A. Ogawa K. Tokunaga L. Lin K. Kashiwase H. Tanaka M.J. Herrero C. Vilches M.H. Park G.J. Jia N.-0. Chimge E.W. Sideltseva Y. lshikawa T. Akaza K. Tadokoro T. Juji

Diversity of HLA-B61 alleles and haplotypes in East Asians and Spanish Gypsies

Key words: association analysis; Buryat; Chinese; HLA-661 allele; South Korean; Mongolian; East Asian; PCR-SSOP method; Spanish Gypsy

Abstract: The dishibution of KLA-BGl alleles and their association with HLAC and DRBl alleles were investigated in six East kian popdations (South Korean, Chinese Korean, Man (Manchu), Northern Han, Mongolian and Buryat) and Spanish Gypsies and compared to our previous report on the Japanese population. The alleles were identified using a group-specific polymerase chain reaction (PCR) and genomic DNA followed by hybridiza- tion with sequence-specific oligonucleotide probes (SSOP). Both HLA- B*4002 and B*4006 were commonly detected in the South Korean, Chinese

Acknowledgment: This study was partly supported by grants from Ministry of Education, Science, and Culture, Innan __r-...

Korean, Man, Northern Han and Japanese populations, while HLA-B*4002 was predominant in the Mongolian and Buryat populations. Strong associ- ations of B*4002 with Cw*0304 and of B*4006 with Cw*0801 were com- monly observed in these East Asian populations. In contrast, in Spanish Gypsies, only HLA-B*4006 was found and the allele exhibited a strong association with Cw*1502. HLA-B*4003 was also identified in the South Ko- rean, Chinese Korean, Northern Han, Mongolian and Japanese populations at relatively low frequencies, and exhibited an association with Cw*0304. Moreover, the association of these B61 alleles with the DRBl alleles re- vealed considerable diversity among the different populations. HLA-B*4004 and B*4009 were not observed in these populations. Consequently, the fre- quencies of the B61 alleles varied among the different East Asian popula- tions, but the individual B61 alleles were carried by specific haplotypes often regardless of the ethnic differences.

Received 11 November 1997. revised. accepted for publication 22 December 1997

Copyright 0 Munksgaard 1998 Tissue AnNgens . ISSN 0001.2815

Tissue Antigens 1998: 51: 356366 Printed in Denmark .Al l rights reserved

HLA-B61 is an antigen that belongs to the HLA-I340 cross-reacting group and is encoded by several distinct alleles. The antigens en- coded by the B*4001, B*4005, B*4007 and B*4008 alleles have been serologically identified as HLA-BGO (1, 2), BN21 (3), BFU' (4) and a EM0 variant (5), respectively. In contrast, the B*4002 and B*4006 alleles were shown to encode the HLA-B61 antigen (2, 6, 7). HLA- B*4003 and B*4004 were originally isolated from Brazilian Indians possessing the B40 antigens (8), and their gene products were also typed as the B61 antigen ((9) and our unpublished results). Recently,

Authors' afWatlons:

A. Ogawa'. K. Tokunaga'.', L. Lin', K. Kashiwase'. H. Tanaka', MJ. Henero', C. Vilches', M. Hee Park". G. Jun Jias. N..O. Chirngee. E.W. Sldeltseva7, Y. Ishlkawa', T. AkaZa', K, Tadokoro'. T. Juji'

%Japanese Red Cross Central Blood Center, Tokyo. Japan.

9epartment of Human Genetics, School of International Health, University of Tokyo. Tokyo. Japan,

3Servicio de Inmunologla, Clinlca PueRa de Hierro. Madrid, Spain,

%epartment of Clinical Pathology, Seoul Natlonal University College of Medicine, Seoul. Korea,

"arbin Red Cross Blood Center, Harbln. China.

BNational Center of Anthropology, Medical University, Ulaanbaatar, Mongolia,

'Immunogenetics Research Centre of Civil Health Service, Angarsk. Irkutsk, Russia

Correspondence to: Atsuko Ogawa Department of Research Japanese Red Cross Central

4-1.31 Hlroo Shlbuwku Tokyo 150 Japan E-mail:

Fax: +81 3 3406 7892 Tel: + a 1 3 3406 1211

Blood Center

a-ogawa@hla.cDc.Jrc.or.jp

356

Ogawa et at : HLA-561 alleles in East Asians and Spanish Gypsies

two new B61 alleles, B*4009 (10) and B*4011 (ll), have been re- ported. Consequently, at least 6 alleles in the B*40 group have been found to encode the B61 antigen, and the products of these alleles are indistinguishable using serological techniques.

