162

Table 42 Observed and expected genotypes for the 201 samples

P-Value Expected Observed

Genotype Phenotype Percent Number Percent Number

0230 63 126 45 9 AA A

04762 29 583 318 64 AO

07205 29 58 25 5 BB B

09496 197 396 199 40 BO

05915 85 1708 75 15 AB AB

09613 336 676 338 68 OO O

100 201 100 201 Total

The statistical analysis ( using chi square test ) indicated that molecular data

were in good agreement with the ratio calculated from the estimated gene frequencies of

the ABO blood group system in the Gaza Strip population

11

44 PCR Results

441 Quality of the isolated DNA

Regarding the method of DNA isolation that was described in chapter 3 the

quality and quantity of DNA were suitable for PCR processing The quality of the

isolated DNA from human subjects is represented in the following figure (42)

Figure 42 A representative photograph of DNA isolated from human blood The samples were

run on 1 agarose gel stained with ethidium bromide

442 Blood group genotyping by Allele Specific PCR

Multiplex Allele Specific PCR (ASPCR) assay contains four independent PCR

reactions (Table 31) In the first reaction 261G-int6 primer pair was used to amplify

the 205 bp fragment to detect the A101 A102 B101 cis-ABO1 alleles and the 467C-

803G primer pair was selected to amplify the 381 bp fragment to detect the A101 O01

and O02 alleles The 297A-int6 primer pair and the 467C-803C primer pair in the

second reaction were selected to amplify the 381 bp fragment to detect the B101 allele

In the third reaction the 261A-int6 primer pair was selected to amplify the 205 bp

product to detect the O01 and O02 alleles and the 467T-803G primer pair was selected

to amplify the 381 bp product to detect the A102 allele Finally in the fourth reaction

the 297G-int6 primer pair were applied to produce the 164 bp fragment to detect the

B101 and O02 alleles and the 467T-803C primer pair to produce the 381 bp fragment

to recognize the cis-ABO1 allele Table 43 show the products of PCR reactions

according to the genotype

14

Table 43 PCR products according to the genotype

PCR Reactions Genotype

R1 R2 R3 R4

381 164 205 _ O1O1

381 _ 205 164 O2O2

381 164 205 164 O1O2

205 381 _ 164 B1B1

381205 381164 205 164 B1O1

381205 381 205 164 B1O2

381205 381164 _ 164 A1B1

205 381164 381 164 A2B1

381205 164 _ _ A1A1

381205 164 205 _ A1O1

381205 164 205 164 A1O2

381205 164 381 _ A1A2

205 164 381 _ A2A2

381205 164 381205 _ A2O1

381205 164 381205 164 A2O2

Random mating with the six different alleles at the ABO locus can result in 21

different genotype combinations Only 15 different genotype combinations were

detected from the 201 investigated samples (Figure 43)

A- 1 O1O1 2 O2O2

15

B- 1 B1B1 2 O1O2

C- 1 B1O1

2 A1B

D- 1 A1A2 2 A2O2

E- 1 A1O1 2 A1A1

16

F- 1 A1O2 2 B1O2 3 A2A2

G- 1 A2O1

2 A2B1

Figure 43 The electrophoresis pattern of recognized genotypes using the multiplex ASPCR

method This figure shows 15 different genotypes lane M indicates the 50 bp ladder The R1-

R4 show the PCR reaction number

443 Genotype Frequencies

The frequency of ABO recognized genotypes belonging to the population

residing in Gaza Strip are shown in table 44 As shown in the table the highest allele

frequencies in A B AB and O phenotypes were A1O2 (124) B1O1 (129) A1B

(55) and O1O1 (154) respectively

17

Table 44 The frequency of recognized genotypes using multiplex ASPCR method for a

population residing in Gaza Strip

Genotype Frequency Percent

A1A1 3 15

A1O1 24 119

A1O2 25 124

A1A2 4 20

A2A2 2 10

A2O1 13 65

A2O2 2 10

B1B1 5 25

B1O1 26 129

B1O2 14 70

A1B 11 55

A2B 4 20

O1O1 31 154

O1O2 26 129

O2O2 11 55

Total 201 1000

18

CHAPTER 5

DISCUSSION

19

CHAPTER 5

DISCUSSION

Since the first delineation of the molecular basis of the ABO blood group by

Yamamoto et al (11 79) it has become possible to determine the ABO genotypes using

molecular methods without the need for family investigations ABO genotyping is

commonly used in cases of an ABO discrepancy between cell typing and serum typing

as well as in forensic practices for personal identification and paternity testing (72) The

ABH antigens are ubiquitously expressed in humans and are present primarily as

glycoproteins and partly as glycolipids The ABO locus is found on the long arm

of chromosome 9 (9q341-q342)

Multiplex allele specific PCR (the technique we used here) has advantages over

PCR-RFLP in terms of cost effectiveness reaction time and simplicity of handling It is

a rapid method that is based on detecting four single nucleotide polymorphisms at

nucleotides 261 297 796 and 803 of the ABO locus which in turn discriminate the

major ABO alleles

In this study the ABO genotypes of 201 samples recruited from the Islamic

University ndashGenetics Diagnosis Laboratory were determined using multiplex AS-PCR

method The objectives of this study were to correlate ABO genotypes with phenotypes

in blood samples in a Gaza Strip population and to compare the frequency of ABO

genotypes with other populations

51 The Allele Frequencies of ABO Antigens in Gaza Strip

The frequency of the alleles in the total samples were 0250 for the I

A allele

0170 for the IB allele and 0580 for the I

O allele This distribution is in agreement with

the distribution with the samples reported in an Iranian study were they reported

023974 for IA allele 018147 for I

B allele and 057879 for I

O allele (73) The

frequencies of IA I

B and I

O alleles in our subjects are also comparable to those

reported in Iraqi and Jordanian studies where they found 0212 for IA allele 0177 for

IB allele and 06611 for I

O allele and 0270 for I

A allele 0130 for I

B allele and 060 for

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

22

Bugert et al (2012) determined the major ABO alleles by PCR amplification

with sequence-specific primers (PCR-SSP) in a representative sample of 1335 blood

donors in Germany The genotypes were compared to the ABO blood groups registered

in the blood donor files Then the ABO phenotypes and genotypes were determined in

95 paternity trio cases that have been investigated They found that the prevalence of

the major ABO alleles and genotypes corresponded to the expected occurrence of ABO

blood groups in a Caucasian population In 12 of 35 exclusion cases (343) the ABO

genotype also excluded the alleged father whereas the ABO phenotype excluded the

alleged father only in 7 cases (20) (74)

27 ABO Genotyping and Susceptibility to Diseases

The presence of the A and B blood group antigens expressed on red blood cells

and other cells and molecules within the body has been associated with susceptibility to

diseases like cancer leukemia cardiovascular disease and risk of both arterial and

venous thrombosis Most studies indicated an increased risk of thrombosis associated

with the non-O blood group (7576) Nonndashgroup O patients have a greater risk of

venous thromboembolism (VTE) than patients of group O and have greater levels of

von Willebrand factor (vWF) and factor VIII (7577) The risk of VTE is probably

related to the level of vWF and factor VIII because patients of group A2 have lower

levels of these proteins than A1 B and AB and have a lower risk of VTE (76) A B

and H blood group antigens are expressed on N-glycans of vWF and influence the half-

life of the protein providing an explanation for the greater levels in non-O patients

which increase clot formation in non-O patients (78)

21

CHAPTER 3

MATERIALS amp METHODS

24

CHAPTER 3

MATERIALS amp METHODS

31 Materials

311 PCR primers

The nucleotide sequence of the PCR primers used in the current study was as

described by Yamamoto et al (79) The nucleotide substitutions in the primers are

focused on the nucleotide positions 261 297 467 and 803 so as to discriminate

between the A101 A102 B101 O01 O02 and cis-ABO1 alleles Table 31 shows the

oligonucleotide primer sequences their combinations amplification product lengths

and allele specificities The 3prime base of each primer (except int6) was designed to

correspond to the nucleotides at positions 261 297 467 and 803 which define the

polymorphisms

Table 31 PCR primers sequence used for ABO genotype

PCR

reaction

Primer pair Fragment

size (bp)

Allele specificity

1 261G 5prime-GCAGTAGGAAGGATGTCCTCGTGTTG-3prime 205 A101 A102 B101 cis-ABO1

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467C 5prime-CCACTACTATGTCTTCACCGACCATCC-3prime 381 A101 O01 O02

803G 5prime-CACCGACCCCCCGAAGATCC-3prime

2 297A 5prime-CCATTGTCTGGGAGGGCCCA-3prime 164 A101 A102 O01 cis-ABO1

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467C 5prime-CCACTACTATGTCTTCACCGACCATCC-3prime 381 B101

803C 5prime-CACCGACCCCCCGAAGATCG-3prime

3 261A 5prime-GCAGTAGGAAGGATGTCCTCGTGTTA-3prime 205 O01 O02

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467T 5prime-CCACTACTATGTCTTCACCGACCATCT-3prime 381 A102

803G 5prime-CACCGACCCCCCGAAGATCC-3prime

4 297G 5prime-CCATTGTCTGGGAGGGCCCG-3prime 164 B101 O02

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467T 5prime-CCACTACTATGTCTTCACCGACCATCT-3prime 381 cis-ABO1

803C 5prime-CACCGACCCCCCGAAGATCG-3prime

The primers are named based on the position of their 3prime end relative to the cDNA sequence of A101 allele

25

312 Kits

DNA extraction kit (Promega USA)

313 Reagents and Chemicals

Anti A (Plasmatec Monoclonal UK)

Anti B (Plasmatec Monoclonal UK)

Agarose Molecular biology grade (Promega USA)

DNA molecular weight marker 50 bp-ladder (Promega USA)

EDTA disodium salt (Promega USA)

Absolute Ethanol (Sigma USA)

Ethidium bromide (Promega USA)

Absolute Isopropanol (Sigma USA)

Tris base hydroxyl methyl amino methane (Promega USA)

DNAse RNAse free water (Promega USA)

Acetic acid (Sigma USA)

PCR Master mix (Promega USA)

314 Apparatus and Equipments

Thermal Cycler (Biometra Germany)

LG Microwave Oven

Electrophoresis Apparatus

Vortex Mixer

Digital Camera

Power Supply (Biorad)

Freezer Refrigerator

Micro-Centrifuge

Hoefer shortwave UV light table (Transilluminator)

Computer

Electrical Balance

Automatic Micropipettes

26

32 Methods

321 Study population

322 Sample collection

Blood samples were collected from 201 subjects recruited from the Islamic

University ndashGenetics Laboratory Each sample was collected into EDTA tube The

EDTA samples were kept at 4degC and were used within 24 hours for forward blood

grouping and DNA extraction and subsequent PCR analysis

323 Ethical Considerations

An authorization to carry out the study was obtained from a local ethics

committee using an agreement letter prepared by the Islamic University of Gaza All the

information that were obtained about the subjects were kept confidential

324 Data Analysis

The data were entered stored and analyzed by personal computer using the

Statistical Package for Social Sciences (SPSS) version 160 Allele frequencies were

calculated under the assumption of HardyndashWeinberg equilibrium and expressed as

percentages Chi-square test was used to compare observed allelic and genotypic

frequency distributions of the blood group antigens to that expected under the Hardyndash

Weinberg equation P values gt005 were considered statistically significant

The frequency of ABO in the studied population were compared with the

frequency of ABO in some neighbor countries

33 Blood ABO-typing

331 Forward blood group

Whole blood sample (50 microl) was mixed with Anti A and Anti B reagents by

using slide method in the proportions shown in Table 32 below

27

Table 32 Sample Anti A Anti B reaction for forward blood grouping

Sample Anti -A Anti -B

50 microl 50 microl ــــــ

50 microl 50 ــــــ microl

The contents of each slide were rotated then the agglutination (if present) was

read by naked eye after 30 seconds

34 ABO Genotyping

341 DNA Extraction DNA was isolated from fresh EDTA whole blood cells by using Promega kit for

human DNA isolation The kit contains the following components that are enough for

purifying genomic DNA from 200 samples of human blood

Cell lysis solution

Nuclei lysis solution

Protein precipitation solution

DNA rehydration solution

RNase solution

The human genomic DNA was isolated from human blood sample according to

the kit instructions and was as follows

Three hundred microl EDTA blood were transferred into a sterile 15 ml micro-

centrifuge tube containing 900 microl of cell lysis solution The tube was inverted 5-6 times

to mix the components The mixture was incubated for 10 minutes at room temperature

(with gentle mixing once during the incubation) to lyse the red blood cells The tube

was then centrifuged at 13000 rpm for 20 seconds at room temperature

Supernatant was removed and discarded as much as possible without disturbing

the visible white pellet Approximately 10-20 microl of residual liquid should be left in tube

The tube was vortexed vigorously until the white blood cells were completely

resuspended Three hundred microl of nuclei lysis solution was then added to the tube

28

containing the resuspended cells and the suspension was mixed by pipetting the solution

5-6 times to lyse the white blood cells the solution should become very viscous

RNase solution (15 microl) was added to nuclear lysate and the sample was mixed

by inverting the tube 2-5 times The mixture was incubated at 37degC for 15 minutes and

then cooled to room temperature One hundred microl of protein precipitation solution was

added to the nuclear lysate and the mixture was vortexed vigorously for 10ndash20

seconds Small protein clumps may be visible after vortexing and were removed by

centrifuge precipitation at 13000 rpm for 3 minutes at room temperature The

supernatant was transferred to a clean 15 ml micro-centrifuge tube containing 300microl

isopropanol at room temperature The mixture was gently mixed by inversion until the

white thread-like strands of DNA form a visible mass The DNA was then precipitated

by centrifugation at 13000 rpm for 1 minute at room temperature The DNA would be

visible as a small white pellet

The supernatant decanted and one volume of 70 ethanol was added to the

DNA and kept at room temperature and gently inverted several times to wash the DNA

pellet and the sides of the micro-centrifuge tube After centrifugation at 13000 rpm for

1 minute the ethanol was aspirated using a suitable pipette The DNA pellet is very

loose at this point and care must be used to avoid aspirating the pellet into the pipette

The tube was inverted on a clean absorbent paper for 10ndash15 minutes in order to air-dry

the pellet DNA rehydration solution was added to the dry pellet and the DNA was

rehydrated by incubation at 65degC for 1 hour Periodically the solution was mixed by

gently tapping the tube The DNA was then stored at 2-8degC

342 Detection of extracted DNA

The quality of the isolated DNA was determined by running 5 microl of each sample

on ethidium bromide stained 30 agarose gels and the DNA was visualized on a short

wave UV transilluminator the results were documented by photography

35 PCR reactions

PCR was performed using the primers listed in Table 31 in 4 micro-tubes as

described by Sung Ho Lee et al (70) For each PCR 5 μl master mix (Promega) 2 μl

deionized water 1 μl DNA template and 05 μl of each allele specific primer (5 pmol) in

29

one micro-tube (02 ml) were mixed The volume and concentration of a typical PCR

reaction are shown in Table 32

PCR was performed in a thermal cycler The cycling conditions were as

described below In each PCR MTHFR gene specific primers with the following

sequences (Forward 5prime-ACGATGGGGCAAGTGATGCCC -3 and reverse 5-

GAGAAGGTGTCTGCGGGATC-3) were used as a positive control that produces a 95

bp fragment Upon completion of PCR the products were analyzed by electrophoresis

on 2 ethidium bromide stained agarose gel or stored at 4degC until analysis

Table 33 Composition of PCR master mix

Reagent Composition

dNTPs 400microM each dATP dGTP dCTP dTTP

Taq DNA Polymerase 50 units ml

MgCl2 2mM

351 Temperature cycling program

The thermal cycler program was set as follows

Step 1 Denaturation for 3 minutes at 95degC

Step 2 35 cycles of

21 Melting for 40 seconds at 95degC

22 Annealing for 40 seconds at 585degC

23 Extension for 40 seconds at 72degC

Step3 Final elongation for 5 minutes at72degC

352 Expected PCR results

The PCR product size was estimated by comparing it with DNA molecular size

marker (50 bp ladder DNA ) run on the same gel

11

CHAPTER 4

RESULTS

13

CHAPTER 4

RESULTS

41 Study Population

The study population consisted of 201 subjects (50 males 151 females ) The

percentage of males was 249 while that of females was 751 ( figure 41)

0

10

20

30

40

50

60

70

80

maleFemale

Figure 41 Distribution of the study subjects according to gender

42 Phenotypic Frequency of ABO Blood Groups

Blood grouping was done by antigen antibody agglutination test by using

commercial monoclonal antisera The distribution of phenotypes in the total sample

were 363 (73) 224 (45) 75 (15) 338 (68) for groups A B AB and O

respectively Group A was the dominant one in both genders and AB was the rarest in

both males and females ( table 41)

Table 41 Phenotypic frequencies of various blood groups in the study population

Subject Sex Phenotype

Total A B AB O

Male 16 10 4 20 50

Female 57 35 11 48 151

Total 73 45 15 68 201

753

249

12

43 The Allele Frequencies of ABO Antigens

Allele frequency for the antigens was computed by the Hardy-Weinberg law on

the basis of the number of subjects with blood groups ABO The distribution of the

alleles in the total samples was 025 017 058 for IA I

B and I

O respectively

All genotyping results were compatible with the determined phenotypes by

serological method The observed genotypes as compared with the expected genotypes

are shown in Table 42 The frequencies of the five alleles in the our sample population

were O1 and O2 alleles 0376 and 0221 respectively while A1 0174 A2 0067

B 0162

Table 42 Observed and expected genotypes for the 201 samples

P-Value Expected Observed

Genotype Phenotype Percent Number Percent Number

0230 63 126 45 9 AA A

04762 29 583 318 64 AO

07205 29 58 25 5 BB B

09496 197 396 199 40 BO

05915 85 1708 75 15 AB AB

09613 336 676 338 68 OO O

100 201 100 201 Total

The statistical analysis ( using chi square test ) indicated that molecular data

were in good agreement with the ratio calculated from the estimated gene frequencies of

the ABO blood group system in the Gaza Strip population

11

44 PCR Results

441 Quality of the isolated DNA

Regarding the method of DNA isolation that was described in chapter 3 the

quality and quantity of DNA were suitable for PCR processing The quality of the

isolated DNA from human subjects is represented in the following figure (42)

Figure 42 A representative photograph of DNA isolated from human blood The samples were

run on 1 agarose gel stained with ethidium bromide

442 Blood group genotyping by Allele Specific PCR

Multiplex Allele Specific PCR (ASPCR) assay contains four independent PCR

reactions (Table 31) In the first reaction 261G-int6 primer pair was used to amplify

the 205 bp fragment to detect the A101 A102 B101 cis-ABO1 alleles and the 467C-

803G primer pair was selected to amplify the 381 bp fragment to detect the A101 O01

and O02 alleles The 297A-int6 primer pair and the 467C-803C primer pair in the

second reaction were selected to amplify the 381 bp fragment to detect the B101 allele

In the third reaction the 261A-int6 primer pair was selected to amplify the 205 bp

product to detect the O01 and O02 alleles and the 467T-803G primer pair was selected

to amplify the 381 bp product to detect the A102 allele Finally in the fourth reaction

the 297G-int6 primer pair were applied to produce the 164 bp fragment to detect the

B101 and O02 alleles and the 467T-803C primer pair to produce the 381 bp fragment

to recognize the cis-ABO1 allele Table 43 show the products of PCR reactions

according to the genotype

14

Table 43 PCR products according to the genotype

PCR Reactions Genotype

R1 R2 R3 R4

381 164 205 _ O1O1

381 _ 205 164 O2O2

381 164 205 164 O1O2

205 381 _ 164 B1B1

381205 381164 205 164 B1O1

381205 381 205 164 B1O2

381205 381164 _ 164 A1B1

205 381164 381 164 A2B1

381205 164 _ _ A1A1

381205 164 205 _ A1O1

381205 164 205 164 A1O2

381205 164 381 _ A1A2

205 164 381 _ A2A2

381205 164 381205 _ A2O1

381205 164 381205 164 A2O2

Random mating with the six different alleles at the ABO locus can result in 21

different genotype combinations Only 15 different genotype combinations were

detected from the 201 investigated samples (Figure 43)

A- 1 O1O1 2 O2O2

15

B- 1 B1B1 2 O1O2

C- 1 B1O1

2 A1B

D- 1 A1A2 2 A2O2

E- 1 A1O1 2 A1A1

16

F- 1 A1O2 2 B1O2 3 A2A2

G- 1 A2O1

2 A2B1

Figure 43 The electrophoresis pattern of recognized genotypes using the multiplex ASPCR

method This figure shows 15 different genotypes lane M indicates the 50 bp ladder The R1-

R4 show the PCR reaction number

443 Genotype Frequencies

The frequency of ABO recognized genotypes belonging to the population

residing in Gaza Strip are shown in table 44 As shown in the table the highest allele

frequencies in A B AB and O phenotypes were A1O2 (124) B1O1 (129) A1B

(55) and O1O1 (154) respectively

17

Table 44 The frequency of recognized genotypes using multiplex ASPCR method for a

population residing in Gaza Strip

Genotype Frequency Percent

A1A1 3 15

A1O1 24 119

A1O2 25 124

A1A2 4 20

A2A2 2 10

A2O1 13 65

A2O2 2 10

B1B1 5 25

B1O1 26 129

B1O2 14 70

A1B 11 55

A2B 4 20

O1O1 31 154

O1O2 26 129

O2O2 11 55

Total 201 1000

18

CHAPTER 5

DISCUSSION

19

CHAPTER 5

DISCUSSION

Since the first delineation of the molecular basis of the ABO blood group by

Yamamoto et al (11 79) it has become possible to determine the ABO genotypes using

molecular methods without the need for family investigations ABO genotyping is

commonly used in cases of an ABO discrepancy between cell typing and serum typing

as well as in forensic practices for personal identification and paternity testing (72) The

ABH antigens are ubiquitously expressed in humans and are present primarily as

glycoproteins and partly as glycolipids The ABO locus is found on the long arm

of chromosome 9 (9q341-q342)

Multiplex allele specific PCR (the technique we used here) has advantages over

PCR-RFLP in terms of cost effectiveness reaction time and simplicity of handling It is

a rapid method that is based on detecting four single nucleotide polymorphisms at

nucleotides 261 297 796 and 803 of the ABO locus which in turn discriminate the

major ABO alleles

In this study the ABO genotypes of 201 samples recruited from the Islamic

University ndashGenetics Diagnosis Laboratory were determined using multiplex AS-PCR

method The objectives of this study were to correlate ABO genotypes with phenotypes

in blood samples in a Gaza Strip population and to compare the frequency of ABO

genotypes with other populations

51 The Allele Frequencies of ABO Antigens in Gaza Strip

The frequency of the alleles in the total samples were 0250 for the I

A allele

0170 for the IB allele and 0580 for the I

O allele This distribution is in agreement with

the distribution with the samples reported in an Iranian study were they reported