The HLA-B61 antigen is widely distributed in Asian populations with frequencies of approximately 10% (12). In the Japanese popula- tion, both B*4002 and B*4006 commonly exist and show strong association with Cw*0304 and Cw*0801, respectively (13, 14). B*4003 also occurs at a low frequency (9). In the South Korean population, similar frequencies of B61 alleles were reported (15). Therefore, B61 alleles are expected to exhibit an extensive diversity in various East Asian populations. In contrast, the antigen is also observed with a high frequency (21%) in Spanish Gypsies (16) and exhibits a strong association with Cw* 1502 (17).

To investigate the distribution of the B61 alleles in six East Asian populations, i.e., South Korean, three Chinese populations (Korean, Man and Northern Han), Mongolian, and Buryat and in Spanish Gypsies, we identified HLA-B61 alleles using the PCR- S O P method employing genomic DNA. We also determined the distributions of %A-C and -DRB1 alleles and analyzed their associ- ation with the B61 alleles in these populations.

Material and methods

Populations

Peripheral blood was collected from the following unrelated healthy individuals belonging to the 7 populations: 212 unrelated parents of 106 South Korean families studied previously (18); 197 Korean, 171 Man and 196 Northern Han individuals living in the suburbs of Harbin city in the northeast of China (Heilongjiang province); 187 Mongolian individuals who belong to the Khalkha group living in the central part of Mongolia as previously described (19); 148 Bury- at individuals living in the suburbs of Angarsk city in East Siberia as previously reported (20); and 81 Spanish Gypsy individuals pre- viously reported (17).

Serological typing

The peripheral blood samples were used for typing of HLA-A, -C, and -B by the standard NLH microlymphocytotoxicity technique with local qualified antisera. We confirmed that the antigens en- coded by the alleles HLA-B*4001, B*4005 and B*4007 were typed as B60, B50 and B’FU‘, respectively, and those antigens encoded by B*4002, B*4003, B*4004 and B*4006 were typed as B61. Samples that possessed B*4008 and B*4009 were not available in this study.

The HLA-Cw3 antigen was further subdivided into two split anti- gens, Cw9 and CwlO, in the South Korean, Chinese Korean, North- ern Han and Buryat populations. However, samples heterozygous for Cw9 and CwlO could not be distinguished from Cw9 homo- zygous (Cw9/-) samples by serological typing. Antigens encoded by the alleles Cw*08, Cw*12, Cw*13, Cw*14, Cw*15, Cw*16, Cw*17 and Cw*18 were classified into the HLA-C ‘blank’ group using our local antisera for population studies. A summary report of the class I serological studies using these East Asian population samples ap- peared in the proceedings of the 12th International Histocompat- ibility Workshop (21). HLA-Cw6.2 (Cw*1502), defined by Vilches et al. (17, was also classified into the ‘blank’ group in this study.

Samples selected for PCR-SSOP typing

Based on the results of serological typing, B61-positive samples were selected and subjected to DNA typing to identify the B61 and C locus alleles. Apparent B60 homozygous (J360/-) samples were also examined, because samples heterozygous for B60 and B61 alleles were also found to be included in B60/- samples by serological typ- ing.

DNA amplification

Genomic DNA was prepared from the peripheral blood samples using a DNA extraction kit (QIAamp blood kit, Qiagen, Chatsworth, CA).