023974 for IA allele 018147 for I

B allele and 057879 for I

O allele (73) The

frequencies of IA I

B and I

O alleles in our subjects are also comparable to those

reported in Iraqi and Jordanian studies where they found 0212 for IA allele 0177 for

IB allele and 06611 for I

O allele and 0270 for I

A allele 0130 for I

B allele and 060 for

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

21

CHAPTER 3

MATERIALS amp METHODS

24

CHAPTER 3

MATERIALS amp METHODS

31 Materials

311 PCR primers

The nucleotide sequence of the PCR primers used in the current study was as

described by Yamamoto et al (79) The nucleotide substitutions in the primers are

focused on the nucleotide positions 261 297 467 and 803 so as to discriminate

between the A101 A102 B101 O01 O02 and cis-ABO1 alleles Table 31 shows the

oligonucleotide primer sequences their combinations amplification product lengths

and allele specificities The 3prime base of each primer (except int6) was designed to

correspond to the nucleotides at positions 261 297 467 and 803 which define the

polymorphisms

Table 31 PCR primers sequence used for ABO genotype

PCR

reaction

Primer pair Fragment

size (bp)

Allele specificity

1 261G 5prime-GCAGTAGGAAGGATGTCCTCGTGTTG-3prime 205 A101 A102 B101 cis-ABO1

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467C 5prime-CCACTACTATGTCTTCACCGACCATCC-3prime 381 A101 O01 O02

803G 5prime-CACCGACCCCCCGAAGATCC-3prime

2 297A 5prime-CCATTGTCTGGGAGGGCCCA-3prime 164 A101 A102 O01 cis-ABO1

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467C 5prime-CCACTACTATGTCTTCACCGACCATCC-3prime 381 B101

803C 5prime-CACCGACCCCCCGAAGATCG-3prime

3 261A 5prime-GCAGTAGGAAGGATGTCCTCGTGTTA-3prime 205 O01 O02

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467T 5prime-CCACTACTATGTCTTCACCGACCATCT-3prime 381 A102

803G 5prime-CACCGACCCCCCGAAGATCC-3prime

4 297G 5prime-CCATTGTCTGGGAGGGCCCG-3prime 164 B101 O02

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467T 5prime-CCACTACTATGTCTTCACCGACCATCT-3prime 381 cis-ABO1

803C 5prime-CACCGACCCCCCGAAGATCG-3prime

The primers are named based on the position of their 3prime end relative to the cDNA sequence of A101 allele

25

312 Kits

DNA extraction kit (Promega USA)

313 Reagents and Chemicals

Anti A (Plasmatec Monoclonal UK)

Anti B (Plasmatec Monoclonal UK)

Agarose Molecular biology grade (Promega USA)

DNA molecular weight marker 50 bp-ladder (Promega USA)

EDTA disodium salt (Promega USA)

Absolute Ethanol (Sigma USA)

Ethidium bromide (Promega USA)

Absolute Isopropanol (Sigma USA)

Tris base hydroxyl methyl amino methane (Promega USA)

DNAse RNAse free water (Promega USA)

Acetic acid (Sigma USA)

PCR Master mix (Promega USA)

314 Apparatus and Equipments

Thermal Cycler (Biometra Germany)

LG Microwave Oven

Electrophoresis Apparatus

Vortex Mixer

Digital Camera

Power Supply (Biorad)

Freezer Refrigerator

Micro-Centrifuge

Hoefer shortwave UV light table (Transilluminator)

Computer

Electrical Balance

Automatic Micropipettes

26

32 Methods

321 Study population

322 Sample collection

Blood samples were collected from 201 subjects recruited from the Islamic

University ndashGenetics Laboratory Each sample was collected into EDTA tube The

EDTA samples were kept at 4degC and were used within 24 hours for forward blood

grouping and DNA extraction and subsequent PCR analysis

323 Ethical Considerations

An authorization to carry out the study was obtained from a local ethics

committee using an agreement letter prepared by the Islamic University of Gaza All the

information that were obtained about the subjects were kept confidential

324 Data Analysis

The data were entered stored and analyzed by personal computer using the

Statistical Package for Social Sciences (SPSS) version 160 Allele frequencies were

calculated under the assumption of HardyndashWeinberg equilibrium and expressed as

percentages Chi-square test was used to compare observed allelic and genotypic

frequency distributions of the blood group antigens to that expected under the Hardyndash

Weinberg equation P values gt005 were considered statistically significant

The frequency of ABO in the studied population were compared with the

frequency of ABO in some neighbor countries

33 Blood ABO-typing

331 Forward blood group

Whole blood sample (50 microl) was mixed with Anti A and Anti B reagents by

using slide method in the proportions shown in Table 32 below

27

Table 32 Sample Anti A Anti B reaction for forward blood grouping

Sample Anti -A Anti -B

50 microl 50 microl ــــــ

50 microl 50 ــــــ microl

The contents of each slide were rotated then the agglutination (if present) was

read by naked eye after 30 seconds

34 ABO Genotyping

341 DNA Extraction DNA was isolated from fresh EDTA whole blood cells by using Promega kit for

human DNA isolation The kit contains the following components that are enough for

purifying genomic DNA from 200 samples of human blood

Cell lysis solution

Nuclei lysis solution

Protein precipitation solution

DNA rehydration solution

RNase solution

The human genomic DNA was isolated from human blood sample according to

the kit instructions and was as follows

Three hundred microl EDTA blood were transferred into a sterile 15 ml micro-

centrifuge tube containing 900 microl of cell lysis solution The tube was inverted 5-6 times

to mix the components The mixture was incubated for 10 minutes at room temperature

(with gentle mixing once during the incubation) to lyse the red blood cells The tube

was then centrifuged at 13000 rpm for 20 seconds at room temperature

Supernatant was removed and discarded as much as possible without disturbing

the visible white pellet Approximately 10-20 microl of residual liquid should be left in tube

The tube was vortexed vigorously until the white blood cells were completely

resuspended Three hundred microl of nuclei lysis solution was then added to the tube

28

containing the resuspended cells and the suspension was mixed by pipetting the solution

5-6 times to lyse the white blood cells the solution should become very viscous

RNase solution (15 microl) was added to nuclear lysate and the sample was mixed

by inverting the tube 2-5 times The mixture was incubated at 37degC for 15 minutes and

then cooled to room temperature One hundred microl of protein precipitation solution was

added to the nuclear lysate and the mixture was vortexed vigorously for 10ndash20

seconds Small protein clumps may be visible after vortexing and were removed by

centrifuge precipitation at 13000 rpm for 3 minutes at room temperature The

supernatant was transferred to a clean 15 ml micro-centrifuge tube containing 300microl

isopropanol at room temperature The mixture was gently mixed by inversion until the

white thread-like strands of DNA form a visible mass The DNA was then precipitated

by centrifugation at 13000 rpm for 1 minute at room temperature The DNA would be

visible as a small white pellet

The supernatant decanted and one volume of 70 ethanol was added to the

DNA and kept at room temperature and gently inverted several times to wash the DNA

pellet and the sides of the micro-centrifuge tube After centrifugation at 13000 rpm for

1 minute the ethanol was aspirated using a suitable pipette The DNA pellet is very

loose at this point and care must be used to avoid aspirating the pellet into the pipette

The tube was inverted on a clean absorbent paper for 10ndash15 minutes in order to air-dry

the pellet DNA rehydration solution was added to the dry pellet and the DNA was

rehydrated by incubation at 65degC for 1 hour Periodically the solution was mixed by

gently tapping the tube The DNA was then stored at 2-8degC

342 Detection of extracted DNA

The quality of the isolated DNA was determined by running 5 microl of each sample

on ethidium bromide stained 30 agarose gels and the DNA was visualized on a short

wave UV transilluminator the results were documented by photography

35 PCR reactions

PCR was performed using the primers listed in Table 31 in 4 micro-tubes as

described by Sung Ho Lee et al (70) For each PCR 5 μl master mix (Promega) 2 μl

deionized water 1 μl DNA template and 05 μl of each allele specific primer (5 pmol) in

29

one micro-tube (02 ml) were mixed The volume and concentration of a typical PCR

reaction are shown in Table 32

PCR was performed in a thermal cycler The cycling conditions were as

described below In each PCR MTHFR gene specific primers with the following

sequences (Forward 5prime-ACGATGGGGCAAGTGATGCCC -3 and reverse 5-

GAGAAGGTGTCTGCGGGATC-3) were used as a positive control that produces a 95

bp fragment Upon completion of PCR the products were analyzed by electrophoresis

on 2 ethidium bromide stained agarose gel or stored at 4degC until analysis

Table 33 Composition of PCR master mix

Reagent Composition

dNTPs 400microM each dATP dGTP dCTP dTTP

Taq DNA Polymerase 50 units ml

MgCl2 2mM

351 Temperature cycling program

The thermal cycler program was set as follows

Step 1 Denaturation for 3 minutes at 95degC

Step 2 35 cycles of

21 Melting for 40 seconds at 95degC

22 Annealing for 40 seconds at 585degC

23 Extension for 40 seconds at 72degC

Step3 Final elongation for 5 minutes at72degC

352 Expected PCR results

The PCR product size was estimated by comparing it with DNA molecular size

marker (50 bp ladder DNA ) run on the same gel

11

CHAPTER 4

RESULTS

13

CHAPTER 4

RESULTS

41 Study Population

The study population consisted of 201 subjects (50 males 151 females ) The

percentage of males was 249 while that of females was 751 ( figure 41)

0

10

20

30

40

50

60

70

80

maleFemale

Figure 41 Distribution of the study subjects according to gender

42 Phenotypic Frequency of ABO Blood Groups

Blood grouping was done by antigen antibody agglutination test by using

commercial monoclonal antisera The distribution of phenotypes in the total sample

were 363 (73) 224 (45) 75 (15) 338 (68) for groups A B AB and O

respectively Group A was the dominant one in both genders and AB was the rarest in

both males and females ( table 41)

Table 41 Phenotypic frequencies of various blood groups in the study population

Subject Sex Phenotype

Total A B AB O

Male 16 10 4 20 50

Female 57 35 11 48 151

Total 73 45 15 68 201

753

249

12

43 The Allele Frequencies of ABO Antigens

Allele frequency for the antigens was computed by the Hardy-Weinberg law on

the basis of the number of subjects with blood groups ABO The distribution of the

alleles in the total samples was 025 017 058 for IA I

B and I

O respectively

All genotyping results were compatible with the determined phenotypes by

serological method The observed genotypes as compared with the expected genotypes

are shown in Table 42 The frequencies of the five alleles in the our sample population

were O1 and O2 alleles 0376 and 0221 respectively while A1 0174 A2 0067

B 0162

Table 42 Observed and expected genotypes for the 201 samples

P-Value Expected Observed

Genotype Phenotype Percent Number Percent Number

0230 63 126 45 9 AA A

04762 29 583 318 64 AO

07205 29 58 25 5 BB B

09496 197 396 199 40 BO

05915 85 1708 75 15 AB AB

09613 336 676 338 68 OO O

100 201 100 201 Total

The statistical analysis ( using chi square test ) indicated that molecular data

were in good agreement with the ratio calculated from the estimated gene frequencies of

the ABO blood group system in the Gaza Strip population

11

44 PCR Results

441 Quality of the isolated DNA

Regarding the method of DNA isolation that was described in chapter 3 the

quality and quantity of DNA were suitable for PCR processing The quality of the

isolated DNA from human subjects is represented in the following figure (42)

Figure 42 A representative photograph of DNA isolated from human blood The samples were

run on 1 agarose gel stained with ethidium bromide

442 Blood group genotyping by Allele Specific PCR

Multiplex Allele Specific PCR (ASPCR) assay contains four independent PCR

reactions (Table 31) In the first reaction 261G-int6 primer pair was used to amplify

the 205 bp fragment to detect the A101 A102 B101 cis-ABO1 alleles and the 467C-

803G primer pair was selected to amplify the 381 bp fragment to detect the A101 O01

and O02 alleles The 297A-int6 primer pair and the 467C-803C primer pair in the

second reaction were selected to amplify the 381 bp fragment to detect the B101 allele

In the third reaction the 261A-int6 primer pair was selected to amplify the 205 bp

product to detect the O01 and O02 alleles and the 467T-803G primer pair was selected

to amplify the 381 bp product to detect the A102 allele Finally in the fourth reaction

the 297G-int6 primer pair were applied to produce the 164 bp fragment to detect the

B101 and O02 alleles and the 467T-803C primer pair to produce the 381 bp fragment

to recognize the cis-ABO1 allele Table 43 show the products of PCR reactions

according to the genotype

14

Table 43 PCR products according to the genotype

PCR Reactions Genotype

R1 R2 R3 R4

381 164 205 _ O1O1

381 _ 205 164 O2O2

381 164 205 164 O1O2

205 381 _ 164 B1B1

381205 381164 205 164 B1O1

381205 381 205 164 B1O2

381205 381164 _ 164 A1B1

205 381164 381 164 A2B1

381205 164 _ _ A1A1

381205 164 205 _ A1O1

381205 164 205 164 A1O2

381205 164 381 _ A1A2

205 164 381 _ A2A2

381205 164 381205 _ A2O1

381205 164 381205 164 A2O2

Random mating with the six different alleles at the ABO locus can result in 21

different genotype combinations Only 15 different genotype combinations were

detected from the 201 investigated samples (Figure 43)

A- 1 O1O1 2 O2O2

15

B- 1 B1B1 2 O1O2

C- 1 B1O1

2 A1B

D- 1 A1A2 2 A2O2

E- 1 A1O1 2 A1A1

16

F- 1 A1O2 2 B1O2 3 A2A2

G- 1 A2O1

2 A2B1

Figure 43 The electrophoresis pattern of recognized genotypes using the multiplex ASPCR

method This figure shows 15 different genotypes lane M indicates the 50 bp ladder The R1-

R4 show the PCR reaction number

443 Genotype Frequencies

The frequency of ABO recognized genotypes belonging to the population

residing in Gaza Strip are shown in table 44 As shown in the table the highest allele

frequencies in A B AB and O phenotypes were A1O2 (124) B1O1 (129) A1B

(55) and O1O1 (154) respectively

17

Table 44 The frequency of recognized genotypes using multiplex ASPCR method for a

population residing in Gaza Strip

Genotype Frequency Percent

A1A1 3 15

A1O1 24 119

A1O2 25 124

A1A2 4 20

A2A2 2 10

A2O1 13 65

A2O2 2 10

B1B1 5 25

B1O1 26 129

B1O2 14 70

A1B 11 55

A2B 4 20

O1O1 31 154

O1O2 26 129

O2O2 11 55

Total 201 1000

18

CHAPTER 5

DISCUSSION

19

CHAPTER 5

DISCUSSION

Since the first delineation of the molecular basis of the ABO blood group by

Yamamoto et al (11 79) it has become possible to determine the ABO genotypes using

molecular methods without the need for family investigations ABO genotyping is

commonly used in cases of an ABO discrepancy between cell typing and serum typing

as well as in forensic practices for personal identification and paternity testing (72) The

ABH antigens are ubiquitously expressed in humans and are present primarily as

glycoproteins and partly as glycolipids The ABO locus is found on the long arm

of chromosome 9 (9q341-q342)

Multiplex allele specific PCR (the technique we used here) has advantages over

PCR-RFLP in terms of cost effectiveness reaction time and simplicity of handling It is

a rapid method that is based on detecting four single nucleotide polymorphisms at

nucleotides 261 297 796 and 803 of the ABO locus which in turn discriminate the

major ABO alleles

In this study the ABO genotypes of 201 samples recruited from the Islamic

University ndashGenetics Diagnosis Laboratory were determined using multiplex AS-PCR

method The objectives of this study were to correlate ABO genotypes with phenotypes

in blood samples in a Gaza Strip population and to compare the frequency of ABO

genotypes with other populations

51 The Allele Frequencies of ABO Antigens in Gaza Strip

The frequency of the alleles in the total samples were 0250 for the I

A allele

0170 for the IB allele and 0580 for the I

O allele This distribution is in agreement with

the distribution with the samples reported in an Iranian study were they reported

023974 for IA allele 018147 for I

B allele and 057879 for I

O allele (73) The

frequencies of IA I

B and I

O alleles in our subjects are also comparable to those

reported in Iraqi and Jordanian studies where they found 0212 for IA allele 0177 for

IB allele and 06611 for I

O allele and 0270 for I

A allele 0130 for I

B allele and 060 for

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

24

CHAPTER 3

MATERIALS amp METHODS

31 Materials

311 PCR primers

The nucleotide sequence of the PCR primers used in the current study was as

described by Yamamoto et al (79) The nucleotide substitutions in the primers are

focused on the nucleotide positions 261 297 467 and 803 so as to discriminate

between the A101 A102 B101 O01 O02 and cis-ABO1 alleles Table 31 shows the

oligonucleotide primer sequences their combinations amplification product lengths

and allele specificities The 3prime base of each primer (except int6) was designed to

correspond to the nucleotides at positions 261 297 467 and 803 which define the

polymorphisms

Table 31 PCR primers sequence used for ABO genotype

PCR

reaction

Primer pair Fragment

size (bp)

Allele specificity

1 261G 5prime-GCAGTAGGAAGGATGTCCTCGTGTTG-3prime 205 A101 A102 B101 cis-ABO1

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467C 5prime-CCACTACTATGTCTTCACCGACCATCC-3prime 381 A101 O01 O02

803G 5prime-CACCGACCCCCCGAAGATCC-3prime

2 297A 5prime-CCATTGTCTGGGAGGGCCCA-3prime 164 A101 A102 O01 cis-ABO1

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467C 5prime-CCACTACTATGTCTTCACCGACCATCC-3prime 381 B101

803C 5prime-CACCGACCCCCCGAAGATCG-3prime

3 261A 5prime-GCAGTAGGAAGGATGTCCTCGTGTTA-3prime 205 O01 O02

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467T 5prime-CCACTACTATGTCTTCACCGACCATCT-3prime 381 A102

803G 5prime-CACCGACCCCCCGAAGATCC-3prime

4 297G 5prime-CCATTGTCTGGGAGGGCCCG-3prime 164 B101 O02

int6 5prime-AGACCTCAATGTCCACAGTCACTCG-3prime

467T 5prime-CCACTACTATGTCTTCACCGACCATCT-3prime 381 cis-ABO1

803C 5prime-CACCGACCCCCCGAAGATCG-3prime

The primers are named based on the position of their 3prime end relative to the cDNA sequence of A101 allele

25

312 Kits

DNA extraction kit (Promega USA)

313 Reagents and Chemicals

Anti A (Plasmatec Monoclonal UK)

Anti B (Plasmatec Monoclonal UK)

Agarose Molecular biology grade (Promega USA)

DNA molecular weight marker 50 bp-ladder (Promega USA)

EDTA disodium salt (Promega USA)

Absolute Ethanol (Sigma USA)

Ethidium bromide (Promega USA)

Absolute Isopropanol (Sigma USA)

Tris base hydroxyl methyl amino methane (Promega USA)

DNAse RNAse free water (Promega USA)

Acetic acid (Sigma USA)

PCR Master mix (Promega USA)

314 Apparatus and Equipments

Thermal Cycler (Biometra Germany)

LG Microwave Oven

Electrophoresis Apparatus

Vortex Mixer

Digital Camera

Power Supply (Biorad)

Freezer Refrigerator

Micro-Centrifuge

Hoefer shortwave UV light table (Transilluminator)

Computer

Electrical Balance

Automatic Micropipettes

26

32 Methods

321 Study population

322 Sample collection

Blood samples were collected from 201 subjects recruited from the Islamic

University ndashGenetics Laboratory Each sample was collected into EDTA tube The

EDTA samples were kept at 4degC and were used within 24 hours for forward blood

grouping and DNA extraction and subsequent PCR analysis

323 Ethical Considerations

An authorization to carry out the study was obtained from a local ethics

committee using an agreement letter prepared by the Islamic University of Gaza All the

information that were obtained about the subjects were kept confidential

324 Data Analysis

The data were entered stored and analyzed by personal computer using the

Statistical Package for Social Sciences (SPSS) version 160 Allele frequencies were

calculated under the assumption of HardyndashWeinberg equilibrium and expressed as

percentages Chi-square test was used to compare observed allelic and genotypic

frequency distributions of the blood group antigens to that expected under the Hardyndash

Weinberg equation P values gt005 were considered statistically significant

The frequency of ABO in the studied population were compared with the

frequency of ABO in some neighbor countries

33 Blood ABO-typing

331 Forward blood group

Whole blood sample (50 microl) was mixed with Anti A and Anti B reagents by

using slide method in the proportions shown in Table 32 below

27

Table 32 Sample Anti A Anti B reaction for forward blood grouping

Sample Anti -A Anti -B

50 microl 50 microl ــــــ

50 microl 50 ــــــ microl

The contents of each slide were rotated then the agglutination (if present) was

read by naked eye after 30 seconds

34 ABO Genotyping

341 DNA Extraction DNA was isolated from fresh EDTA whole blood cells by using Promega kit for

human DNA isolation The kit contains the following components that are enough for

purifying genomic DNA from 200 samples of human blood

Cell lysis solution

Nuclei lysis solution

Protein precipitation solution

DNA rehydration solution

RNase solution

The human genomic DNA was isolated from human blood sample according to

the kit instructions and was as follows

Three hundred microl EDTA blood were transferred into a sterile 15 ml micro-

centrifuge tube containing 900 microl of cell lysis solution The tube was inverted 5-6 times

to mix the components The mixture was incubated for 10 minutes at room temperature

(with gentle mixing once during the incubation) to lyse the red blood cells The tube

was then centrifuged at 13000 rpm for 20 seconds at room temperature

Supernatant was removed and discarded as much as possible without disturbing

the visible white pellet Approximately 10-20 microl of residual liquid should be left in tube

The tube was vortexed vigorously until the white blood cells were completely

resuspended Three hundred microl of nuclei lysis solution was then added to the tube

28

containing the resuspended cells and the suspension was mixed by pipetting the solution

5-6 times to lyse the white blood cells the solution should become very viscous

RNase solution (15 microl) was added to nuclear lysate and the sample was mixed

by inverting the tube 2-5 times The mixture was incubated at 37degC for 15 minutes and

then cooled to room temperature One hundred microl of protein precipitation solution was

added to the nuclear lysate and the mixture was vortexed vigorously for 10ndash20

seconds Small protein clumps may be visible after vortexing and were removed by

centrifuge precipitation at 13000 rpm for 3 minutes at room temperature The

supernatant was transferred to a clean 15 ml micro-centrifuge tube containing 300microl

isopropanol at room temperature The mixture was gently mixed by inversion until the

white thread-like strands of DNA form a visible mass The DNA was then precipitated

by centrifugation at 13000 rpm for 1 minute at room temperature The DNA would be

visible as a small white pellet

The supernatant decanted and one volume of 70 ethanol was added to the

DNA and kept at room temperature and gently inverted several times to wash the DNA

pellet and the sides of the micro-centrifuge tube After centrifugation at 13000 rpm for

1 minute the ethanol was aspirated using a suitable pipette The DNA pellet is very

loose at this point and care must be used to avoid aspirating the pellet into the pipette

The tube was inverted on a clean absorbent paper for 10ndash15 minutes in order to air-dry

the pellet DNA rehydration solution was added to the dry pellet and the DNA was

rehydrated by incubation at 65degC for 1 hour Periodically the solution was mixed by

gently tapping the tube The DNA was then stored at 2-8degC

342 Detection of extracted DNA

The quality of the isolated DNA was determined by running 5 microl of each sample

on ethidium bromide stained 30 agarose gels and the DNA was visualized on a short

wave UV transilluminator the results were documented by photography

35 PCR reactions

PCR was performed using the primers listed in Table 31 in 4 micro-tubes as

described by Sung Ho Lee et al (70) For each PCR 5 μl master mix (Promega) 2 μl

deionized water 1 μl DNA template and 05 μl of each allele specific primer (5 pmol) in

29

one micro-tube (02 ml) were mixed The volume and concentration of a typical PCR

reaction are shown in Table 32

PCR was performed in a thermal cycler The cycling conditions were as

described below In each PCR MTHFR gene specific primers with the following

sequences (Forward 5prime-ACGATGGGGCAAGTGATGCCC -3 and reverse 5-

GAGAAGGTGTCTGCGGGATC-3) were used as a positive control that produces a 95

bp fragment Upon completion of PCR the products were analyzed by electrophoresis

on 2 ethidium bromide stained agarose gel or stored at 4degC until analysis

Table 33 Composition of PCR master mix

Reagent Composition

dNTPs 400microM each dATP dGTP dCTP dTTP

Taq DNA Polymerase 50 units ml

MgCl2 2mM

351 Temperature cycling program

The thermal cycler program was set as follows

Step 1 Denaturation for 3 minutes at 95degC

Step 2 35 cycles of

21 Melting for 40 seconds at 95degC

22 Annealing for 40 seconds at 585degC

23 Extension for 40 seconds at 72degC

Step3 Final elongation for 5 minutes at72degC

352 Expected PCR results

The PCR product size was estimated by comparing it with DNA molecular size

marker (50 bp ladder DNA ) run on the same gel

11

CHAPTER 4

RESULTS

13

CHAPTER 4

RESULTS

41 Study Population

The study population consisted of 201 subjects (50 males 151 females ) The

percentage of males was 249 while that of females was 751 ( figure 41)

0

10

20

30

40

50

60

70

80

maleFemale

Figure 41 Distribution of the study subjects according to gender

42 Phenotypic Frequency of ABO Blood Groups

Blood grouping was done by antigen antibody agglutination test by using

commercial monoclonal antisera The distribution of phenotypes in the total sample

were 363 (73) 224 (45) 75 (15) 338 (68) for groups A B AB and O

respectively Group A was the dominant one in both genders and AB was the rarest in

both males and females ( table 41)

Table 41 Phenotypic frequencies of various blood groups in the study population

Subject Sex Phenotype

Total A B AB O

Male 16 10 4 20 50

Female 57 35 11 48 151

Total 73 45 15 68 201

753

249

12

43 The Allele Frequencies of ABO Antigens

Allele frequency for the antigens was computed by the Hardy-Weinberg law on

the basis of the number of subjects with blood groups ABO The distribution of the

alleles in the total samples was 025 017 058 for IA I

B and I

O respectively

All genotyping results were compatible with the determined phenotypes by

serological method The observed genotypes as compared with the expected genotypes

are shown in Table 42 The frequencies of the five alleles in the our sample population

were O1 and O2 alleles 0376 and 0221 respectively while A1 0174 A2 0067

B 0162

Table 42 Observed and expected genotypes for the 201 samples

P-Value Expected Observed

Genotype Phenotype Percent Number Percent Number

0230 63 126 45 9 AA A

04762 29 583 318 64 AO

07205 29 58 25 5 BB B

09496 197 396 199 40 BO

05915 85 1708 75 15 AB AB

09613 336 676 338 68 OO O

100 201 100 201 Total

The statistical analysis ( using chi square test ) indicated that molecular data

were in good agreement with the ratio calculated from the estimated gene frequencies of

the ABO blood group system in the Gaza Strip population

11

44 PCR Results

441 Quality of the isolated DNA

Regarding the method of DNA isolation that was described in chapter 3 the

quality and quantity of DNA were suitable for PCR processing The quality of the

isolated DNA from human subjects is represented in the following figure (42)

Figure 42 A representative photograph of DNA isolated from human blood The samples were

run on 1 agarose gel stained with ethidium bromide

442 Blood group genotyping by Allele Specific PCR

Multiplex Allele Specific PCR (ASPCR) assay contains four independent PCR

reactions (Table 31) In the first reaction 261G-int6 primer pair was used to amplify

the 205 bp fragment to detect the A101 A102 B101 cis-ABO1 alleles and the 467C-

803G primer pair was selected to amplify the 381 bp fragment to detect the A101 O01

and O02 alleles The 297A-int6 primer pair and the 467C-803C primer pair in the

second reaction were selected to amplify the 381 bp fragment to detect the B101 allele

In the third reaction the 261A-int6 primer pair was selected to amplify the 205 bp

product to detect the O01 and O02 alleles and the 467T-803G primer pair was selected

to amplify the 381 bp product to detect the A102 allele Finally in the fourth reaction

the 297G-int6 primer pair were applied to produce the 164 bp fragment to detect the

B101 and O02 alleles and the 467T-803C primer pair to produce the 381 bp fragment

to recognize the cis-ABO1 allele Table 43 show the products of PCR reactions

according to the genotype

14

Table 43 PCR products according to the genotype

PCR Reactions Genotype

R1 R2 R3 R4

381 164 205 _ O1O1

381 _ 205 164 O2O2

381 164 205 164 O1O2

205 381 _ 164 B1B1

381205 381164 205 164 B1O1

381205 381 205 164 B1O2

381205 381164 _ 164 A1B1

205 381164 381 164 A2B1

381205 164 _ _ A1A1

381205 164 205 _ A1O1

381205 164 205 164 A1O2

381205 164 381 _ A1A2

205 164 381 _ A2A2

381205 164 381205 _ A2O1

381205 164 381205 164 A2O2

Random mating with the six different alleles at the ABO locus can result in 21

different genotype combinations Only 15 different genotype combinations were

detected from the 201 investigated samples (Figure 43)

A- 1 O1O1 2 O2O2

15

B- 1 B1B1 2 O1O2

C- 1 B1O1

2 A1B

D- 1 A1A2 2 A2O2

E- 1 A1O1 2 A1A1

16

F- 1 A1O2 2 B1O2 3 A2A2

G- 1 A2O1

2 A2B1

Figure 43 The electrophoresis pattern of recognized genotypes using the multiplex ASPCR

method This figure shows 15 different genotypes lane M indicates the 50 bp ladder The R1-

R4 show the PCR reaction number

443 Genotype Frequencies

The frequency of ABO recognized genotypes belonging to the population

residing in Gaza Strip are shown in table 44 As shown in the table the highest allele

frequencies in A B AB and O phenotypes were A1O2 (124) B1O1 (129) A1B

(55) and O1O1 (154) respectively

17

Table 44 The frequency of recognized genotypes using multiplex ASPCR method for a

population residing in Gaza Strip

Genotype Frequency Percent

A1A1 3 15

A1O1 24 119

A1O2 25 124

A1A2 4 20

A2A2 2 10

A2O1 13 65

A2O2 2 10

B1B1 5 25

B1O1 26 129

B1O2 14 70

A1B 11 55

A2B 4 20

O1O1 31 154

O1O2 26 129

O2O2 11 55

Total 201 1000

18

CHAPTER 5

DISCUSSION

19

CHAPTER 5

DISCUSSION

Since the first delineation of the molecular basis of the ABO blood group by

Yamamoto et al (11 79) it has become possible to determine the ABO genotypes using

molecular methods without the need for family investigations ABO genotyping is

commonly used in cases of an ABO discrepancy between cell typing and serum typing

as well as in forensic practices for personal identification and paternity testing (72) The

ABH antigens are ubiquitously expressed in humans and are present primarily as

glycoproteins and partly as glycolipids The ABO locus is found on the long arm

of chromosome 9 (9q341-q342)

Multiplex allele specific PCR (the technique we used here) has advantages over

PCR-RFLP in terms of cost effectiveness reaction time and simplicity of handling It is

a rapid method that is based on detecting four single nucleotide polymorphisms at

nucleotides 261 297 796 and 803 of the ABO locus which in turn discriminate the

major ABO alleles

In this study the ABO genotypes of 201 samples recruited from the Islamic

University ndashGenetics Diagnosis Laboratory were determined using multiplex AS-PCR

method The objectives of this study were to correlate ABO genotypes with phenotypes

in blood samples in a Gaza Strip population and to compare the frequency of ABO

genotypes with other populations

51 The Allele Frequencies of ABO Antigens in Gaza Strip

The frequency of the alleles in the total samples were 0250 for the I

A allele

0170 for the IB allele and 0580 for the I

O allele This distribution is in agreement with

the distribution with the samples reported in an Iranian study were they reported

023974 for IA allele 018147 for I

B allele and 057879 for I

O allele (73) The

frequencies of IA I

B and I

O alleles in our subjects are also comparable to those

reported in Iraqi and Jordanian studies where they found 0212 for IA allele 0177 for

IB allele and 06611 for I

O allele and 0270 for I

A allele 0130 for I

B allele and 060 for

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

25

312 Kits

DNA extraction kit (Promega USA)

313 Reagents and Chemicals

Anti A (Plasmatec Monoclonal UK)

Anti B (Plasmatec Monoclonal UK)

Agarose Molecular biology grade (Promega USA)

DNA molecular weight marker 50 bp-ladder (Promega USA)

EDTA disodium salt (Promega USA)

Absolute Ethanol (Sigma USA)

Ethidium bromide (Promega USA)

Absolute Isopropanol (Sigma USA)

Tris base hydroxyl methyl amino methane (Promega USA)

DNAse RNAse free water (Promega USA)

Acetic acid (Sigma USA)

PCR Master mix (Promega USA)

314 Apparatus and Equipments

Thermal Cycler (Biometra Germany)

LG Microwave Oven

Electrophoresis Apparatus

Vortex Mixer

Digital Camera

Power Supply (Biorad)

Freezer Refrigerator

Micro-Centrifuge

Hoefer shortwave UV light table (Transilluminator)

Computer

Electrical Balance

Automatic Micropipettes

26

32 Methods

321 Study population

322 Sample collection

Blood samples were collected from 201 subjects recruited from the Islamic

University ndashGenetics Laboratory Each sample was collected into EDTA tube The

EDTA samples were kept at 4degC and were used within 24 hours for forward blood

grouping and DNA extraction and subsequent PCR analysis

323 Ethical Considerations

An authorization to carry out the study was obtained from a local ethics

committee using an agreement letter prepared by the Islamic University of Gaza All the

information that were obtained about the subjects were kept confidential

324 Data Analysis

The data were entered stored and analyzed by personal computer using the

Statistical Package for Social Sciences (SPSS) version 160 Allele frequencies were

calculated under the assumption of HardyndashWeinberg equilibrium and expressed as

percentages Chi-square test was used to compare observed allelic and genotypic

frequency distributions of the blood group antigens to that expected under the Hardyndash

Weinberg equation P values gt005 were considered statistically significant

The frequency of ABO in the studied population were compared with the

frequency of ABO in some neighbor countries

33 Blood ABO-typing

331 Forward blood group

Whole blood sample (50 microl) was mixed with Anti A and Anti B reagents by

using slide method in the proportions shown in Table 32 below

27

Table 32 Sample Anti A Anti B reaction for forward blood grouping

Sample Anti -A Anti -B

50 microl 50 microl ــــــ

50 microl 50 ــــــ microl

The contents of each slide were rotated then the agglutination (if present) was

read by naked eye after 30 seconds

34 ABO Genotyping

341 DNA Extraction DNA was isolated from fresh EDTA whole blood cells by using Promega kit for

human DNA isolation The kit contains the following components that are enough for

purifying genomic DNA from 200 samples of human blood

Cell lysis solution

Nuclei lysis solution

Protein precipitation solution

DNA rehydration solution

RNase solution

The human genomic DNA was isolated from human blood sample according to

the kit instructions and was as follows

Three hundred microl EDTA blood were transferred into a sterile 15 ml micro-

centrifuge tube containing 900 microl of cell lysis solution The tube was inverted 5-6 times

to mix the components The mixture was incubated for 10 minutes at room temperature

(with gentle mixing once during the incubation) to lyse the red blood cells The tube

was then centrifuged at 13000 rpm for 20 seconds at room temperature

Supernatant was removed and discarded as much as possible without disturbing

the visible white pellet Approximately 10-20 microl of residual liquid should be left in tube

The tube was vortexed vigorously until the white blood cells were completely

resuspended Three hundred microl of nuclei lysis solution was then added to the tube

28

containing the resuspended cells and the suspension was mixed by pipetting the solution

5-6 times to lyse the white blood cells the solution should become very viscous

RNase solution (15 microl) was added to nuclear lysate and the sample was mixed

by inverting the tube 2-5 times The mixture was incubated at 37degC for 15 minutes and

then cooled to room temperature One hundred microl of protein precipitation solution was

added to the nuclear lysate and the mixture was vortexed vigorously for 10ndash20

seconds Small protein clumps may be visible after vortexing and were removed by

centrifuge precipitation at 13000 rpm for 3 minutes at room temperature The

supernatant was transferred to a clean 15 ml micro-centrifuge tube containing 300microl

isopropanol at room temperature The mixture was gently mixed by inversion until the

white thread-like strands of DNA form a visible mass The DNA was then precipitated

by centrifugation at 13000 rpm for 1 minute at room temperature The DNA would be

visible as a small white pellet

The supernatant decanted and one volume of 70 ethanol was added to the

DNA and kept at room temperature and gently inverted several times to wash the DNA

pellet and the sides of the micro-centrifuge tube After centrifugation at 13000 rpm for

1 minute the ethanol was aspirated using a suitable pipette The DNA pellet is very

loose at this point and care must be used to avoid aspirating the pellet into the pipette

The tube was inverted on a clean absorbent paper for 10ndash15 minutes in order to air-dry

the pellet DNA rehydration solution was added to the dry pellet and the DNA was

rehydrated by incubation at 65degC for 1 hour Periodically the solution was mixed by

gently tapping the tube The DNA was then stored at 2-8degC

342 Detection of extracted DNA

The quality of the isolated DNA was determined by running 5 microl of each sample

on ethidium bromide stained 30 agarose gels and the DNA was visualized on a short

wave UV transilluminator the results were documented by photography

35 PCR reactions

PCR was performed using the primers listed in Table 31 in 4 micro-tubes as

described by Sung Ho Lee et al (70) For each PCR 5 μl master mix (Promega) 2 μl

deionized water 1 μl DNA template and 05 μl of each allele specific primer (5 pmol) in

29

one micro-tube (02 ml) were mixed The volume and concentration of a typical PCR

reaction are shown in Table 32

PCR was performed in a thermal cycler The cycling conditions were as

described below In each PCR MTHFR gene specific primers with the following

sequences (Forward 5prime-ACGATGGGGCAAGTGATGCCC -3 and reverse 5-

GAGAAGGTGTCTGCGGGATC-3) were used as a positive control that produces a 95

bp fragment Upon completion of PCR the products were analyzed by electrophoresis

on 2 ethidium bromide stained agarose gel or stored at 4degC until analysis

Table 33 Composition of PCR master mix

Reagent Composition

dNTPs 400microM each dATP dGTP dCTP dTTP

Taq DNA Polymerase 50 units ml

MgCl2 2mM

351 Temperature cycling program

The thermal cycler program was set as follows

Step 1 Denaturation for 3 minutes at 95degC

Step 2 35 cycles of

21 Melting for 40 seconds at 95degC

22 Annealing for 40 seconds at 585degC

23 Extension for 40 seconds at 72degC

Step3 Final elongation for 5 minutes at72degC

352 Expected PCR results

The PCR product size was estimated by comparing it with DNA molecular size

marker (50 bp ladder DNA ) run on the same gel

11

CHAPTER 4

RESULTS

13

CHAPTER 4

RESULTS

41 Study Population

The study population consisted of 201 subjects (50 males 151 females ) The

percentage of males was 249 while that of females was 751 ( figure 41)

0

10

20

30

40

50

60

70

80

maleFemale

Figure 41 Distribution of the study subjects according to gender

42 Phenotypic Frequency of ABO Blood Groups

Blood grouping was done by antigen antibody agglutination test by using

commercial monoclonal antisera The distribution of phenotypes in the total sample

were 363 (73) 224 (45) 75 (15) 338 (68) for groups A B AB and O

respectively Group A was the dominant one in both genders and AB was the rarest in

both males and females ( table 41)

Table 41 Phenotypic frequencies of various blood groups in the study population

Subject Sex Phenotype

Total A B AB O

Male 16 10 4 20 50

Female 57 35 11 48 151

Total 73 45 15 68 201

753

249

12

43 The Allele Frequencies of ABO Antigens

Allele frequency for the antigens was computed by the Hardy-Weinberg law on

the basis of the number of subjects with blood groups ABO The distribution of the

alleles in the total samples was 025 017 058 for IA I

B and I

O respectively

All genotyping results were compatible with the determined phenotypes by

serological method The observed genotypes as compared with the expected genotypes

are shown in Table 42 The frequencies of the five alleles in the our sample population

were O1 and O2 alleles 0376 and 0221 respectively while A1 0174 A2 0067

B 0162

Table 42 Observed and expected genotypes for the 201 samples

P-Value Expected Observed

Genotype Phenotype Percent Number Percent Number

0230 63 126 45 9 AA A

04762 29 583 318 64 AO

07205 29 58 25 5 BB B

09496 197 396 199 40 BO

05915 85 1708 75 15 AB AB

09613 336 676 338 68 OO O

100 201 100 201 Total

The statistical analysis ( using chi square test ) indicated that molecular data

were in good agreement with the ratio calculated from the estimated gene frequencies of

the ABO blood group system in the Gaza Strip population

11

44 PCR Results

441 Quality of the isolated DNA

Regarding the method of DNA isolation that was described in chapter 3 the

quality and quantity of DNA were suitable for PCR processing The quality of the

isolated DNA from human subjects is represented in the following figure (42)

Figure 42 A representative photograph of DNA isolated from human blood The samples were

run on 1 agarose gel stained with ethidium bromide

442 Blood group genotyping by Allele Specific PCR

Multiplex Allele Specific PCR (ASPCR) assay contains four independent PCR

reactions (Table 31) In the first reaction 261G-int6 primer pair was used to amplify

the 205 bp fragment to detect the A101 A102 B101 cis-ABO1 alleles and the 467C-

803G primer pair was selected to amplify the 381 bp fragment to detect the A101 O01

and O02 alleles The 297A-int6 primer pair and the 467C-803C primer pair in the

second reaction were selected to amplify the 381 bp fragment to detect the B101 allele

In the third reaction the 261A-int6 primer pair was selected to amplify the 205 bp

product to detect the O01 and O02 alleles and the 467T-803G primer pair was selected

to amplify the 381 bp product to detect the A102 allele Finally in the fourth reaction

the 297G-int6 primer pair were applied to produce the 164 bp fragment to detect the

B101 and O02 alleles and the 467T-803C primer pair to produce the 381 bp fragment

to recognize the cis-ABO1 allele Table 43 show the products of PCR reactions

according to the genotype

14

Table 43 PCR products according to the genotype

PCR Reactions Genotype

R1 R2 R3 R4

381 164 205 _ O1O1

381 _ 205 164 O2O2

381 164 205 164 O1O2

205 381 _ 164 B1B1

381205 381164 205 164 B1O1

381205 381 205 164 B1O2

381205 381164 _ 164 A1B1

205 381164 381 164 A2B1

381205 164 _ _ A1A1

381205 164 205 _ A1O1

381205 164 205 164 A1O2

381205 164 381 _ A1A2

205 164 381 _ A2A2

381205 164 381205 _ A2O1

381205 164 381205 164 A2O2

Random mating with the six different alleles at the ABO locus can result in 21

different genotype combinations Only 15 different genotype combinations were

detected from the 201 investigated samples (Figure 43)

A- 1 O1O1 2 O2O2

15

B- 1 B1B1 2 O1O2

C- 1 B1O1

2 A1B

D- 1 A1A2 2 A2O2

E- 1 A1O1 2 A1A1

16

F- 1 A1O2 2 B1O2 3 A2A2

G- 1 A2O1

2 A2B1

Figure 43 The electrophoresis pattern of recognized genotypes using the multiplex ASPCR

method This figure shows 15 different genotypes lane M indicates the 50 bp ladder The R1-

R4 show the PCR reaction number

443 Genotype Frequencies

The frequency of ABO recognized genotypes belonging to the population

residing in Gaza Strip are shown in table 44 As shown in the table the highest allele

frequencies in A B AB and O phenotypes were A1O2 (124) B1O1 (129) A1B

(55) and O1O1 (154) respectively

17

Table 44 The frequency of recognized genotypes using multiplex ASPCR method for a

population residing in Gaza Strip

Genotype Frequency Percent

A1A1 3 15

A1O1 24 119

A1O2 25 124

A1A2 4 20

A2A2 2 10

A2O1 13 65

A2O2 2 10

B1B1 5 25

B1O1 26 129

B1O2 14 70

A1B 11 55

A2B 4 20

O1O1 31 154

O1O2 26 129

O2O2 11 55

Total 201 1000

18

CHAPTER 5

DISCUSSION

19

CHAPTER 5

DISCUSSION

Since the first delineation of the molecular basis of the ABO blood group by

Yamamoto et al (11 79) it has become possible to determine the ABO genotypes using

molecular methods without the need for family investigations ABO genotyping is

commonly used in cases of an ABO discrepancy between cell typing and serum typing

as well as in forensic practices for personal identification and paternity testing (72) The

ABH antigens are ubiquitously expressed in humans and are present primarily as

glycoproteins and partly as glycolipids The ABO locus is found on the long arm

of chromosome 9 (9q341-q342)

Multiplex allele specific PCR (the technique we used here) has advantages over

PCR-RFLP in terms of cost effectiveness reaction time and simplicity of handling It is

a rapid method that is based on detecting four single nucleotide polymorphisms at

nucleotides 261 297 796 and 803 of the ABO locus which in turn discriminate the

major ABO alleles

In this study the ABO genotypes of 201 samples recruited from the Islamic

University ndashGenetics Diagnosis Laboratory were determined using multiplex AS-PCR

method The objectives of this study were to correlate ABO genotypes with phenotypes

in blood samples in a Gaza Strip population and to compare the frequency of ABO

genotypes with other populations

51 The Allele Frequencies of ABO Antigens in Gaza Strip

The frequency of the alleles in the total samples were 0250 for the I

A allele

0170 for the IB allele and 0580 for the I

O allele This distribution is in agreement with

the distribution with the samples reported in an Iranian study were they reported

023974 for IA allele 018147 for I

B allele and 057879 for I

O allele (73) The

frequencies of IA I

B and I

O alleles in our subjects are also comparable to those

reported in Iraqi and Jordanian studies where they found 0212 for IA allele 0177 for

IB allele and 06611 for I

O allele and 0270 for I

A allele 0130 for I

B allele and 060 for

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

26

32 Methods

321 Study population

322 Sample collection

Blood samples were collected from 201 subjects recruited from the Islamic

University ndashGenetics Laboratory Each sample was collected into EDTA tube The

EDTA samples were kept at 4degC and were used within 24 hours for forward blood

grouping and DNA extraction and subsequent PCR analysis

323 Ethical Considerations

An authorization to carry out the study was obtained from a local ethics

committee using an agreement letter prepared by the Islamic University of Gaza All the

information that were obtained about the subjects were kept confidential

324 Data Analysis

The data were entered stored and analyzed by personal computer using the

Statistical Package for Social Sciences (SPSS) version 160 Allele frequencies were

calculated under the assumption of HardyndashWeinberg equilibrium and expressed as

percentages Chi-square test was used to compare observed allelic and genotypic

frequency distributions of the blood group antigens to that expected under the Hardyndash

Weinberg equation P values gt005 were considered statistically significant

The frequency of ABO in the studied population were compared with the

frequency of ABO in some neighbor countries

33 Blood ABO-typing

331 Forward blood group

Whole blood sample (50 microl) was mixed with Anti A and Anti B reagents by

using slide method in the proportions shown in Table 32 below

27

Table 32 Sample Anti A Anti B reaction for forward blood grouping

Sample Anti -A Anti -B

50 microl 50 microl ــــــ

50 microl 50 ــــــ microl

The contents of each slide were rotated then the agglutination (if present) was

read by naked eye after 30 seconds

34 ABO Genotyping

341 DNA Extraction DNA was isolated from fresh EDTA whole blood cells by using Promega kit for

human DNA isolation The kit contains the following components that are enough for

purifying genomic DNA from 200 samples of human blood

Cell lysis solution

Nuclei lysis solution

Protein precipitation solution

DNA rehydration solution

RNase solution

The human genomic DNA was isolated from human blood sample according to

the kit instructions and was as follows

Three hundred microl EDTA blood were transferred into a sterile 15 ml micro-

centrifuge tube containing 900 microl of cell lysis solution The tube was inverted 5-6 times

to mix the components The mixture was incubated for 10 minutes at room temperature

(with gentle mixing once during the incubation) to lyse the red blood cells The tube

was then centrifuged at 13000 rpm for 20 seconds at room temperature

Supernatant was removed and discarded as much as possible without disturbing

the visible white pellet Approximately 10-20 microl of residual liquid should be left in tube

The tube was vortexed vigorously until the white blood cells were completely

resuspended Three hundred microl of nuclei lysis solution was then added to the tube

28

containing the resuspended cells and the suspension was mixed by pipetting the solution

5-6 times to lyse the white blood cells the solution should become very viscous

RNase solution (15 microl) was added to nuclear lysate and the sample was mixed

by inverting the tube 2-5 times The mixture was incubated at 37degC for 15 minutes and

then cooled to room temperature One hundred microl of protein precipitation solution was

added to the nuclear lysate and the mixture was vortexed vigorously for 10ndash20

seconds Small protein clumps may be visible after vortexing and were removed by

centrifuge precipitation at 13000 rpm for 3 minutes at room temperature The

supernatant was transferred to a clean 15 ml micro-centrifuge tube containing 300microl

isopropanol at room temperature The mixture was gently mixed by inversion until the

white thread-like strands of DNA form a visible mass The DNA was then precipitated

by centrifugation at 13000 rpm for 1 minute at room temperature The DNA would be

visible as a small white pellet

The supernatant decanted and one volume of 70 ethanol was added to the

DNA and kept at room temperature and gently inverted several times to wash the DNA

pellet and the sides of the micro-centrifuge tube After centrifugation at 13000 rpm for

1 minute the ethanol was aspirated using a suitable pipette The DNA pellet is very

loose at this point and care must be used to avoid aspirating the pellet into the pipette

The tube was inverted on a clean absorbent paper for 10ndash15 minutes in order to air-dry

the pellet DNA rehydration solution was added to the dry pellet and the DNA was

rehydrated by incubation at 65degC for 1 hour Periodically the solution was mixed by

gently tapping the tube The DNA was then stored at 2-8degC

342 Detection of extracted DNA

The quality of the isolated DNA was determined by running 5 microl of each sample

on ethidium bromide stained 30 agarose gels and the DNA was visualized on a short

wave UV transilluminator the results were documented by photography

35 PCR reactions

PCR was performed using the primers listed in Table 31 in 4 micro-tubes as

described by Sung Ho Lee et al (70) For each PCR 5 μl master mix (Promega) 2 μl

deionized water 1 μl DNA template and 05 μl of each allele specific primer (5 pmol) in

29

one micro-tube (02 ml) were mixed The volume and concentration of a typical PCR

reaction are shown in Table 32

PCR was performed in a thermal cycler The cycling conditions were as

described below In each PCR MTHFR gene specific primers with the following

sequences (Forward 5prime-ACGATGGGGCAAGTGATGCCC -3 and reverse 5-

GAGAAGGTGTCTGCGGGATC-3) were used as a positive control that produces a 95

bp fragment Upon completion of PCR the products were analyzed by electrophoresis

on 2 ethidium bromide stained agarose gel or stored at 4degC until analysis

Table 33 Composition of PCR master mix

Reagent Composition

dNTPs 400microM each dATP dGTP dCTP dTTP

Taq DNA Polymerase 50 units ml

MgCl2 2mM

351 Temperature cycling program

The thermal cycler program was set as follows

Step 1 Denaturation for 3 minutes at 95degC

Step 2 35 cycles of

21 Melting for 40 seconds at 95degC

22 Annealing for 40 seconds at 585degC

23 Extension for 40 seconds at 72degC

Step3 Final elongation for 5 minutes at72degC

352 Expected PCR results

The PCR product size was estimated by comparing it with DNA molecular size

marker (50 bp ladder DNA ) run on the same gel

11

CHAPTER 4

RESULTS

13

CHAPTER 4

RESULTS

41 Study Population

The study population consisted of 201 subjects (50 males 151 females ) The

percentage of males was 249 while that of females was 751 ( figure 41)

0

10

20

30

40

50

60

70

80

maleFemale

Figure 41 Distribution of the study subjects according to gender

42 Phenotypic Frequency of ABO Blood Groups

Blood grouping was done by antigen antibody agglutination test by using

commercial monoclonal antisera The distribution of phenotypes in the total sample

were 363 (73) 224 (45) 75 (15) 338 (68) for groups A B AB and O

respectively Group A was the dominant one in both genders and AB was the rarest in

both males and females ( table 41)

Table 41 Phenotypic frequencies of various blood groups in the study population

Subject Sex Phenotype

Total A B AB O

Male 16 10 4 20 50

Female 57 35 11 48 151

Total 73 45 15 68 201

753

249

12

43 The Allele Frequencies of ABO Antigens

Allele frequency for the antigens was computed by the Hardy-Weinberg law on

the basis of the number of subjects with blood groups ABO The distribution of the

alleles in the total samples was 025 017 058 for IA I

B and I

O respectively

All genotyping results were compatible with the determined phenotypes by

serological method The observed genotypes as compared with the expected genotypes

are shown in Table 42 The frequencies of the five alleles in the our sample population

were O1 and O2 alleles 0376 and 0221 respectively while A1 0174 A2 0067

B 0162

Table 42 Observed and expected genotypes for the 201 samples

P-Value Expected Observed

Genotype Phenotype Percent Number Percent Number

0230 63 126 45 9 AA A

04762 29 583 318 64 AO

07205 29 58 25 5 BB B

09496 197 396 199 40 BO

05915 85 1708 75 15 AB AB

09613 336 676 338 68 OO O

100 201 100 201 Total

The statistical analysis ( using chi square test ) indicated that molecular data

were in good agreement with the ratio calculated from the estimated gene frequencies of

the ABO blood group system in the Gaza Strip population

11

44 PCR Results

441 Quality of the isolated DNA

Regarding the method of DNA isolation that was described in chapter 3 the

quality and quantity of DNA were suitable for PCR processing The quality of the

isolated DNA from human subjects is represented in the following figure (42)

Figure 42 A representative photograph of DNA isolated from human blood The samples were

run on 1 agarose gel stained with ethidium bromide

442 Blood group genotyping by Allele Specific PCR

Multiplex Allele Specific PCR (ASPCR) assay contains four independent PCR

reactions (Table 31) In the first reaction 261G-int6 primer pair was used to amplify

the 205 bp fragment to detect the A101 A102 B101 cis-ABO1 alleles and the 467C-

803G primer pair was selected to amplify the 381 bp fragment to detect the A101 O01

and O02 alleles The 297A-int6 primer pair and the 467C-803C primer pair in the

second reaction were selected to amplify the 381 bp fragment to detect the B101 allele

In the third reaction the 261A-int6 primer pair was selected to amplify the 205 bp

product to detect the O01 and O02 alleles and the 467T-803G primer pair was selected

to amplify the 381 bp product to detect the A102 allele Finally in the fourth reaction

the 297G-int6 primer pair were applied to produce the 164 bp fragment to detect the

B101 and O02 alleles and the 467T-803C primer pair to produce the 381 bp fragment

to recognize the cis-ABO1 allele Table 43 show the products of PCR reactions

according to the genotype

14

Table 43 PCR products according to the genotype

PCR Reactions Genotype

R1 R2 R3 R4

381 164 205 _ O1O1

381 _ 205 164 O2O2

381 164 205 164 O1O2

205 381 _ 164 B1B1

381205 381164 205 164 B1O1

381205 381 205 164 B1O2

381205 381164 _ 164 A1B1

205 381164 381 164 A2B1

381205 164 _ _ A1A1

381205 164 205 _ A1O1

381205 164 205 164 A1O2

381205 164 381 _ A1A2

205 164 381 _ A2A2

381205 164 381205 _ A2O1

381205 164 381205 164 A2O2

Random mating with the six different alleles at the ABO locus can result in 21

different genotype combinations Only 15 different genotype combinations were

detected from the 201 investigated samples (Figure 43)

A- 1 O1O1 2 O2O2

15

B- 1 B1B1 2 O1O2

C- 1 B1O1

2 A1B

D- 1 A1A2 2 A2O2

E- 1 A1O1 2 A1A1

16

F- 1 A1O2 2 B1O2 3 A2A2

G- 1 A2O1

2 A2B1

Figure 43 The electrophoresis pattern of recognized genotypes using the multiplex ASPCR

method This figure shows 15 different genotypes lane M indicates the 50 bp ladder The R1-

R4 show the PCR reaction number

443 Genotype Frequencies

The frequency of ABO recognized genotypes belonging to the population

residing in Gaza Strip are shown in table 44 As shown in the table the highest allele

frequencies in A B AB and O phenotypes were A1O2 (124) B1O1 (129) A1B

(55) and O1O1 (154) respectively

17

Table 44 The frequency of recognized genotypes using multiplex ASPCR method for a

population residing in Gaza Strip

Genotype Frequency Percent

A1A1 3 15

A1O1 24 119

A1O2 25 124

A1A2 4 20

A2A2 2 10

A2O1 13 65

A2O2 2 10

B1B1 5 25

B1O1 26 129

B1O2 14 70

A1B 11 55

A2B 4 20

O1O1 31 154

O1O2 26 129

O2O2 11 55

Total 201 1000

18

CHAPTER 5

DISCUSSION

19

CHAPTER 5

DISCUSSION

Since the first delineation of the molecular basis of the ABO blood group by

Yamamoto et al (11 79) it has become possible to determine the ABO genotypes using

molecular methods without the need for family investigations ABO genotyping is

commonly used in cases of an ABO discrepancy between cell typing and serum typing

as well as in forensic practices for personal identification and paternity testing (72) The

ABH antigens are ubiquitously expressed in humans and are present primarily as

glycoproteins and partly as glycolipids The ABO locus is found on the long arm

of chromosome 9 (9q341-q342)

Multiplex allele specific PCR (the technique we used here) has advantages over

PCR-RFLP in terms of cost effectiveness reaction time and simplicity of handling It is

a rapid method that is based on detecting four single nucleotide polymorphisms at

nucleotides 261 297 796 and 803 of the ABO locus which in turn discriminate the

major ABO alleles

In this study the ABO genotypes of 201 samples recruited from the Islamic

University ndashGenetics Diagnosis Laboratory were determined using multiplex AS-PCR

method The objectives of this study were to correlate ABO genotypes with phenotypes

in blood samples in a Gaza Strip population and to compare the frequency of ABO

genotypes with other populations

51 The Allele Frequencies of ABO Antigens in Gaza Strip

The frequency of the alleles in the total samples were 0250 for the I

A allele

0170 for the IB allele and 0580 for the I

O allele This distribution is in agreement with

the distribution with the samples reported in an Iranian study were they reported

023974 for IA allele 018147 for I

B allele and 057879 for I

O allele (73) The

frequencies of IA I

B and I

O alleles in our subjects are also comparable to those

reported in Iraqi and Jordanian studies where they found 0212 for IA allele 0177 for

IB allele and 06611 for I

O allele and 0270 for I

A allele 0130 for I

B allele and 060 for

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

27

Table 32 Sample Anti A Anti B reaction for forward blood grouping

Sample Anti -A Anti -B

50 microl 50 microl ــــــ

50 microl 50 ــــــ microl

The contents of each slide were rotated then the agglutination (if present) was

read by naked eye after 30 seconds

34 ABO Genotyping

341 DNA Extraction DNA was isolated from fresh EDTA whole blood cells by using Promega kit for

human DNA isolation The kit contains the following components that are enough for

purifying genomic DNA from 200 samples of human blood

Cell lysis solution

Nuclei lysis solution

Protein precipitation solution

DNA rehydration solution

RNase solution

The human genomic DNA was isolated from human blood sample according to

the kit instructions and was as follows

Three hundred microl EDTA blood were transferred into a sterile 15 ml micro-

centrifuge tube containing 900 microl of cell lysis solution The tube was inverted 5-6 times

to mix the components The mixture was incubated for 10 minutes at room temperature

(with gentle mixing once during the incubation) to lyse the red blood cells The tube

was then centrifuged at 13000 rpm for 20 seconds at room temperature

Supernatant was removed and discarded as much as possible without disturbing

the visible white pellet Approximately 10-20 microl of residual liquid should be left in tube

The tube was vortexed vigorously until the white blood cells were completely

resuspended Three hundred microl of nuclei lysis solution was then added to the tube

28

containing the resuspended cells and the suspension was mixed by pipetting the solution

5-6 times to lyse the white blood cells the solution should become very viscous

RNase solution (15 microl) was added to nuclear lysate and the sample was mixed

by inverting the tube 2-5 times The mixture was incubated at 37degC for 15 minutes and

then cooled to room temperature One hundred microl of protein precipitation solution was

added to the nuclear lysate and the mixture was vortexed vigorously for 10ndash20

seconds Small protein clumps may be visible after vortexing and were removed by

centrifuge precipitation at 13000 rpm for 3 minutes at room temperature The

supernatant was transferred to a clean 15 ml micro-centrifuge tube containing 300microl

isopropanol at room temperature The mixture was gently mixed by inversion until the

white thread-like strands of DNA form a visible mass The DNA was then precipitated

by centrifugation at 13000 rpm for 1 minute at room temperature The DNA would be

visible as a small white pellet

The supernatant decanted and one volume of 70 ethanol was added to the

DNA and kept at room temperature and gently inverted several times to wash the DNA

pellet and the sides of the micro-centrifuge tube After centrifugation at 13000 rpm for

1 minute the ethanol was aspirated using a suitable pipette The DNA pellet is very

loose at this point and care must be used to avoid aspirating the pellet into the pipette

The tube was inverted on a clean absorbent paper for 10ndash15 minutes in order to air-dry

the pellet DNA rehydration solution was added to the dry pellet and the DNA was

rehydrated by incubation at 65degC for 1 hour Periodically the solution was mixed by

gently tapping the tube The DNA was then stored at 2-8degC

342 Detection of extracted DNA

The quality of the isolated DNA was determined by running 5 microl of each sample

on ethidium bromide stained 30 agarose gels and the DNA was visualized on a short

wave UV transilluminator the results were documented by photography

35 PCR reactions

PCR was performed using the primers listed in Table 31 in 4 micro-tubes as

described by Sung Ho Lee et al (70) For each PCR 5 μl master mix (Promega) 2 μl

deionized water 1 μl DNA template and 05 μl of each allele specific primer (5 pmol) in

29

one micro-tube (02 ml) were mixed The volume and concentration of a typical PCR

reaction are shown in Table 32

PCR was performed in a thermal cycler The cycling conditions were as

described below In each PCR MTHFR gene specific primers with the following

sequences (Forward 5prime-ACGATGGGGCAAGTGATGCCC -3 and reverse 5-

GAGAAGGTGTCTGCGGGATC-3) were used as a positive control that produces a 95

bp fragment Upon completion of PCR the products were analyzed by electrophoresis

on 2 ethidium bromide stained agarose gel or stored at 4degC until analysis

Table 33 Composition of PCR master mix

Reagent Composition

dNTPs 400microM each dATP dGTP dCTP dTTP

Taq DNA Polymerase 50 units ml

MgCl2 2mM

351 Temperature cycling program

The thermal cycler program was set as follows

Step 1 Denaturation for 3 minutes at 95degC

Step 2 35 cycles of

21 Melting for 40 seconds at 95degC

22 Annealing for 40 seconds at 585degC

23 Extension for 40 seconds at 72degC

Step3 Final elongation for 5 minutes at72degC

352 Expected PCR results

The PCR product size was estimated by comparing it with DNA molecular size

marker (50 bp ladder DNA ) run on the same gel

11

CHAPTER 4

RESULTS

13

CHAPTER 4

RESULTS

41 Study Population

The study population consisted of 201 subjects (50 males 151 females ) The

percentage of males was 249 while that of females was 751 ( figure 41)

0

10

20

30

40

50

60

70

80

maleFemale

Figure 41 Distribution of the study subjects according to gender

42 Phenotypic Frequency of ABO Blood Groups

Blood grouping was done by antigen antibody agglutination test by using

commercial monoclonal antisera The distribution of phenotypes in the total sample

were 363 (73) 224 (45) 75 (15) 338 (68) for groups A B AB and O

respectively Group A was the dominant one in both genders and AB was the rarest in

both males and females ( table 41)

Table 41 Phenotypic frequencies of various blood groups in the study population

Subject Sex Phenotype

Total A B AB O

Male 16 10 4 20 50

Female 57 35 11 48 151

Total 73 45 15 68 201

753

249

12

43 The Allele Frequencies of ABO Antigens

Allele frequency for the antigens was computed by the Hardy-Weinberg law on

the basis of the number of subjects with blood groups ABO The distribution of the

alleles in the total samples was 025 017 058 for IA I

B and I

O respectively

All genotyping results were compatible with the determined phenotypes by

serological method The observed genotypes as compared with the expected genotypes

are shown in Table 42 The frequencies of the five alleles in the our sample population

were O1 and O2 alleles 0376 and 0221 respectively while A1 0174 A2 0067

B 0162

Table 42 Observed and expected genotypes for the 201 samples

P-Value Expected Observed

Genotype Phenotype Percent Number Percent Number

0230 63 126 45 9 AA A

04762 29 583 318 64 AO

07205 29 58 25 5 BB B

09496 197 396 199 40 BO

05915 85 1708 75 15 AB AB

09613 336 676 338 68 OO O

100 201 100 201 Total

The statistical analysis ( using chi square test ) indicated that molecular data

were in good agreement with the ratio calculated from the estimated gene frequencies of

the ABO blood group system in the Gaza Strip population

11

44 PCR Results

441 Quality of the isolated DNA

Regarding the method of DNA isolation that was described in chapter 3 the

quality and quantity of DNA were suitable for PCR processing The quality of the

isolated DNA from human subjects is represented in the following figure (42)

Figure 42 A representative photograph of DNA isolated from human blood The samples were

run on 1 agarose gel stained with ethidium bromide

442 Blood group genotyping by Allele Specific PCR

Multiplex Allele Specific PCR (ASPCR) assay contains four independent PCR

reactions (Table 31) In the first reaction 261G-int6 primer pair was used to amplify

the 205 bp fragment to detect the A101 A102 B101 cis-ABO1 alleles and the 467C-

803G primer pair was selected to amplify the 381 bp fragment to detect the A101 O01

and O02 alleles The 297A-int6 primer pair and the 467C-803C primer pair in the

second reaction were selected to amplify the 381 bp fragment to detect the B101 allele

In the third reaction the 261A-int6 primer pair was selected to amplify the 205 bp

product to detect the O01 and O02 alleles and the 467T-803G primer pair was selected

to amplify the 381 bp product to detect the A102 allele Finally in the fourth reaction

the 297G-int6 primer pair were applied to produce the 164 bp fragment to detect the

B101 and O02 alleles and the 467T-803C primer pair to produce the 381 bp fragment

to recognize the cis-ABO1 allele Table 43 show the products of PCR reactions

according to the genotype

14

Table 43 PCR products according to the genotype

PCR Reactions Genotype

R1 R2 R3 R4

381 164 205 _ O1O1

381 _ 205 164 O2O2

381 164 205 164 O1O2

205 381 _ 164 B1B1

381205 381164 205 164 B1O1

381205 381 205 164 B1O2

381205 381164 _ 164 A1B1

205 381164 381 164 A2B1

381205 164 _ _ A1A1

381205 164 205 _ A1O1

381205 164 205 164 A1O2

381205 164 381 _ A1A2

205 164 381 _ A2A2

381205 164 381205 _ A2O1

381205 164 381205 164 A2O2

Random mating with the six different alleles at the ABO locus can result in 21

different genotype combinations Only 15 different genotype combinations were

detected from the 201 investigated samples (Figure 43)

A- 1 O1O1 2 O2O2

15

B- 1 B1B1 2 O1O2

C- 1 B1O1

2 A1B

D- 1 A1A2 2 A2O2

E- 1 A1O1 2 A1A1

16

F- 1 A1O2 2 B1O2 3 A2A2

G- 1 A2O1

2 A2B1

Figure 43 The electrophoresis pattern of recognized genotypes using the multiplex ASPCR

method This figure shows 15 different genotypes lane M indicates the 50 bp ladder The R1-

R4 show the PCR reaction number

443 Genotype Frequencies

The frequency of ABO recognized genotypes belonging to the population

residing in Gaza Strip are shown in table 44 As shown in the table the highest allele

frequencies in A B AB and O phenotypes were A1O2 (124) B1O1 (129) A1B

(55) and O1O1 (154) respectively

17

Table 44 The frequency of recognized genotypes using multiplex ASPCR method for a

population residing in Gaza Strip

Genotype Frequency Percent

A1A1 3 15

A1O1 24 119

A1O2 25 124

A1A2 4 20

A2A2 2 10

A2O1 13 65

A2O2 2 10

B1B1 5 25

B1O1 26 129

B1O2 14 70

A1B 11 55

A2B 4 20

O1O1 31 154

O1O2 26 129

O2O2 11 55

Total 201 1000

18

CHAPTER 5

DISCUSSION

19

CHAPTER 5

DISCUSSION

Since the first delineation of the molecular basis of the ABO blood group by

Yamamoto et al (11 79) it has become possible to determine the ABO genotypes using

molecular methods without the need for family investigations ABO genotyping is

commonly used in cases of an ABO discrepancy between cell typing and serum typing

as well as in forensic practices for personal identification and paternity testing (72) The

ABH antigens are ubiquitously expressed in humans and are present primarily as

glycoproteins and partly as glycolipids The ABO locus is found on the long arm

of chromosome 9 (9q341-q342)

Multiplex allele specific PCR (the technique we used here) has advantages over

PCR-RFLP in terms of cost effectiveness reaction time and simplicity of handling It is

a rapid method that is based on detecting four single nucleotide polymorphisms at

nucleotides 261 297 796 and 803 of the ABO locus which in turn discriminate the

major ABO alleles

In this study the ABO genotypes of 201 samples recruited from the Islamic

University ndashGenetics Diagnosis Laboratory were determined using multiplex AS-PCR

method The objectives of this study were to correlate ABO genotypes with phenotypes

in blood samples in a Gaza Strip population and to compare the frequency of ABO

genotypes with other populations

51 The Allele Frequencies of ABO Antigens in Gaza Strip

The frequency of the alleles in the total samples were 0250 for the I

A allele

0170 for the IB allele and 0580 for the I

O allele This distribution is in agreement with

the distribution with the samples reported in an Iranian study were they reported

023974 for IA allele 018147 for I

B allele and 057879 for I

O allele (73) The

frequencies of IA I

B and I

O alleles in our subjects are also comparable to those

reported in Iraqi and Jordanian studies where they found 0212 for IA allele 0177 for

IB allele and 06611 for I

O allele and 0270 for I

A allele 0130 for I

B allele and 060 for

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

28

containing the resuspended cells and the suspension was mixed by pipetting the solution

5-6 times to lyse the white blood cells the solution should become very viscous

RNase solution (15 microl) was added to nuclear lysate and the sample was mixed

by inverting the tube 2-5 times The mixture was incubated at 37degC for 15 minutes and

then cooled to room temperature One hundred microl of protein precipitation solution was

added to the nuclear lysate and the mixture was vortexed vigorously for 10ndash20

seconds Small protein clumps may be visible after vortexing and were removed by

centrifuge precipitation at 13000 rpm for 3 minutes at room temperature The

supernatant was transferred to a clean 15 ml micro-centrifuge tube containing 300microl

isopropanol at room temperature The mixture was gently mixed by inversion until the

white thread-like strands of DNA form a visible mass The DNA was then precipitated

by centrifugation at 13000 rpm for 1 minute at room temperature The DNA would be

visible as a small white pellet

The supernatant decanted and one volume of 70 ethanol was added to the

DNA and kept at room temperature and gently inverted several times to wash the DNA

pellet and the sides of the micro-centrifuge tube After centrifugation at 13000 rpm for

1 minute the ethanol was aspirated using a suitable pipette The DNA pellet is very

loose at this point and care must be used to avoid aspirating the pellet into the pipette

The tube was inverted on a clean absorbent paper for 10ndash15 minutes in order to air-dry

the pellet DNA rehydration solution was added to the dry pellet and the DNA was

rehydrated by incubation at 65degC for 1 hour Periodically the solution was mixed by

gently tapping the tube The DNA was then stored at 2-8degC

342 Detection of extracted DNA

The quality of the isolated DNA was determined by running 5 microl of each sample

on ethidium bromide stained 30 agarose gels and the DNA was visualized on a short

wave UV transilluminator the results were documented by photography

35 PCR reactions

PCR was performed using the primers listed in Table 31 in 4 micro-tubes as

described by Sung Ho Lee et al (70) For each PCR 5 μl master mix (Promega) 2 μl

deionized water 1 μl DNA template and 05 μl of each allele specific primer (5 pmol) in

29

one micro-tube (02 ml) were mixed The volume and concentration of a typical PCR

reaction are shown in Table 32

PCR was performed in a thermal cycler The cycling conditions were as

described below In each PCR MTHFR gene specific primers with the following

sequences (Forward 5prime-ACGATGGGGCAAGTGATGCCC -3 and reverse 5-

GAGAAGGTGTCTGCGGGATC-3) were used as a positive control that produces a 95

bp fragment Upon completion of PCR the products were analyzed by electrophoresis

on 2 ethidium bromide stained agarose gel or stored at 4degC until analysis

Table 33 Composition of PCR master mix

Reagent Composition

dNTPs 400microM each dATP dGTP dCTP dTTP

Taq DNA Polymerase 50 units ml

MgCl2 2mM

351 Temperature cycling program

The thermal cycler program was set as follows

Step 1 Denaturation for 3 minutes at 95degC

Step 2 35 cycles of

21 Melting for 40 seconds at 95degC

22 Annealing for 40 seconds at 585degC

23 Extension for 40 seconds at 72degC

Step3 Final elongation for 5 minutes at72degC

352 Expected PCR results

The PCR product size was estimated by comparing it with DNA molecular size

marker (50 bp ladder DNA ) run on the same gel

11

CHAPTER 4

RESULTS

13

CHAPTER 4

RESULTS

41 Study Population

The study population consisted of 201 subjects (50 males 151 females ) The

percentage of males was 249 while that of females was 751 ( figure 41)

0

10

20

30

40

50

60

70

80

maleFemale

Figure 41 Distribution of the study subjects according to gender

42 Phenotypic Frequency of ABO Blood Groups

Blood grouping was done by antigen antibody agglutination test by using

commercial monoclonal antisera The distribution of phenotypes in the total sample

were 363 (73) 224 (45) 75 (15) 338 (68) for groups A B AB and O

respectively Group A was the dominant one in both genders and AB was the rarest in

both males and females ( table 41)

Table 41 Phenotypic frequencies of various blood groups in the study population

Subject Sex Phenotype

Total A B AB O

Male 16 10 4 20 50

Female 57 35 11 48 151

Total 73 45 15 68 201

753

249

12

43 The Allele Frequencies of ABO Antigens

Allele frequency for the antigens was computed by the Hardy-Weinberg law on

the basis of the number of subjects with blood groups ABO The distribution of the

alleles in the total samples was 025 017 058 for IA I

B and I

O respectively

All genotyping results were compatible with the determined phenotypes by

serological method The observed genotypes as compared with the expected genotypes

are shown in Table 42 The frequencies of the five alleles in the our sample population

were O1 and O2 alleles 0376 and 0221 respectively while A1 0174 A2 0067

B 0162

Table 42 Observed and expected genotypes for the 201 samples

P-Value Expected Observed

Genotype Phenotype Percent Number Percent Number

0230 63 126 45 9 AA A

04762 29 583 318 64 AO

07205 29 58 25 5 BB B

09496 197 396 199 40 BO

05915 85 1708 75 15 AB AB

09613 336 676 338 68 OO O

100 201 100 201 Total

The statistical analysis ( using chi square test ) indicated that molecular data

were in good agreement with the ratio calculated from the estimated gene frequencies of

the ABO blood group system in the Gaza Strip population

11

44 PCR Results

441 Quality of the isolated DNA

Regarding the method of DNA isolation that was described in chapter 3 the

quality and quantity of DNA were suitable for PCR processing The quality of the

isolated DNA from human subjects is represented in the following figure (42)

Figure 42 A representative photograph of DNA isolated from human blood The samples were

run on 1 agarose gel stained with ethidium bromide

442 Blood group genotyping by Allele Specific PCR

Multiplex Allele Specific PCR (ASPCR) assay contains four independent PCR

reactions (Table 31) In the first reaction 261G-int6 primer pair was used to amplify

the 205 bp fragment to detect the A101 A102 B101 cis-ABO1 alleles and the 467C-

803G primer pair was selected to amplify the 381 bp fragment to detect the A101 O01

and O02 alleles The 297A-int6 primer pair and the 467C-803C primer pair in the

second reaction were selected to amplify the 381 bp fragment to detect the B101 allele

In the third reaction the 261A-int6 primer pair was selected to amplify the 205 bp

product to detect the O01 and O02 alleles and the 467T-803G primer pair was selected

to amplify the 381 bp product to detect the A102 allele Finally in the fourth reaction

the 297G-int6 primer pair were applied to produce the 164 bp fragment to detect the

B101 and O02 alleles and the 467T-803C primer pair to produce the 381 bp fragment

to recognize the cis-ABO1 allele Table 43 show the products of PCR reactions

according to the genotype

14

Table 43 PCR products according to the genotype

PCR Reactions Genotype

R1 R2 R3 R4

381 164 205 _ O1O1

381 _ 205 164 O2O2

381 164 205 164 O1O2

205 381 _ 164 B1B1

381205 381164 205 164 B1O1

381205 381 205 164 B1O2

381205 381164 _ 164 A1B1

205 381164 381 164 A2B1

381205 164 _ _ A1A1

381205 164 205 _ A1O1

381205 164 205 164 A1O2

381205 164 381 _ A1A2

205 164 381 _ A2A2

381205 164 381205 _ A2O1

381205 164 381205 164 A2O2

Random mating with the six different alleles at the ABO locus can result in 21

different genotype combinations Only 15 different genotype combinations were

detected from the 201 investigated samples (Figure 43)

A- 1 O1O1 2 O2O2

15

B- 1 B1B1 2 O1O2

C- 1 B1O1

2 A1B

D- 1 A1A2 2 A2O2

E- 1 A1O1 2 A1A1

16

F- 1 A1O2 2 B1O2 3 A2A2

G- 1 A2O1

2 A2B1

Figure 43 The electrophoresis pattern of recognized genotypes using the multiplex ASPCR

method This figure shows 15 different genotypes lane M indicates the 50 bp ladder The R1-

R4 show the PCR reaction number

443 Genotype Frequencies

The frequency of ABO recognized genotypes belonging to the population

residing in Gaza Strip are shown in table 44 As shown in the table the highest allele

frequencies in A B AB and O phenotypes were A1O2 (124) B1O1 (129) A1B

(55) and O1O1 (154) respectively

17

Table 44 The frequency of recognized genotypes using multiplex ASPCR method for a

population residing in Gaza Strip

Genotype Frequency Percent

A1A1 3 15

A1O1 24 119

A1O2 25 124

A1A2 4 20

A2A2 2 10

A2O1 13 65

A2O2 2 10

B1B1 5 25

B1O1 26 129

B1O2 14 70

A1B 11 55

A2B 4 20

O1O1 31 154

O1O2 26 129

O2O2 11 55

Total 201 1000

18

CHAPTER 5

DISCUSSION

19

CHAPTER 5

DISCUSSION

Since the first delineation of the molecular basis of the ABO blood group by

Yamamoto et al (11 79) it has become possible to determine the ABO genotypes using

molecular methods without the need for family investigations ABO genotyping is

commonly used in cases of an ABO discrepancy between cell typing and serum typing

as well as in forensic practices for personal identification and paternity testing (72) The

ABH antigens are ubiquitously expressed in humans and are present primarily as

glycoproteins and partly as glycolipids The ABO locus is found on the long arm

of chromosome 9 (9q341-q342)

Multiplex allele specific PCR (the technique we used here) has advantages over

PCR-RFLP in terms of cost effectiveness reaction time and simplicity of handling It is

a rapid method that is based on detecting four single nucleotide polymorphisms at

nucleotides 261 297 796 and 803 of the ABO locus which in turn discriminate the

major ABO alleles

In this study the ABO genotypes of 201 samples recruited from the Islamic

University ndashGenetics Diagnosis Laboratory were determined using multiplex AS-PCR

method The objectives of this study were to correlate ABO genotypes with phenotypes

in blood samples in a Gaza Strip population and to compare the frequency of ABO

genotypes with other populations

51 The Allele Frequencies of ABO Antigens in Gaza Strip

The frequency of the alleles in the total samples were 0250 for the I

A allele

0170 for the IB allele and 0580 for the I

O allele This distribution is in agreement with

the distribution with the samples reported in an Iranian study were they reported

023974 for IA allele 018147 for I

B allele and 057879 for I

O allele (73) The

frequencies of IA I

B and I

O alleles in our subjects are also comparable to those

reported in Iraqi and Jordanian studies where they found 0212 for IA allele 0177 for

IB allele and 06611 for I

O allele and 0270 for I

A allele 0130 for I

B allele and 060 for

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

29

one micro-tube (02 ml) were mixed The volume and concentration of a typical PCR

reaction are shown in Table 32

PCR was performed in a thermal cycler The cycling conditions were as

described below In each PCR MTHFR gene specific primers with the following

sequences (Forward 5prime-ACGATGGGGCAAGTGATGCCC -3 and reverse 5-

GAGAAGGTGTCTGCGGGATC-3) were used as a positive control that produces a 95

bp fragment Upon completion of PCR the products were analyzed by electrophoresis

on 2 ethidium bromide stained agarose gel or stored at 4degC until analysis

Table 33 Composition of PCR master mix

Reagent Composition

dNTPs 400microM each dATP dGTP dCTP dTTP

Taq DNA Polymerase 50 units ml

MgCl2 2mM

351 Temperature cycling program

The thermal cycler program was set as follows

Step 1 Denaturation for 3 minutes at 95degC

Step 2 35 cycles of

21 Melting for 40 seconds at 95degC

22 Annealing for 40 seconds at 585degC

23 Extension for 40 seconds at 72degC

Step3 Final elongation for 5 minutes at72degC

352 Expected PCR results

The PCR product size was estimated by comparing it with DNA molecular size

marker (50 bp ladder DNA ) run on the same gel

11

CHAPTER 4

RESULTS

13

CHAPTER 4

RESULTS

41 Study Population

The study population consisted of 201 subjects (50 males 151 females ) The

percentage of males was 249 while that of females was 751 ( figure 41)

0

10

20

30

40

50

60

70

80

maleFemale

Figure 41 Distribution of the study subjects according to gender

42 Phenotypic Frequency of ABO Blood Groups

Blood grouping was done by antigen antibody agglutination test by using

commercial monoclonal antisera The distribution of phenotypes in the total sample

were 363 (73) 224 (45) 75 (15) 338 (68) for groups A B AB and O

respectively Group A was the dominant one in both genders and AB was the rarest in

both males and females ( table 41)

Table 41 Phenotypic frequencies of various blood groups in the study population

Subject Sex Phenotype

Total A B AB O

Male 16 10 4 20 50

Female 57 35 11 48 151

Total 73 45 15 68 201

753

249

12

43 The Allele Frequencies of ABO Antigens

Allele frequency for the antigens was computed by the Hardy-Weinberg law on

the basis of the number of subjects with blood groups ABO The distribution of the

alleles in the total samples was 025 017 058 for IA I

B and I

O respectively

All genotyping results were compatible with the determined phenotypes by

serological method The observed genotypes as compared with the expected genotypes

are shown in Table 42 The frequencies of the five alleles in the our sample population

were O1 and O2 alleles 0376 and 0221 respectively while A1 0174 A2 0067

B 0162

Table 42 Observed and expected genotypes for the 201 samples

P-Value Expected Observed

Genotype Phenotype Percent Number Percent Number

0230 63 126 45 9 AA A

04762 29 583 318 64 AO

07205 29 58 25 5 BB B

09496 197 396 199 40 BO

05915 85 1708 75 15 AB AB

09613 336 676 338 68 OO O

100 201 100 201 Total

The statistical analysis ( using chi square test ) indicated that molecular data

were in good agreement with the ratio calculated from the estimated gene frequencies of

the ABO blood group system in the Gaza Strip population

11

44 PCR Results

441 Quality of the isolated DNA

Regarding the method of DNA isolation that was described in chapter 3 the

quality and quantity of DNA were suitable for PCR processing The quality of the

isolated DNA from human subjects is represented in the following figure (42)

Figure 42 A representative photograph of DNA isolated from human blood The samples were

run on 1 agarose gel stained with ethidium bromide

442 Blood group genotyping by Allele Specific PCR

Multiplex Allele Specific PCR (ASPCR) assay contains four independent PCR

reactions (Table 31) In the first reaction 261G-int6 primer pair was used to amplify

the 205 bp fragment to detect the A101 A102 B101 cis-ABO1 alleles and the 467C-

803G primer pair was selected to amplify the 381 bp fragment to detect the A101 O01

and O02 alleles The 297A-int6 primer pair and the 467C-803C primer pair in the

second reaction were selected to amplify the 381 bp fragment to detect the B101 allele

In the third reaction the 261A-int6 primer pair was selected to amplify the 205 bp

product to detect the O01 and O02 alleles and the 467T-803G primer pair was selected

to amplify the 381 bp product to detect the A102 allele Finally in the fourth reaction

the 297G-int6 primer pair were applied to produce the 164 bp fragment to detect the

B101 and O02 alleles and the 467T-803C primer pair to produce the 381 bp fragment

to recognize the cis-ABO1 allele Table 43 show the products of PCR reactions

according to the genotype

14

Table 43 PCR products according to the genotype

PCR Reactions Genotype

R1 R2 R3 R4

381 164 205 _ O1O1

381 _ 205 164 O2O2

381 164 205 164 O1O2

205 381 _ 164 B1B1

381205 381164 205 164 B1O1

381205 381 205 164 B1O2

381205 381164 _ 164 A1B1

205 381164 381 164 A2B1

381205 164 _ _ A1A1

381205 164 205 _ A1O1

381205 164 205 164 A1O2

381205 164 381 _ A1A2

205 164 381 _ A2A2

381205 164 381205 _ A2O1

381205 164 381205 164 A2O2

Random mating with the six different alleles at the ABO locus can result in 21

different genotype combinations Only 15 different genotype combinations were

detected from the 201 investigated samples (Figure 43)

A- 1 O1O1 2 O2O2

15

B- 1 B1B1 2 O1O2

C- 1 B1O1

2 A1B

D- 1 A1A2 2 A2O2

E- 1 A1O1 2 A1A1

16

F- 1 A1O2 2 B1O2 3 A2A2

G- 1 A2O1

2 A2B1

Figure 43 The electrophoresis pattern of recognized genotypes using the multiplex ASPCR

method This figure shows 15 different genotypes lane M indicates the 50 bp ladder The R1-

R4 show the PCR reaction number

443 Genotype Frequencies

The frequency of ABO recognized genotypes belonging to the population

residing in Gaza Strip are shown in table 44 As shown in the table the highest allele

frequencies in A B AB and O phenotypes were A1O2 (124) B1O1 (129) A1B

(55) and O1O1 (154) respectively

17

Table 44 The frequency of recognized genotypes using multiplex ASPCR method for a

population residing in Gaza Strip

Genotype Frequency Percent

A1A1 3 15

A1O1 24 119

A1O2 25 124

A1A2 4 20

A2A2 2 10

A2O1 13 65

A2O2 2 10

B1B1 5 25

B1O1 26 129

B1O2 14 70

A1B 11 55

A2B 4 20

O1O1 31 154

O1O2 26 129

O2O2 11 55

Total 201 1000

18

CHAPTER 5

DISCUSSION

19

CHAPTER 5

DISCUSSION

Since the first delineation of the molecular basis of the ABO blood group by

Yamamoto et al (11 79) it has become possible to determine the ABO genotypes using

molecular methods without the need for family investigations ABO genotyping is

commonly used in cases of an ABO discrepancy between cell typing and serum typing

as well as in forensic practices for personal identification and paternity testing (72) The

ABH antigens are ubiquitously expressed in humans and are present primarily as

glycoproteins and partly as glycolipids The ABO locus is found on the long arm

of chromosome 9 (9q341-q342)

Multiplex allele specific PCR (the technique we used here) has advantages over

PCR-RFLP in terms of cost effectiveness reaction time and simplicity of handling It is

a rapid method that is based on detecting four single nucleotide polymorphisms at

nucleotides 261 297 796 and 803 of the ABO locus which in turn discriminate the

major ABO alleles

In this study the ABO genotypes of 201 samples recruited from the Islamic

University ndashGenetics Diagnosis Laboratory were determined using multiplex AS-PCR

method The objectives of this study were to correlate ABO genotypes with phenotypes

in blood samples in a Gaza Strip population and to compare the frequency of ABO

genotypes with other populations

51 The Allele Frequencies of ABO Antigens in Gaza Strip

The frequency of the alleles in the total samples were 0250 for the I

A allele

0170 for the IB allele and 0580 for the I

O allele This distribution is in agreement with

the distribution with the samples reported in an Iranian study were they reported

023974 for IA allele 018147 for I

B allele and 057879 for I

O allele (73) The

frequencies of IA I

B and I

O alleles in our subjects are also comparable to those

reported in Iraqi and Jordanian studies where they found 0212 for IA allele 0177 for

IB allele and 06611 for I

O allele and 0270 for I

A allele 0130 for I

B allele and 060 for

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

11

CHAPTER 4

RESULTS

13

CHAPTER 4

RESULTS

41 Study Population

The study population consisted of 201 subjects (50 males 151 females ) The

percentage of males was 249 while that of females was 751 ( figure 41)

0

10

20

30

40

50

60

70

80

maleFemale

Figure 41 Distribution of the study subjects according to gender

42 Phenotypic Frequency of ABO Blood Groups

Blood grouping was done by antigen antibody agglutination test by using

commercial monoclonal antisera The distribution of phenotypes in the total sample

were 363 (73) 224 (45) 75 (15) 338 (68) for groups A B AB and O

respectively Group A was the dominant one in both genders and AB was the rarest in

both males and females ( table 41)

Table 41 Phenotypic frequencies of various blood groups in the study population

Subject Sex Phenotype

Total A B AB O

Male 16 10 4 20 50

Female 57 35 11 48 151

Total 73 45 15 68 201

753

249

12

43 The Allele Frequencies of ABO Antigens

Allele frequency for the antigens was computed by the Hardy-Weinberg law on

the basis of the number of subjects with blood groups ABO The distribution of the

alleles in the total samples was 025 017 058 for IA I

B and I

O respectively

All genotyping results were compatible with the determined phenotypes by

serological method The observed genotypes as compared with the expected genotypes

are shown in Table 42 The frequencies of the five alleles in the our sample population

were O1 and O2 alleles 0376 and 0221 respectively while A1 0174 A2 0067

B 0162

Table 42 Observed and expected genotypes for the 201 samples

P-Value Expected Observed

Genotype Phenotype Percent Number Percent Number

0230 63 126 45 9 AA A

04762 29 583 318 64 AO

07205 29 58 25 5 BB B

09496 197 396 199 40 BO

05915 85 1708 75 15 AB AB

09613 336 676 338 68 OO O

100 201 100 201 Total

The statistical analysis ( using chi square test ) indicated that molecular data

were in good agreement with the ratio calculated from the estimated gene frequencies of

the ABO blood group system in the Gaza Strip population

11

44 PCR Results

441 Quality of the isolated DNA

Regarding the method of DNA isolation that was described in chapter 3 the

quality and quantity of DNA were suitable for PCR processing The quality of the

isolated DNA from human subjects is represented in the following figure (42)

Figure 42 A representative photograph of DNA isolated from human blood The samples were

run on 1 agarose gel stained with ethidium bromide

442 Blood group genotyping by Allele Specific PCR

Multiplex Allele Specific PCR (ASPCR) assay contains four independent PCR

reactions (Table 31) In the first reaction 261G-int6 primer pair was used to amplify

the 205 bp fragment to detect the A101 A102 B101 cis-ABO1 alleles and the 467C-

803G primer pair was selected to amplify the 381 bp fragment to detect the A101 O01

and O02 alleles The 297A-int6 primer pair and the 467C-803C primer pair in the

second reaction were selected to amplify the 381 bp fragment to detect the B101 allele

In the third reaction the 261A-int6 primer pair was selected to amplify the 205 bp

product to detect the O01 and O02 alleles and the 467T-803G primer pair was selected

to amplify the 381 bp product to detect the A102 allele Finally in the fourth reaction

the 297G-int6 primer pair were applied to produce the 164 bp fragment to detect the

B101 and O02 alleles and the 467T-803C primer pair to produce the 381 bp fragment

to recognize the cis-ABO1 allele Table 43 show the products of PCR reactions

according to the genotype

14

Table 43 PCR products according to the genotype

PCR Reactions Genotype

R1 R2 R3 R4

381 164 205 _ O1O1

381 _ 205 164 O2O2

381 164 205 164 O1O2

205 381 _ 164 B1B1

381205 381164 205 164 B1O1

381205 381 205 164 B1O2

381205 381164 _ 164 A1B1

205 381164 381 164 A2B1

381205 164 _ _ A1A1

381205 164 205 _ A1O1

381205 164 205 164 A1O2

381205 164 381 _ A1A2

205 164 381 _ A2A2

381205 164 381205 _ A2O1

381205 164 381205 164 A2O2

Random mating with the six different alleles at the ABO locus can result in 21

different genotype combinations Only 15 different genotype combinations were

detected from the 201 investigated samples (Figure 43)

A- 1 O1O1 2 O2O2

15

B- 1 B1B1 2 O1O2

C- 1 B1O1

2 A1B

D- 1 A1A2 2 A2O2

E- 1 A1O1 2 A1A1

16

F- 1 A1O2 2 B1O2 3 A2A2

G- 1 A2O1

2 A2B1

Figure 43 The electrophoresis pattern of recognized genotypes using the multiplex ASPCR

method This figure shows 15 different genotypes lane M indicates the 50 bp ladder The R1-

R4 show the PCR reaction number

443 Genotype Frequencies

The frequency of ABO recognized genotypes belonging to the population

residing in Gaza Strip are shown in table 44 As shown in the table the highest allele

frequencies in A B AB and O phenotypes were A1O2 (124) B1O1 (129) A1B

(55) and O1O1 (154) respectively

17

Table 44 The frequency of recognized genotypes using multiplex ASPCR method for a

population residing in Gaza Strip

Genotype Frequency Percent

A1A1 3 15

A1O1 24 119

A1O2 25 124

A1A2 4 20

A2A2 2 10

A2O1 13 65

A2O2 2 10

B1B1 5 25

B1O1 26 129

B1O2 14 70

A1B 11 55

A2B 4 20

O1O1 31 154

O1O2 26 129

O2O2 11 55

Total 201 1000

18

CHAPTER 5

DISCUSSION

19

CHAPTER 5

DISCUSSION

Since the first delineation of the molecular basis of the ABO blood group by

Yamamoto et al (11 79) it has become possible to determine the ABO genotypes using

molecular methods without the need for family investigations ABO genotyping is

commonly used in cases of an ABO discrepancy between cell typing and serum typing

as well as in forensic practices for personal identification and paternity testing (72) The

ABH antigens are ubiquitously expressed in humans and are present primarily as

glycoproteins and partly as glycolipids The ABO locus is found on the long arm

of chromosome 9 (9q341-q342)

Multiplex allele specific PCR (the technique we used here) has advantages over

PCR-RFLP in terms of cost effectiveness reaction time and simplicity of handling It is

a rapid method that is based on detecting four single nucleotide polymorphisms at

nucleotides 261 297 796 and 803 of the ABO locus which in turn discriminate the

major ABO alleles

In this study the ABO genotypes of 201 samples recruited from the Islamic

University ndashGenetics Diagnosis Laboratory were determined using multiplex AS-PCR

method The objectives of this study were to correlate ABO genotypes with phenotypes

in blood samples in a Gaza Strip population and to compare the frequency of ABO

genotypes with other populations

51 The Allele Frequencies of ABO Antigens in Gaza Strip

The frequency of the alleles in the total samples were 0250 for the I

A allele

0170 for the IB allele and 0580 for the I

O allele This distribution is in agreement with

the distribution with the samples reported in an Iranian study were they reported

023974 for IA allele 018147 for I

B allele and 057879 for I

O allele (73) The

frequencies of IA I

B and I

O alleles in our subjects are also comparable to those

reported in Iraqi and Jordanian studies where they found 0212 for IA allele 0177 for

IB allele and 06611 for I

O allele and 0270 for I

A allele 0130 for I

B allele and 060 for

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

13

CHAPTER 4

RESULTS

41 Study Population

The study population consisted of 201 subjects (50 males 151 females ) The

percentage of males was 249 while that of females was 751 ( figure 41)

0

10

20

30

40

50

60

70

80

maleFemale

Figure 41 Distribution of the study subjects according to gender

42 Phenotypic Frequency of ABO Blood Groups

Blood grouping was done by antigen antibody agglutination test by using

commercial monoclonal antisera The distribution of phenotypes in the total sample

were 363 (73) 224 (45) 75 (15) 338 (68) for groups A B AB and O

respectively Group A was the dominant one in both genders and AB was the rarest in

both males and females ( table 41)

Table 41 Phenotypic frequencies of various blood groups in the study population

Subject Sex Phenotype

Total A B AB O

Male 16 10 4 20 50

Female 57 35 11 48 151

Total 73 45 15 68 201

753

249

12

43 The Allele Frequencies of ABO Antigens

Allele frequency for the antigens was computed by the Hardy-Weinberg law on

the basis of the number of subjects with blood groups ABO The distribution of the

alleles in the total samples was 025 017 058 for IA I

B and I

O respectively

All genotyping results were compatible with the determined phenotypes by

serological method The observed genotypes as compared with the expected genotypes

are shown in Table 42 The frequencies of the five alleles in the our sample population

were O1 and O2 alleles 0376 and 0221 respectively while A1 0174 A2 0067

B 0162

Table 42 Observed and expected genotypes for the 201 samples

P-Value Expected Observed

Genotype Phenotype Percent Number Percent Number

0230 63 126 45 9 AA A

04762 29 583 318 64 AO

07205 29 58 25 5 BB B

09496 197 396 199 40 BO

05915 85 1708 75 15 AB AB

09613 336 676 338 68 OO O

100 201 100 201 Total

The statistical analysis ( using chi square test ) indicated that molecular data

were in good agreement with the ratio calculated from the estimated gene frequencies of

the ABO blood group system in the Gaza Strip population

11

44 PCR Results

441 Quality of the isolated DNA

Regarding the method of DNA isolation that was described in chapter 3 the

quality and quantity of DNA were suitable for PCR processing The quality of the

isolated DNA from human subjects is represented in the following figure (42)

Figure 42 A representative photograph of DNA isolated from human blood The samples were

run on 1 agarose gel stained with ethidium bromide

442 Blood group genotyping by Allele Specific PCR

Multiplex Allele Specific PCR (ASPCR) assay contains four independent PCR

reactions (Table 31) In the first reaction 261G-int6 primer pair was used to amplify

the 205 bp fragment to detect the A101 A102 B101 cis-ABO1 alleles and the 467C-

803G primer pair was selected to amplify the 381 bp fragment to detect the A101 O01

and O02 alleles The 297A-int6 primer pair and the 467C-803C primer pair in the

second reaction were selected to amplify the 381 bp fragment to detect the B101 allele

In the third reaction the 261A-int6 primer pair was selected to amplify the 205 bp

product to detect the O01 and O02 alleles and the 467T-803G primer pair was selected

to amplify the 381 bp product to detect the A102 allele Finally in the fourth reaction

the 297G-int6 primer pair were applied to produce the 164 bp fragment to detect the

B101 and O02 alleles and the 467T-803C primer pair to produce the 381 bp fragment

to recognize the cis-ABO1 allele Table 43 show the products of PCR reactions

according to the genotype

14

Table 43 PCR products according to the genotype

PCR Reactions Genotype

R1 R2 R3 R4

381 164 205 _ O1O1

381 _ 205 164 O2O2

381 164 205 164 O1O2

205 381 _ 164 B1B1

381205 381164 205 164 B1O1

381205 381 205 164 B1O2

381205 381164 _ 164 A1B1

205 381164 381 164 A2B1

381205 164 _ _ A1A1

381205 164 205 _ A1O1

381205 164 205 164 A1O2

381205 164 381 _ A1A2

205 164 381 _ A2A2

381205 164 381205 _ A2O1

381205 164 381205 164 A2O2

Random mating with the six different alleles at the ABO locus can result in 21

different genotype combinations Only 15 different genotype combinations were

detected from the 201 investigated samples (Figure 43)

A- 1 O1O1 2 O2O2

15

B- 1 B1B1 2 O1O2

C- 1 B1O1

2 A1B

D- 1 A1A2 2 A2O2

E- 1 A1O1 2 A1A1

16

F- 1 A1O2 2 B1O2 3 A2A2

G- 1 A2O1

2 A2B1

Figure 43 The electrophoresis pattern of recognized genotypes using the multiplex ASPCR

method This figure shows 15 different genotypes lane M indicates the 50 bp ladder The R1-

R4 show the PCR reaction number

443 Genotype Frequencies

The frequency of ABO recognized genotypes belonging to the population

residing in Gaza Strip are shown in table 44 As shown in the table the highest allele

frequencies in A B AB and O phenotypes were A1O2 (124) B1O1 (129) A1B

(55) and O1O1 (154) respectively

17

Table 44 The frequency of recognized genotypes using multiplex ASPCR method for a

population residing in Gaza Strip

Genotype Frequency Percent

A1A1 3 15

A1O1 24 119

A1O2 25 124

A1A2 4 20

A2A2 2 10

A2O1 13 65

A2O2 2 10

B1B1 5 25

B1O1 26 129

B1O2 14 70

A1B 11 55

A2B 4 20

O1O1 31 154

O1O2 26 129

O2O2 11 55

Total 201 1000

18

CHAPTER 5

DISCUSSION

19

CHAPTER 5

DISCUSSION

Since the first delineation of the molecular basis of the ABO blood group by

Yamamoto et al (11 79) it has become possible to determine the ABO genotypes using

molecular methods without the need for family investigations ABO genotyping is

commonly used in cases of an ABO discrepancy between cell typing and serum typing

as well as in forensic practices for personal identification and paternity testing (72) The

ABH antigens are ubiquitously expressed in humans and are present primarily as

glycoproteins and partly as glycolipids The ABO locus is found on the long arm

of chromosome 9 (9q341-q342)

Multiplex allele specific PCR (the technique we used here) has advantages over

PCR-RFLP in terms of cost effectiveness reaction time and simplicity of handling It is

a rapid method that is based on detecting four single nucleotide polymorphisms at

nucleotides 261 297 796 and 803 of the ABO locus which in turn discriminate the

major ABO alleles

In this study the ABO genotypes of 201 samples recruited from the Islamic

University ndashGenetics Diagnosis Laboratory were determined using multiplex AS-PCR

method The objectives of this study were to correlate ABO genotypes with phenotypes

in blood samples in a Gaza Strip population and to compare the frequency of ABO

genotypes with other populations

51 The Allele Frequencies of ABO Antigens in Gaza Strip

The frequency of the alleles in the total samples were 0250 for the I

A allele

0170 for the IB allele and 0580 for the I

O allele This distribution is in agreement with

the distribution with the samples reported in an Iranian study were they reported

023974 for IA allele 018147 for I

B allele and 057879 for I

O allele (73) The

frequencies of IA I

B and I

O alleles in our subjects are also comparable to those

reported in Iraqi and Jordanian studies where they found 0212 for IA allele 0177 for

IB allele and 06611 for I

O allele and 0270 for I

A allele 0130 for I

B allele and 060 for

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

12

43 The Allele Frequencies of ABO Antigens

Allele frequency for the antigens was computed by the Hardy-Weinberg law on

the basis of the number of subjects with blood groups ABO The distribution of the

alleles in the total samples was 025 017 058 for IA I

B and I

O respectively

All genotyping results were compatible with the determined phenotypes by

serological method The observed genotypes as compared with the expected genotypes

are shown in Table 42 The frequencies of the five alleles in the our sample population

were O1 and O2 alleles 0376 and 0221 respectively while A1 0174 A2 0067

B 0162

Table 42 Observed and expected genotypes for the 201 samples

P-Value Expected Observed

Genotype Phenotype Percent Number Percent Number

0230 63 126 45 9 AA A

04762 29 583 318 64 AO

07205 29 58 25 5 BB B

09496 197 396 199 40 BO

05915 85 1708 75 15 AB AB

09613 336 676 338 68 OO O

100 201 100 201 Total

The statistical analysis ( using chi square test ) indicated that molecular data

were in good agreement with the ratio calculated from the estimated gene frequencies of

the ABO blood group system in the Gaza Strip population

11

44 PCR Results

441 Quality of the isolated DNA

Regarding the method of DNA isolation that was described in chapter 3 the

quality and quantity of DNA were suitable for PCR processing The quality of the

isolated DNA from human subjects is represented in the following figure (42)

Figure 42 A representative photograph of DNA isolated from human blood The samples were

run on 1 agarose gel stained with ethidium bromide

442 Blood group genotyping by Allele Specific PCR

Multiplex Allele Specific PCR (ASPCR) assay contains four independent PCR

reactions (Table 31) In the first reaction 261G-int6 primer pair was used to amplify

the 205 bp fragment to detect the A101 A102 B101 cis-ABO1 alleles and the 467C-

803G primer pair was selected to amplify the 381 bp fragment to detect the A101 O01

and O02 alleles The 297A-int6 primer pair and the 467C-803C primer pair in the

second reaction were selected to amplify the 381 bp fragment to detect the B101 allele

In the third reaction the 261A-int6 primer pair was selected to amplify the 205 bp

product to detect the O01 and O02 alleles and the 467T-803G primer pair was selected

to amplify the 381 bp product to detect the A102 allele Finally in the fourth reaction

the 297G-int6 primer pair were applied to produce the 164 bp fragment to detect the

B101 and O02 alleles and the 467T-803C primer pair to produce the 381 bp fragment

to recognize the cis-ABO1 allele Table 43 show the products of PCR reactions

according to the genotype

14

Table 43 PCR products according to the genotype

PCR Reactions Genotype

R1 R2 R3 R4

381 164 205 _ O1O1

381 _ 205 164 O2O2

381 164 205 164 O1O2

205 381 _ 164 B1B1

381205 381164 205 164 B1O1

381205 381 205 164 B1O2

381205 381164 _ 164 A1B1

205 381164 381 164 A2B1

381205 164 _ _ A1A1

381205 164 205 _ A1O1

381205 164 205 164 A1O2

381205 164 381 _ A1A2

205 164 381 _ A2A2

381205 164 381205 _ A2O1

381205 164 381205 164 A2O2

Random mating with the six different alleles at the ABO locus can result in 21

different genotype combinations Only 15 different genotype combinations were

detected from the 201 investigated samples (Figure 43)

A- 1 O1O1 2 O2O2

15

B- 1 B1B1 2 O1O2

C- 1 B1O1

2 A1B

D- 1 A1A2 2 A2O2

E- 1 A1O1 2 A1A1

16

F- 1 A1O2 2 B1O2 3 A2A2

G- 1 A2O1

2 A2B1

Figure 43 The electrophoresis pattern of recognized genotypes using the multiplex ASPCR

method This figure shows 15 different genotypes lane M indicates the 50 bp ladder The R1-

R4 show the PCR reaction number

443 Genotype Frequencies

The frequency of ABO recognized genotypes belonging to the population

residing in Gaza Strip are shown in table 44 As shown in the table the highest allele

frequencies in A B AB and O phenotypes were A1O2 (124) B1O1 (129) A1B

(55) and O1O1 (154) respectively

17

Table 44 The frequency of recognized genotypes using multiplex ASPCR method for a

population residing in Gaza Strip

Genotype Frequency Percent

A1A1 3 15

A1O1 24 119

A1O2 25 124

A1A2 4 20

A2A2 2 10

A2O1 13 65

A2O2 2 10

B1B1 5 25

B1O1 26 129

B1O2 14 70

A1B 11 55

A2B 4 20

O1O1 31 154

O1O2 26 129

O2O2 11 55

Total 201 1000

18

CHAPTER 5

DISCUSSION

19

CHAPTER 5

DISCUSSION

Since the first delineation of the molecular basis of the ABO blood group by

Yamamoto et al (11 79) it has become possible to determine the ABO genotypes using

molecular methods without the need for family investigations ABO genotyping is

commonly used in cases of an ABO discrepancy between cell typing and serum typing

as well as in forensic practices for personal identification and paternity testing (72) The

ABH antigens are ubiquitously expressed in humans and are present primarily as

glycoproteins and partly as glycolipids The ABO locus is found on the long arm

of chromosome 9 (9q341-q342)

Multiplex allele specific PCR (the technique we used here) has advantages over

PCR-RFLP in terms of cost effectiveness reaction time and simplicity of handling It is

a rapid method that is based on detecting four single nucleotide polymorphisms at

nucleotides 261 297 796 and 803 of the ABO locus which in turn discriminate the

major ABO alleles

In this study the ABO genotypes of 201 samples recruited from the Islamic

University ndashGenetics Diagnosis Laboratory were determined using multiplex AS-PCR

method The objectives of this study were to correlate ABO genotypes with phenotypes

in blood samples in a Gaza Strip population and to compare the frequency of ABO

genotypes with other populations

51 The Allele Frequencies of ABO Antigens in Gaza Strip

The frequency of the alleles in the total samples were 0250 for the I

A allele

0170 for the IB allele and 0580 for the I

O allele This distribution is in agreement with

the distribution with the samples reported in an Iranian study were they reported

023974 for IA allele 018147 for I

B allele and 057879 for I

O allele (73) The

frequencies of IA I

B and I

O alleles in our subjects are also comparable to those

reported in Iraqi and Jordanian studies where they found 0212 for IA allele 0177 for

IB allele and 06611 for I

O allele and 0270 for I

A allele 0130 for I

B allele and 060 for

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

11

44 PCR Results

441 Quality of the isolated DNA

Regarding the method of DNA isolation that was described in chapter 3 the

quality and quantity of DNA were suitable for PCR processing The quality of the

isolated DNA from human subjects is represented in the following figure (42)

Figure 42 A representative photograph of DNA isolated from human blood The samples were

run on 1 agarose gel stained with ethidium bromide

442 Blood group genotyping by Allele Specific PCR

Multiplex Allele Specific PCR (ASPCR) assay contains four independent PCR

reactions (Table 31) In the first reaction 261G-int6 primer pair was used to amplify

the 205 bp fragment to detect the A101 A102 B101 cis-ABO1 alleles and the 467C-

803G primer pair was selected to amplify the 381 bp fragment to detect the A101 O01

and O02 alleles The 297A-int6 primer pair and the 467C-803C primer pair in the

second reaction were selected to amplify the 381 bp fragment to detect the B101 allele

In the third reaction the 261A-int6 primer pair was selected to amplify the 205 bp

product to detect the O01 and O02 alleles and the 467T-803G primer pair was selected

to amplify the 381 bp product to detect the A102 allele Finally in the fourth reaction

the 297G-int6 primer pair were applied to produce the 164 bp fragment to detect the

B101 and O02 alleles and the 467T-803C primer pair to produce the 381 bp fragment

to recognize the cis-ABO1 allele Table 43 show the products of PCR reactions

according to the genotype

14

Table 43 PCR products according to the genotype

PCR Reactions Genotype

R1 R2 R3 R4

381 164 205 _ O1O1

381 _ 205 164 O2O2

381 164 205 164 O1O2

205 381 _ 164 B1B1

381205 381164 205 164 B1O1

381205 381 205 164 B1O2

381205 381164 _ 164 A1B1

205 381164 381 164 A2B1

381205 164 _ _ A1A1

381205 164 205 _ A1O1

381205 164 205 164 A1O2

381205 164 381 _ A1A2

205 164 381 _ A2A2

381205 164 381205 _ A2O1

381205 164 381205 164 A2O2

Random mating with the six different alleles at the ABO locus can result in 21

different genotype combinations Only 15 different genotype combinations were

detected from the 201 investigated samples (Figure 43)

A- 1 O1O1 2 O2O2

15

B- 1 B1B1 2 O1O2

C- 1 B1O1

2 A1B

D- 1 A1A2 2 A2O2

E- 1 A1O1 2 A1A1

16

F- 1 A1O2 2 B1O2 3 A2A2

G- 1 A2O1

2 A2B1

Figure 43 The electrophoresis pattern of recognized genotypes using the multiplex ASPCR

method This figure shows 15 different genotypes lane M indicates the 50 bp ladder The R1-

R4 show the PCR reaction number

443 Genotype Frequencies

The frequency of ABO recognized genotypes belonging to the population

residing in Gaza Strip are shown in table 44 As shown in the table the highest allele

frequencies in A B AB and O phenotypes were A1O2 (124) B1O1 (129) A1B

(55) and O1O1 (154) respectively

17

Table 44 The frequency of recognized genotypes using multiplex ASPCR method for a

population residing in Gaza Strip

Genotype Frequency Percent

A1A1 3 15

A1O1 24 119

A1O2 25 124

A1A2 4 20

A2A2 2 10

A2O1 13 65

A2O2 2 10

B1B1 5 25

B1O1 26 129

B1O2 14 70

A1B 11 55

A2B 4 20

O1O1 31 154

O1O2 26 129

O2O2 11 55

Total 201 1000

18

CHAPTER 5

DISCUSSION

19

CHAPTER 5

DISCUSSION

Since the first delineation of the molecular basis of the ABO blood group by

Yamamoto et al (11 79) it has become possible to determine the ABO genotypes using

molecular methods without the need for family investigations ABO genotyping is

commonly used in cases of an ABO discrepancy between cell typing and serum typing

as well as in forensic practices for personal identification and paternity testing (72) The

ABH antigens are ubiquitously expressed in humans and are present primarily as

glycoproteins and partly as glycolipids The ABO locus is found on the long arm

of chromosome 9 (9q341-q342)

Multiplex allele specific PCR (the technique we used here) has advantages over

PCR-RFLP in terms of cost effectiveness reaction time and simplicity of handling It is

a rapid method that is based on detecting four single nucleotide polymorphisms at

nucleotides 261 297 796 and 803 of the ABO locus which in turn discriminate the

major ABO alleles

In this study the ABO genotypes of 201 samples recruited from the Islamic

University ndashGenetics Diagnosis Laboratory were determined using multiplex AS-PCR

method The objectives of this study were to correlate ABO genotypes with phenotypes

in blood samples in a Gaza Strip population and to compare the frequency of ABO

genotypes with other populations

51 The Allele Frequencies of ABO Antigens in Gaza Strip

The frequency of the alleles in the total samples were 0250 for the I

A allele

0170 for the IB allele and 0580 for the I

O allele This distribution is in agreement with

the distribution with the samples reported in an Iranian study were they reported

023974 for IA allele 018147 for I

B allele and 057879 for I

O allele (73) The

frequencies of IA I

B and I

O alleles in our subjects are also comparable to those

reported in Iraqi and Jordanian studies where they found 0212 for IA allele 0177 for

IB allele and 06611 for I

O allele and 0270 for I

A allele 0130 for I

B allele and 060 for

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

14

Table 43 PCR products according to the genotype

PCR Reactions Genotype

R1 R2 R3 R4

381 164 205 _ O1O1

381 _ 205 164 O2O2

381 164 205 164 O1O2

205 381 _ 164 B1B1

381205 381164 205 164 B1O1

381205 381 205 164 B1O2

381205 381164 _ 164 A1B1

205 381164 381 164 A2B1

381205 164 _ _ A1A1

381205 164 205 _ A1O1

381205 164 205 164 A1O2

381205 164 381 _ A1A2

205 164 381 _ A2A2

381205 164 381205 _ A2O1

381205 164 381205 164 A2O2

Random mating with the six different alleles at the ABO locus can result in 21

different genotype combinations Only 15 different genotype combinations were

detected from the 201 investigated samples (Figure 43)

A- 1 O1O1 2 O2O2

15

B- 1 B1B1 2 O1O2

C- 1 B1O1

2 A1B

D- 1 A1A2 2 A2O2

E- 1 A1O1 2 A1A1

16

F- 1 A1O2 2 B1O2 3 A2A2

G- 1 A2O1

2 A2B1

Figure 43 The electrophoresis pattern of recognized genotypes using the multiplex ASPCR

method This figure shows 15 different genotypes lane M indicates the 50 bp ladder The R1-

R4 show the PCR reaction number

443 Genotype Frequencies

The frequency of ABO recognized genotypes belonging to the population

residing in Gaza Strip are shown in table 44 As shown in the table the highest allele

frequencies in A B AB and O phenotypes were A1O2 (124) B1O1 (129) A1B

(55) and O1O1 (154) respectively

17

Table 44 The frequency of recognized genotypes using multiplex ASPCR method for a

population residing in Gaza Strip

Genotype Frequency Percent

A1A1 3 15

A1O1 24 119

A1O2 25 124

A1A2 4 20

A2A2 2 10

A2O1 13 65

A2O2 2 10

B1B1 5 25

B1O1 26 129

B1O2 14 70

A1B 11 55

A2B 4 20

O1O1 31 154

O1O2 26 129

O2O2 11 55

Total 201 1000

18

CHAPTER 5

DISCUSSION

19

CHAPTER 5

DISCUSSION

Since the first delineation of the molecular basis of the ABO blood group by

Yamamoto et al (11 79) it has become possible to determine the ABO genotypes using

molecular methods without the need for family investigations ABO genotyping is

commonly used in cases of an ABO discrepancy between cell typing and serum typing

as well as in forensic practices for personal identification and paternity testing (72) The

ABH antigens are ubiquitously expressed in humans and are present primarily as

glycoproteins and partly as glycolipids The ABO locus is found on the long arm

of chromosome 9 (9q341-q342)

Multiplex allele specific PCR (the technique we used here) has advantages over

PCR-RFLP in terms of cost effectiveness reaction time and simplicity of handling It is

a rapid method that is based on detecting four single nucleotide polymorphisms at

nucleotides 261 297 796 and 803 of the ABO locus which in turn discriminate the

major ABO alleles

In this study the ABO genotypes of 201 samples recruited from the Islamic

University ndashGenetics Diagnosis Laboratory were determined using multiplex AS-PCR

method The objectives of this study were to correlate ABO genotypes with phenotypes

in blood samples in a Gaza Strip population and to compare the frequency of ABO

genotypes with other populations

51 The Allele Frequencies of ABO Antigens in Gaza Strip

The frequency of the alleles in the total samples were 0250 for the I

A allele

0170 for the IB allele and 0580 for the I

O allele This distribution is in agreement with

the distribution with the samples reported in an Iranian study were they reported

023974 for IA allele 018147 for I

B allele and 057879 for I

O allele (73) The

frequencies of IA I

B and I

O alleles in our subjects are also comparable to those

reported in Iraqi and Jordanian studies where they found 0212 for IA allele 0177 for

IB allele and 06611 for I

O allele and 0270 for I

A allele 0130 for I

B allele and 060 for

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

15

B- 1 B1B1 2 O1O2

C- 1 B1O1

2 A1B

D- 1 A1A2 2 A2O2

E- 1 A1O1 2 A1A1

16

F- 1 A1O2 2 B1O2 3 A2A2

G- 1 A2O1

2 A2B1

Figure 43 The electrophoresis pattern of recognized genotypes using the multiplex ASPCR

method This figure shows 15 different genotypes lane M indicates the 50 bp ladder The R1-

R4 show the PCR reaction number

443 Genotype Frequencies

The frequency of ABO recognized genotypes belonging to the population

residing in Gaza Strip are shown in table 44 As shown in the table the highest allele

frequencies in A B AB and O phenotypes were A1O2 (124) B1O1 (129) A1B

(55) and O1O1 (154) respectively

17

Table 44 The frequency of recognized genotypes using multiplex ASPCR method for a

population residing in Gaza Strip

Genotype Frequency Percent

A1A1 3 15

A1O1 24 119

A1O2 25 124

A1A2 4 20

A2A2 2 10

A2O1 13 65

A2O2 2 10

B1B1 5 25

B1O1 26 129

B1O2 14 70

A1B 11 55

A2B 4 20

O1O1 31 154

O1O2 26 129

O2O2 11 55

Total 201 1000

18

CHAPTER 5

DISCUSSION

19

CHAPTER 5

DISCUSSION

Since the first delineation of the molecular basis of the ABO blood group by

Yamamoto et al (11 79) it has become possible to determine the ABO genotypes using

molecular methods without the need for family investigations ABO genotyping is

commonly used in cases of an ABO discrepancy between cell typing and serum typing

as well as in forensic practices for personal identification and paternity testing (72) The

ABH antigens are ubiquitously expressed in humans and are present primarily as

glycoproteins and partly as glycolipids The ABO locus is found on the long arm

of chromosome 9 (9q341-q342)

Multiplex allele specific PCR (the technique we used here) has advantages over

PCR-RFLP in terms of cost effectiveness reaction time and simplicity of handling It is

a rapid method that is based on detecting four single nucleotide polymorphisms at

nucleotides 261 297 796 and 803 of the ABO locus which in turn discriminate the

major ABO alleles

In this study the ABO genotypes of 201 samples recruited from the Islamic

University ndashGenetics Diagnosis Laboratory were determined using multiplex AS-PCR

method The objectives of this study were to correlate ABO genotypes with phenotypes

in blood samples in a Gaza Strip population and to compare the frequency of ABO

genotypes with other populations

51 The Allele Frequencies of ABO Antigens in Gaza Strip

The frequency of the alleles in the total samples were 0250 for the I

A allele

0170 for the IB allele and 0580 for the I

O allele This distribution is in agreement with

the distribution with the samples reported in an Iranian study were they reported

023974 for IA allele 018147 for I

B allele and 057879 for I

O allele (73) The

frequencies of IA I

B and I

O alleles in our subjects are also comparable to those

reported in Iraqi and Jordanian studies where they found 0212 for IA allele 0177 for

IB allele and 06611 for I

O allele and 0270 for I

A allele 0130 for I

B allele and 060 for

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

16

F- 1 A1O2 2 B1O2 3 A2A2

G- 1 A2O1

2 A2B1

Figure 43 The electrophoresis pattern of recognized genotypes using the multiplex ASPCR

method This figure shows 15 different genotypes lane M indicates the 50 bp ladder The R1-

R4 show the PCR reaction number

443 Genotype Frequencies

The frequency of ABO recognized genotypes belonging to the population

residing in Gaza Strip are shown in table 44 As shown in the table the highest allele

frequencies in A B AB and O phenotypes were A1O2 (124) B1O1 (129) A1B

(55) and O1O1 (154) respectively

17

Table 44 The frequency of recognized genotypes using multiplex ASPCR method for a

population residing in Gaza Strip

Genotype Frequency Percent

A1A1 3 15

A1O1 24 119

A1O2 25 124

A1A2 4 20

A2A2 2 10

A2O1 13 65

A2O2 2 10

B1B1 5 25

B1O1 26 129

B1O2 14 70

A1B 11 55

A2B 4 20

O1O1 31 154

O1O2 26 129

O2O2 11 55

Total 201 1000

18

CHAPTER 5

DISCUSSION

19

CHAPTER 5

DISCUSSION

Since the first delineation of the molecular basis of the ABO blood group by

Yamamoto et al (11 79) it has become possible to determine the ABO genotypes using

molecular methods without the need for family investigations ABO genotyping is

commonly used in cases of an ABO discrepancy between cell typing and serum typing

as well as in forensic practices for personal identification and paternity testing (72) The

ABH antigens are ubiquitously expressed in humans and are present primarily as

glycoproteins and partly as glycolipids The ABO locus is found on the long arm

of chromosome 9 (9q341-q342)

Multiplex allele specific PCR (the technique we used here) has advantages over

PCR-RFLP in terms of cost effectiveness reaction time and simplicity of handling It is

a rapid method that is based on detecting four single nucleotide polymorphisms at

nucleotides 261 297 796 and 803 of the ABO locus which in turn discriminate the

major ABO alleles

In this study the ABO genotypes of 201 samples recruited from the Islamic

University ndashGenetics Diagnosis Laboratory were determined using multiplex AS-PCR

method The objectives of this study were to correlate ABO genotypes with phenotypes

in blood samples in a Gaza Strip population and to compare the frequency of ABO

genotypes with other populations

51 The Allele Frequencies of ABO Antigens in Gaza Strip

The frequency of the alleles in the total samples were 0250 for the I

A allele

0170 for the IB allele and 0580 for the I

O allele This distribution is in agreement with

the distribution with the samples reported in an Iranian study were they reported

023974 for IA allele 018147 for I

B allele and 057879 for I

O allele (73) The

frequencies of IA I

B and I

O alleles in our subjects are also comparable to those

reported in Iraqi and Jordanian studies where they found 0212 for IA allele 0177 for

IB allele and 06611 for I

O allele and 0270 for I

A allele 0130 for I

B allele and 060 for

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

17

Table 44 The frequency of recognized genotypes using multiplex ASPCR method for a

population residing in Gaza Strip

Genotype Frequency Percent

A1A1 3 15

A1O1 24 119

A1O2 25 124

A1A2 4 20

A2A2 2 10

A2O1 13 65

A2O2 2 10

B1B1 5 25

B1O1 26 129

B1O2 14 70

A1B 11 55

A2B 4 20

O1O1 31 154

O1O2 26 129

O2O2 11 55

Total 201 1000

18

CHAPTER 5

DISCUSSION

19

CHAPTER 5

DISCUSSION

Since the first delineation of the molecular basis of the ABO blood group by

Yamamoto et al (11 79) it has become possible to determine the ABO genotypes using

molecular methods without the need for family investigations ABO genotyping is

commonly used in cases of an ABO discrepancy between cell typing and serum typing

as well as in forensic practices for personal identification and paternity testing (72) The

ABH antigens are ubiquitously expressed in humans and are present primarily as

glycoproteins and partly as glycolipids The ABO locus is found on the long arm

of chromosome 9 (9q341-q342)

Multiplex allele specific PCR (the technique we used here) has advantages over

PCR-RFLP in terms of cost effectiveness reaction time and simplicity of handling It is

a rapid method that is based on detecting four single nucleotide polymorphisms at

nucleotides 261 297 796 and 803 of the ABO locus which in turn discriminate the

major ABO alleles

In this study the ABO genotypes of 201 samples recruited from the Islamic

University ndashGenetics Diagnosis Laboratory were determined using multiplex AS-PCR

method The objectives of this study were to correlate ABO genotypes with phenotypes

in blood samples in a Gaza Strip population and to compare the frequency of ABO

genotypes with other populations

51 The Allele Frequencies of ABO Antigens in Gaza Strip

The frequency of the alleles in the total samples were 0250 for the I

A allele

0170 for the IB allele and 0580 for the I

O allele This distribution is in agreement with

the distribution with the samples reported in an Iranian study were they reported

023974 for IA allele 018147 for I

B allele and 057879 for I

O allele (73) The

frequencies of IA I

B and I

O alleles in our subjects are also comparable to those

reported in Iraqi and Jordanian studies where they found 0212 for IA allele 0177 for

IB allele and 06611 for I

O allele and 0270 for I

A allele 0130 for I

B allele and 060 for

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

18

CHAPTER 5

DISCUSSION

19

CHAPTER 5

DISCUSSION

Since the first delineation of the molecular basis of the ABO blood group by

Yamamoto et al (11 79) it has become possible to determine the ABO genotypes using

molecular methods without the need for family investigations ABO genotyping is

commonly used in cases of an ABO discrepancy between cell typing and serum typing

as well as in forensic practices for personal identification and paternity testing (72) The

ABH antigens are ubiquitously expressed in humans and are present primarily as

glycoproteins and partly as glycolipids The ABO locus is found on the long arm

of chromosome 9 (9q341-q342)

Multiplex allele specific PCR (the technique we used here) has advantages over

PCR-RFLP in terms of cost effectiveness reaction time and simplicity of handling It is

a rapid method that is based on detecting four single nucleotide polymorphisms at

nucleotides 261 297 796 and 803 of the ABO locus which in turn discriminate the

major ABO alleles

In this study the ABO genotypes of 201 samples recruited from the Islamic

University ndashGenetics Diagnosis Laboratory were determined using multiplex AS-PCR

method The objectives of this study were to correlate ABO genotypes with phenotypes

in blood samples in a Gaza Strip population and to compare the frequency of ABO

genotypes with other populations

51 The Allele Frequencies of ABO Antigens in Gaza Strip

The frequency of the alleles in the total samples were 0250 for the I

A allele

0170 for the IB allele and 0580 for the I

O allele This distribution is in agreement with

the distribution with the samples reported in an Iranian study were they reported

023974 for IA allele 018147 for I

B allele and 057879 for I

O allele (73) The

frequencies of IA I

B and I

O alleles in our subjects are also comparable to those

reported in Iraqi and Jordanian studies where they found 0212 for IA allele 0177 for

IB allele and 06611 for I

O allele and 0270 for I

A allele 0130 for I

B allele and 060 for

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

19

CHAPTER 5

DISCUSSION

Since the first delineation of the molecular basis of the ABO blood group by

Yamamoto et al (11 79) it has become possible to determine the ABO genotypes using

molecular methods without the need for family investigations ABO genotyping is

commonly used in cases of an ABO discrepancy between cell typing and serum typing

as well as in forensic practices for personal identification and paternity testing (72) The

ABH antigens are ubiquitously expressed in humans and are present primarily as

glycoproteins and partly as glycolipids The ABO locus is found on the long arm

of chromosome 9 (9q341-q342)

Multiplex allele specific PCR (the technique we used here) has advantages over

PCR-RFLP in terms of cost effectiveness reaction time and simplicity of handling It is

a rapid method that is based on detecting four single nucleotide polymorphisms at

nucleotides 261 297 796 and 803 of the ABO locus which in turn discriminate the

major ABO alleles

In this study the ABO genotypes of 201 samples recruited from the Islamic

University ndashGenetics Diagnosis Laboratory were determined using multiplex AS-PCR

method The objectives of this study were to correlate ABO genotypes with phenotypes

in blood samples in a Gaza Strip population and to compare the frequency of ABO

genotypes with other populations

51 The Allele Frequencies of ABO Antigens in Gaza Strip

The frequency of the alleles in the total samples were 0250 for the I

A allele

0170 for the IB allele and 0580 for the I

O allele This distribution is in agreement with

the distribution with the samples reported in an Iranian study were they reported

023974 for IA allele 018147 for I

B allele and 057879 for I

O allele (73) The

frequencies of IA I

B and I

O alleles in our subjects are also comparable to those

reported in Iraqi and Jordanian studies where they found 0212 for IA allele 0177 for

IB allele and 06611 for I

O allele and 0270 for I

A allele 0130 for I

B allele and 060 for

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

41

IO allele respectively (8283) The results showed that the frequency of I

O allele is

higher than either IA or I

B and that of I

A is higher than I

B ie the trend is I

o ˃ I

A ˃ I

B

Our results showed that the distribution of ABO alleles in Gaza Strip is similar

to that reported for other Arabian and Iranian populations as shown in Table 44 which

represents the distribution of ABO alleles in different human populations This result

may imply that those populations share common ancestry

52 ABO Genotype Frequencies

The method employed here for blood group genotyping discriminates A1 A2 O1

O2 and B alleles All expected alleles and allelic combinations were observed in this

group except cis-ABO1 allele and its pertinent genotypes

The results obtained from AS-PCR for the investigated 201 samples showed that

the frequencies of the various ABO genotypes were AA 9 (448) AO 64 (3184)

BB 5 (249) BO 40 (199) AB 15 (746) and OO68 (3383) When compared

to other studies these percentages are close to those reported by Irshaid et al (2007) in

Jordan where they have reported the following frequencies AA571 AO33333

BB0953 BO13333 AB 952 and OO 37143 (83)

According to our results it was found that the highest frequency of A phenotype

(64738767) had the AO genotype while (9731233) were A phenotype

homozygotes (AA) Forty samples with phenotype B were recognized as heterozygous

(BO) and 5 samples with phenotype B recognized as homozygous (BB) On the other

hand (68201 338) were homozygous (OO) and 15 (746) samples were AB

heterozygotes The high heterozygosity observed in this study is mainly due to the high

frequency of the IO allele (058) in our population as compared to that of I

A (0250) and

IB (0170) alleles Additionally the samples investigated here were collected from

unrelated individuals and that allowed for the random assortment of the alleles

according to their frequencies in the population This is further confirmed by finding

that the observed genotypes did not deviate significantly from Hardy-Weinberg

equilibrium

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

43

Regarding the genotypes observed (Table 44) the highest frequency belonged

to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a

frequency of 154 (31201) and the lowest frequency belonged to A2A2 and A2O2

genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 10

(2201) each In addition 145 (7214) out of all the samples were heterozygous and 56

samples (2786) were homozygous

In our population the calculated frequencies of the O1 and O2 alleles were

03756 and 02214 respectively These values differ from those reported in other

populations eg in Kuwaiti population the reported frequencies were 06831 and

00155 for O1 and O2 respectively (80) Our findings are however consistent with

many other studies where they reported higher prevalence of the O1 allele (7380)

(Table 51)

Table 51 Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries

Reference Allele frequencies

Population r

O q

B p

A

Current study 0580 0170 0250 Gaza Strip

Mokhtar and Yunus 2008 (80) 06986 01676 01338 Kuwait

Al-Aarrayed et al 2001 (81) 0704 0157 0141 Bahrain

Tills et al 1983 (82) 06611 0177 0212 Iraq

Irashaid et al 2002 (83) 060 0130 0270 Jordan

Bashwar et al 2001 (84) 0714 01197 01663 Saudi Arabia

Khalil et al 1989 (85) 0663 0149 0188 Egypt

Khalil et al 1989 (85) 0668 0140 0192 Sudan

Nojavan et al 2012 (73) 057879 018147 023974 Iran

Meanwhile the calculated frequencies of A1 A2 and B alleles were A1

017413 A2 006716 B 016171 where these are also different in frequency from

those of the Kuwaiti study

cis-ABO1 allele was not observed in this study It is a rare allele which was

reported in Korea (00354) among blood donors (86) This allele also was not detected

in Kuwaiti or Iranian populations

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

42

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

41

CHAPTER 6

CONCLUSION and RECOMMENDATIONS

The present study focused on detection of the major ABO genotypes in a Palestinian

population residing in Gaza Strip The results of this study can be summarized as

follows

In Gaza Strip the A phenotype was the most common blood group followed by

OAB and B

The frequencies of ABO alleles in the investigated subjects were 025 for IA

017 for IB and 058 for I

O These frequencies are comparable to those obtained

from ABO genotyping

No statistically significant differences were found between the frequency of

observed and expected genotypes This proved that the ABO genotypes of the

randomly collected samples were in Hardy- Weinberg equilibrium data

Molecular data indicated that Hardy-Weinberg equation can be used to detect

the percentage of the major blood group in our population

The distribution of the ABO genotypes in Gaza Strip population is similar to that

of many Asian populations

There is no significant difference between male and female in terms of ABO

phenotypes

Our study indicated that the most common genotype is O1O1 and the lowest are

A2A2 and A2O2 The cis ABO1 was not encountered

The homozygous genotype of A and B alleles (AA BB ) were less than the

heterozygous(AO BO) genotype

The frequency of O2 allele in Gaza strip seems to be higher than that reported in

many neighboring countries

This study determined the exact phenotypic frequency of ABO blood groups in Gaza

Strip and the frequencies of the prevalent ABO alleles namely A1 A2 B1 O1 and O2

When both serological typing and ABO genotyping are performed and full

compatibility between a phenotype and a genotype is observed examiners can

determine the ABO phenotype with an even higher level of confidence Therefore we

recommend that the health care system in Palestine adopt ABO genotyping particularly

for cases of discrepant blood phenotypes

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

44

CHAPTER 7

REFERENCES

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

45

CHAPTER 7

REFERENCES

1 Hosseini-Maaf B and Pahlsson P Genetic Characterization of Human ABO Blood

Group Variants with a Focus on Subgroups and Hybrid Alleles ISBN 978-912007

2Watkins WM Biochemistry and genetics of the ABO Lewis and P blood group

systems In Harris H Hirschhorn K eds Advances in Human Genetics Vol 10 New

York Plenum Press1ndash1361981

3Larsen RD Ernst LK Nair RP Lowe JB Molecular cloning sequence and

expression of a human GDP-L-fucose β -D-galactoside 2- alpha-L-fucosyltransferase

cDNA that can form the H blood group antigen Proc Natl Acad Sci USA 87(17)

6674-66781990

4Yamamoto F Hakomori S Sugar-nucleotide donor specificity of histo-blood group A

and B transferases is based on amino acid substitution J Biol Chem 265 (31) 19257-

192621990

5 Bennett EP Steffensen R Clausen H weghuis DO Geurts van kessel A Genomic

cloning of the human histo-blood group and locus Biochem Biophys Res Commun 206

1 318-3251995

6 Watkins WM The glycosyltransferase products of the A B H and Le genes and their

relationship to the structure of the blood group antigens In Mohn JF Plunkett RW

Cunningham RK Lambert RM eds Human blood groups Basel S Karger134-42

1977

7 Hakomori SI Blood group ABH and Ii antigens of human erythrocytes chemistry

polymorphism and their developmental change Sernin Hematol1839-471981

8 Beattie KM Discrepancies in ABO grouping In A seminar on problems encountered

in pretranfusion tests Washington DC American Association of Blood Banks12965

1972

9 Yamamoto F McNeill PD Hakomori S Genomic organization of human histo

blood group ABO genes Glycobiology 51 51- 581995

10 Seltsam A Hallensleben M Kollmann A and Blasczyk R The nature of diversity

and diversification at the ABO locus Blood 102 3035-30422003

11 Yamamoto F Clausen H White T Marken J Hakamori S Molecular genetic basis

of the histoblood group system Nature 345229-2331990

12 Wade PA Gegonne A Jones PL Ballestar E Aubry F and Wolffe AP The Mi-2

histone deacetylase complex couples DNA methylation to chromatin remodeling and

histone deacetylation Nature Genetics 2362-661999

46

13 Lee HY Park MJ Kim NY Yang WI Shin KJ Rapid direct PCR for ABO blood

typing J Forensic Sci 56 Suppl 1S179-822011

14Storry JR Olsson ML Genetic basis of blood group diversityBr J Haematol

126759-7712004

15Jick H Slone D Westerholm B Inman WH Vessey MP Shapiro S Lewis GP

Worcester J Venous thromboembolic disease and ABO blood type A cooperative

study Lancet1539-5421969

16Clausen H Hakomori S ABH and related histo-blood group antigens

immunochemical differences in carrier isotypes and their distribution Vox Sang 561-

201989

17Schenkel-Brunner H In human blood groups Springer-Verlag New York2000

18Schiffman G Kabat E A and Thompson W Immunochemical studies on blood

groups XCX Cleavage of A B and H blood-group substances by alkali Biochemistry

3113-1201964

19Rege VP Painter TJ Watkins WM and Morgan W T J Isolation of serologically

active fucose-containing oligosaccharides from human blood-group H substance Nature

(London) 203360-3631964

20Kobata A Yamashita K amp Tachibana Y Oligosaccharides from human milk

Methods in Enzymology Vol 50 pp 216-220 ISSN 0076-68791978

21Lundblad A Oligosaccharides from human urine Methods in Enzymology Vol 50

pp 226-235 ISSN 0076-68791978

22Daniels G The molecular genetics of blood group polymorphism Transpl Immunol

14 143-532005

23Blumenfeld OO Patnaik SK Allelic genes of blood group antigens a source of

human mutations and cSNPs documented in the Blood Group Antigen Gene Mutation

Database Hum Mutat23 8-162004

24Yamamoto F McNeill P Hakomori S Human histo-blood group A2 transferase

coded by A2 allele one of the A subtypes is characterized by a single base deletion in

the coding sequence which results in an additional domain at the carboxyl terminal

Biochem Biophys Res Commun187366ndash741992

25Skacel PO Watkins WM Fucosyltransferase expression in human platelets and

leucocytes Glycocon J4267ndash721987

26 Eshleman JR Shakin-Eshleman SH Church A Kant JA Spitalnik SL DNA typing

of the human MN and Ss blood group antigens in amniotic fluid and following massive

transfusion Am J Clin Pathol103353ndash71995

27 Storry JR Reid ME Synthetic peptide inhibition of anti-STransfusion3655S1996

47

28 Lowe JB Red cell membrane antigens in G Stamatoyannopoulos Perlmutter

RM Majerus PW Varmus H The Molecular Basis of Blood Diseases 3rd ed

WB Saunders Orlando FL1999

29Rege VP Painter TJ Watkins WM Morgan WT Three New Trisaccharides

Obtained from Human Blood-Group a B H and Lea Substances Possible Sugar

Sequences in the Carbohydrate Chains Nature 200 532-41963

30Varki A Cummings R Esko J Freeze H Hart G and Marth J Essentials of

Glycobiology Cold Spring Harbor (NY) Cold Spring Harbor Laboratory Press ISBN-

10 0-87969-559-51999

31Jick H Slone D Westerholm B Inman WH Vessey MP Shapiro S Lewis GP

Worcester J Venous thromboembolic disease and ABO blood type A cooperative

study Lancet 1 539ndash 421969

32Taniguchi N Honke K and Fukuda M Handbook of Glycosyltransferase and

Related Genes SpringerTokyo 2002

33Henrissat B Glycosidase families Biochem Soc Trans 26153-1561998

34Schenkel-Brunner H Chester MA Watkins WM Alpha-L-fucosyltransferases in

human serum from donors of different ABO secretor and Lewis blood-group

phenotypes Eur J Biochem 30269-2771972

35Boix E Swaminathan GJ Zhang Y Natesh R Brew K Acharya KR Structure of

UDP complex of UDP galactose beta-galactoside-alpha -13-galactosyltransferase at

153-A resolution reveals a conformational change in the catalytically important C

terminus J Biol Chem 27648608-486142001

36 Breton C Snajdrova L Jeanneau C Koca J Imberty A Structures and mechanisms

of glycosyltransferases Glycobiology 1629R-37R2006

37Ramakrishnan B Boeggeman E Ramasamy V and Qasba PK Structure and

catalytic cycle of beta-14 galactosyltransferase Curr Opin Struct Biol14593

6002004

38 Barbolla L Mojena M Cienfuegos JA Escartiacuten P Presence of an inhibitor of

glycosyltransferase activity in a patient following an ABO incompatible liver transplant

Br J Haematol6993ndash61988

39 Barbolla L Mojena M Boscaacute L Presence of antibody to A- and B-transferases in

minor incompatible bone marrow transplants Br J Haematol 70471ndash61988

40 Rydberg L Samuelsson BE Presence of glycosyltransferase inhibitors in the sera of

patients with long-term surviving ABO incompatible (A2 to O) kidney grafts Transfus

Med 1177ndash821991

47

28 Lowe JB Red cell membrane antigens in G Stamatoyannopoulos Perlmutter

RM Majerus PW Varmus H The Molecular Basis of Blood Diseases 3rd ed

WB Saunders Orlando FL1999

29Rege VP Painter TJ Watkins WM Morgan WT Three New Trisaccharides

Obtained from Human Blood-Group a B H and Lea Substances Possible Sugar

Sequences in the Carbohydrate Chains Nature 200 532-41963

30Varki A Cummings R Esko J Freeze H Hart G and Marth J Essentials of

Glycobiology Cold Spring Harbor (NY) Cold Spring Harbor Laboratory Press ISBN-

10 0-87969-559-51999

31Jick H Slone D Westerholm B Inman WH Vessey MP Shapiro S Lewis GP

Worcester J Venous thromboembolic disease and ABO blood type A cooperative

study Lancet 1 539ndash 421969

32Taniguchi N Honke K and Fukuda M Handbook of Glycosyltransferase and

Related Genes SpringerTokyo 2002

33Henrissat B Glycosidase families Biochem Soc Trans 26153-1561998

34Schenkel-Brunner H Chester MA Watkins WM Alpha-L-fucosyltransferases in

human serum from donors of different ABO secretor and Lewis blood-group

phenotypes Eur J Biochem 30269-2771972

35Boix E Swaminathan GJ Zhang Y Natesh R Brew K Acharya KR Structure of

UDP complex of UDP galactose beta-galactoside-alpha -13-galactosyltransferase at

153-A resolution reveals a conformational change in the catalytically important C

terminus J Biol Chem 27648608-486142001

36 Breton C Snajdrova L Jeanneau C Koca J Imberty A Structures and mechanisms

of glycosyltransferases Glycobiology 1629R-37R2006

37Ramakrishnan B Boeggeman E Ramasamy V and Qasba PK Structure and

catalytic cycle of beta-14 galactosyltransferase Curr Opin Struct Biol14593

6002004

38 Barbolla L Mojena M Cienfuegos JA Escartiacuten P Presence of an inhibitor of

glycosyltransferase activity in a patient following an ABO incompatible liver transplant

Br J Haematol6993ndash61988

39 Barbolla L Mojena M Boscaacute L Presence of antibody to A- and B-transferases in

minor incompatible bone marrow transplants Br J Haematol 70471ndash61988

40 Rydberg L Samuelsson BE Presence of glycosyltransferase inhibitors in the sera of

patients with long-term surviving ABO incompatible (A2 to O) kidney grafts Transfus

Med 1177ndash821991

48

41Tirado I Mateo J Soria JM Oliver A Martinez-Sanchez E Vallve C Borrell M

Urrutia T Fontcuberta J The ABO blood group genotype and factor VIII levels as

independent risk factors for venous thromboembolism Thromb Haemost93468-474

2005

42 Schachter H Michaels MA Tilley CA Crookston MC Crookston JH Qualitative

differences in the N-acetyl-D-galactosaminyltransferases produced by human A1 and

A2 genes Proc Natl Acad Sci U S A70 220-41973

43Olsson ML Irshaid NM Hosseini-Maaf B Hellberg A Moulds MK Sareneva H

Chester MA Genomic analysis of clinical samples with serologic ABO blood grouping

discrepancies identification of 15 novel A and B subgroup alleles Blood 98 1585- 93

2001

44Ogasawara K Bannai M Saitou N Extensive polymorphism of ABO blood group

gene three major lineages of the alleles for the common ABO phenotypes Hum Genet

97777-7831996

45Ogasawara K Yabe R Uchikawa M Nakata K Watanabe J Takahashi Y

Recombination and gene conversion-like events may contribute to ABO gene diversity

causing various phenotypes Immunogenetics 53 190-1992001

46Ogasawara K Yabe R Uchikawa M Bannai M Nakata K Takenaka M Takahashi

Y Juji T Tokunaga K Different alleles cause an imbalance in A2 and A2B phenotypes

of the ABO blood group Vox Sang 74242- 2471998

47Rosendaal FR Risk factors for venous thrombotic disease Thromb Haemost

82610-6191999

48Lewis JD Meehan RR Henzel WJ Maurer-Fogy I Jeppesen P Klein F Bird A

Purification sequence and cellular localization of a novel chromosomal protein that

binds to methylated DNA Cell Jun 1269(6)905ndash9141992

49Mifsud NA Watt JM Condon JA A novel cis-AB variant allele arising from a

nucleotide substitution A796C in the B transferase gene Transfusion 401276-1277

2000

50Olsson ML Chester MA Frequent occurrence of a variant O1 gene at the blood

group ABO locus Vox Sang 7026-301996

51Ki K Iwata M Tsuji H Takagi T Tamura A Ishimoto G Ito S Matsui K Miyazaki

T A de novo ecombination in the ABO blood group gene and evidence for the

occurence of recombination products Hum Genet 99454-4611997

52Hosseini-Maaf B Hellberg A Rodrigues MJ ABO Exon and intron analysis in

individuals with the A weak B phenotype reveals a novel O1v-A2 hybrid allele that

causes four missense mutations in the A transferase BMC Genet 4172003

49

53Zago MA Tavella MH Simoes BP Racial heterogeneity of DNA polymorphisms

linked to the A and the O alleles of the ABO blood group gene Ann Hum Genet6067-

721996

54Olsson ML Santos SE Guerreiro JF Zago MA Chester MA Heterogeneity of the

O alleles at the blood group ABO locus in Amerindians Vox Sang7446-501998

55Yamamoto F McNeill PD Yamamoto M Hakomori S Bromilow IM Duguid JK

Molecular genetic analysis of the ABO blood group system 4 Another type of O allele

Vox Sang64175-81993

56 Schwyzer M and Hill R L J Biol Chem 252 2346ndash23551977

57Pusztai A and Morgan W T J Studies in immunochemistry 22 Amino acid

composition of human blood-group AB H and Le-a specific substances Biochem J

88546-5551963

58Yamamoto F McNeill PD Amino acid residue at codon 268 determines both

activity and nucleotide-sugar donor substrate specificity of human histo-blood group A

and B transferases in vitro mutagenesis study J Biol Chem 271 10515-201996

59Yamamoto F McNeill PD Yamamoto M Hakomori S Harris T Judd WJ

Davenport RD Molecular genetic analysis of the ABO blood group system 1 Weak

subgroups A3 and B3 alleles Vox Sang 64116- 1191993

60 Olsson ML Thuresson B Chester MA An Ael allele-specific nucleotide insertion

at the blood group ABO locus and its detection using a sequence-specific polymerase

chain reaction Biochem Biophys Res Commun 216642-6471995

61Ogasawara K Yabe R Uchikawa M Saitou N Bannai M Nakata K Takenaka M

Fujisawa K Ishikawa Y Juji T Tokunaga K Molecular genetic analysis of variant

phenotypes of the ABO blood group system Blood 882732-27371996

62Yamamoto F McNeill PD Kominato Y Yamamoto M Hakomori S Ishimoto S

Nishida S Shima M Fujimura Y Molecular genetic analysis of the ABO blood group

system 2 cis-AB alleles Vox Sang 64120-1231993

63Coutinho A Kazatchkine MD Avrameas S Natural autoantibodies Curr Opin

Immunol 78121995

64Olsson ML Chester MA A rapid and simple ABO genotype screening method using

a novel BO2 versus AO1 discriminating nucleotide substitution at the ABO locus Vox

Sang 69242-2471995

65Fukumori Y Ohnoki S Shibata H Yamaguchi H and Nishimukai H Genotyping

of ABO blood groups by PCR and RFLP analysis of 5 nucleotide positions Medicine

International Journal of Legal Medicine Volume 107 Number 4 179-1821995

49

53Zago MA Tavella MH Simoes BP Racial heterogeneity of DNA polymorphisms

linked to the A and the O alleles of the ABO blood group gene Ann Hum Genet6067-

721996

54Olsson ML Santos SE Guerreiro JF Zago MA Chester MA Heterogeneity of the

O alleles at the blood group ABO locus in Amerindians Vox Sang7446-501998

55Yamamoto F McNeill PD Yamamoto M Hakomori S Bromilow IM Duguid JK

Molecular genetic analysis of the ABO blood group system 4 Another type of O allele

Vox Sang64175-81993

56 Schwyzer M and Hill R L J Biol Chem 252 2346ndash23551977

57Pusztai A and Morgan W T J Studies in immunochemistry 22 Amino acid

composition of human blood-group AB H and Le-a specific substances Biochem J

88546-5551963

58Yamamoto F McNeill PD Amino acid residue at codon 268 determines both

activity and nucleotide-sugar donor substrate specificity of human histo-blood group A

and B transferases in vitro mutagenesis study J Biol Chem 271 10515-201996

59Yamamoto F McNeill PD Yamamoto M Hakomori S Harris T Judd WJ

Davenport RD Molecular genetic analysis of the ABO blood group system 1 Weak

subgroups A3 and B3 alleles Vox Sang 64116- 1191993

60 Olsson ML Thuresson B Chester MA An Ael allele-specific nucleotide insertion

at the blood group ABO locus and its detection using a sequence-specific polymerase

chain reaction Biochem Biophys Res Commun 216642-6471995

61Ogasawara K Yabe R Uchikawa M Saitou N Bannai M Nakata K Takenaka M

Fujisawa K Ishikawa Y Juji T Tokunaga K Molecular genetic analysis of variant

phenotypes of the ABO blood group system Blood 882732-27371996

62Yamamoto F McNeill PD Kominato Y Yamamoto M Hakomori S Ishimoto S

Nishida S Shima M Fujimura Y Molecular genetic analysis of the ABO blood group

system 2 cis-AB alleles Vox Sang 64120-1231993

63Coutinho A Kazatchkine MD Avrameas S Natural autoantibodies Curr Opin

Immunol 78121995

64Olsson ML Chester MA A rapid and simple ABO genotype screening method using

a novel BO2 versus AO1 discriminating nucleotide substitution at the ABO locus Vox

Sang 69242-2471995

65Fukumori Y Ohnoki S Shibata H Yamaguchi H and Nishimukai H Genotyping

of ABO blood groups by PCR and RFLP analysis of 5 nucleotide positions Medicine

International Journal of Legal Medicine Volume 107 Number 4 179-1821995

51

66Akane A Yoshimura S Yoshida M Okii Y Watabiki T Matsubara K and Kimura

K ABO Genotyping Following a Single PCR Amplification Journal of Forensic

Sciences Vol 41 No 2 March pp 272-2741996

67Al-Bustan S El-Zawahri M Al-Azmi D and Al-Bashir A Allele Frequencies and

Molecular Genotyping of the ABO Blood Group System in a Kuwaiti Population

International Journal of Hematology Volume 75 Number 2 147-1532002

68Mizuno N Ohmori T Sekiguchi K Kato T Fujii T Fujii K Shiraishi T Kasai K

and Sato H Alleles Responsible for ABO Phenotype-Genotype Discrepancy and Alleles

in Individuals with a Weak Expression of A or B Antigens Forensic Sci Jan Vol 49

No 12004

69Honda K Nakamuraa T Tanakaa E Yamazakib K Tuna Z Misawab S Graphic

SSCP analysis of ABO genotypes for forensic application International Congress

Series 1261 586ndash5882004

70 Hanania SS Hassawi DS Irshaid NM Allele frequency and molecular genotypes

of ABO blood group system in a Jordanian population MedSci January 7(1) 51-58

2007

71EL-zawahri MM Luqmani YA Molecular genotyping and frequencies of A1 A2 B

O1 and O2 alleles of the ABO blood group system in a Kuwaiti population International

Journal of Hematology Apr87(3)303-92008

72Lee SH Park G Yang YG Lee SG and Kim SW Rapid ABO Genotyping Using

Whole Blood without DNA Purification Korean J Lab Med29231-72009

73Nojavan M Shamsasenjan K Movassaghpour AA Akbarzadehlaleh P Torabi SE

Ghojazadeh M Allelic Prevalence of ABO Blood Group Genes in Iranian Azari

Population BioImpacts 2(4) 207-2122012

74 Bugert P Rink G Kemp K Kluumlter H Blood Group ABO Genotyping in Paternity

Testing Transfusion Medicine Hemotherapy June 39(3) 182ndash1862012

75Jenkins PV OrsquoDonnell JS ABO blood group determines plasma von Willebrand

factor levels a biologic function after all Transfusion 46(10)1836-18442006

76Tregouet DA Heath S Saut N Andreani C Schved JF Pernod G Galan P Drouet L

Zelenika D Vague I Alessi MC Tiret L Lathrop M Emmerich J Morange PE

Common susceptibility alleles are unlikely to contribute as strongly as the FV and ABO

loci to VTE risk results from a GWAS approach Blood 113(21)5298-53032009

77 Kamphuisen PW Eikenboom JCJ Bertina RM Elevated factor VIII levels and the

risk of thrombosis Arterioscler Thromb Vasc Biol21(5) 731-7382001

78Gallinaro L Cattini MG Sztukowska M Padrini R Sartorello F Pontara E

Bertomoro A Daidone V Pagnan A Casonato A A shorter von Willebrand factor

survival in O blood group subjects explains how ABO determinants influence plasma

von Willebrand factor Blood111(7)3540-35452008

53

79Yamamoto F Marken J Tsuji T White T Clausen H Hakomori S Cloning and

characterization of DNA complementary to human UDP-GalNAc Fuc alpha 1----2Gal

alpha 1----3GalNAc transferase (histo-blood group A transferase) mRNA J Biol Chem

265 1146-511990

80 EL-zawahri MM Luqmani YA Molecular genotyping and frequencies of A1 A2

B O1 and O2 alleles of the ABO blood group system in a Kuwaiti population

International Journal of Hematology Apr87(3)303-92008

81 Al-Arrayed S Shome DK Hafadh N Amin S Al Mukhareq H Al Mulla M ABO

blood group and Rhd phenotypes in Bahrain Results of screening school children and

blood donors Bahrain Med Bull 23 112-52001

82 Tills D Kopec A and Tills R The distribution of the human blood groups and other

polymorphisms Suppl 1 New York Oxford University Press pp 335-401983

83 Irshaid NM Ramadan S Wester ES Olausson P Hellberg A Merza JY et al

Phenotype prediction by DNA ndashbased typing of clinically significant blood group

systems in Jordanian blood donors Vox Sang 83 55-622002

84 Bashwari LA Al-Mulhim AA Ahmad MS Ahmed MA Frequency of ABO blood

group in the eastern region of Saudi Arabia Saudi Med J 22 1008-122001

85 Khalil I Phrykian S and Farri A Blood group distribution in Sudan Gene Geogr 3

7-101989

86 Cho D Kim SH Jeon MJ Choi KL Kee SJ Shin MG Shin JH Suh SP Yazer MH

and Ryang DW The serological and genetic basis of the Cis-AB blood group in Korea

Vox Sang 87 41-32004