The DNA fragment containing exon 2 (from base 34), intron 2 and exon 3 (to base 215) of B61 alleles was specifically amplified by PCR with a primer set, B61F (5‘-GGT ATT TCC ACA CCT CCG- 3’) and B61R (5’-GCC ACT CCA CGC ACT C-37, using genomic DNA as a template. The PCR was performed in 50 p1 of reaction mixture containing 80 mM Tris buffer (PH 9.0), 2 mM MgC12 20 mM ammonium sulfate, 0.2 mM of each dNTP, 250 nM of each primer, 5% dimethylsulfoxide and 2.5 U Taq polymerase. After an initial denaturation at 95°C for 3 min, amplifications were performed for 30 cycles’ with each cycle consisting of denaturation at 94°C for 30 s, annealing at 58°C for 30 s and extension at 72°C for 60 s. The last cycle was followed by an extension at 72°C for 5 min. Amplifi- cation was checked by the 5% aaylamide gel electrophoresis of 5 p1 of the PCR product. In addition to B61 alleles (B*4002, B*4003, B*4004, B*4006, B*4009 and B*4011), B*4008, B*7301 and B*27 alleles were amplified with the primer set. The alleles B*07, B*48 and B*8101 were also amplified, but the amplification efficiency was much lower than that for the former group. Accordingly, when the samples were heterozygous for B6UB7 or B611B48, the PCR

Tissue Antigens 1998: 51: 356-366 357

Ogawa et al : HLA-B61 alleles in East Asians and Spanish Gypsies

products of the B*07 and B*48 alleles were hardly detected by S O P hybridization.

The DNA fragment containing exon 2, intron 2, and exon 3 was amplified by PCR to also idenhfy the HLA-C locus alleles. The con- ditions for the C locus-specific amplification were identical to those for the B61-specific PCR amplification described above, except for the following points. Each cycle of the amplification consisted of denaturation at 94°C for 30 s, annealing at 66°C for 60 s and exten- sion at 72°C for 30 s, using the primer set, 5CIn 1-61 and 3BCIn3- 12, described by Cereb et al. (22).

SSOP analysis for B 6 1 and Cw alleles

The PCR products (1.5 pl) were denatured and immobilized with 0.5 N NaOH onto a nylon membrane (Hybond N+, Amersham). Oligo- nucleotide probes, shown in Table 1, were 3' end-labeled using di- goxigenin (DIG)-ll-ddUTP (Boehringer-Mannheim, Germany). Fol- lowing prehybridization in a solution containing 6xsaline-sodium phosphate-EDTA (0.9 M NaCI, 60 mM Na&IP04, 6 mM EDTA, pH 7.4), 5xDenhardt's solution, 0.1% N-lauroylsarcosine and 0.02% so- dium dodecyl sulfate, at 42°C for 30 min, the denatured DNA on the membrane was hybridized in the same solution with the DIG- labeled probes at 42°C for 1 h. Any mismatched probes were washed off using tetramethylammonium chloride (TMAC) solution (50 mM Tris, pH 8.0, 3 M TMAC, 2 mM EDTA, 0.1% SDS). The washing temperature was 62°C for probes B40-2 and -9, and 59°C for probes EM-common, B40-3, -4, -6, and -8. The completely matched probes on the membrane were detected using an anti-digoxigenin antibody conjugated to alkaline phosphatase (Boehringer-Mannheim, Ger- many) and a chemiluminescent substrate CSPD (Tropix, Massachu- setts, USA). Using these probes, five HLA-B61 alleles (B*4002, B*4003, B*4004, B*4006 and B*4009) and B*4008 were differen- tiated.

Ollgonucleotlde probes used for the deterrnlnatlon of HLA-BS1 alleles

SSOP analysis of HLA-C alleles was performed under the con- ditions described above except for the probes used and washing temperatures. Thirty probes were used for C-allele typing, of which twenty-four probes (probe no. 1-26 except for 14 and 18) were se- lected based on the report by Kennedy et al. (23). We designed the remaining six probes, to distinguish Cw*03, Cw*O8 and Cw*15 al- leles, as follows: C-27, 5'-ACT CGG TCA GCC TGT GC-3' (washing temperature with TMAC solution, 62°C); C-29, 5'-GAG AAC AGG AAG AAG ACG-3' (59°C); C-30,5'-CTG GAT GAT GTG AGA CCC- 3' (56"C), C-31, 5'-GCA CAG ACT GAC CGA GT-3' (59°C); C-32, 5'- GGA GCA GCG GAG AGC CTAS' (62°C); and C-33, 5'-TCA CCG GCC TCG CTC TGG- 3' (59OC). Using these 30 probes, the 42 HLA- C alleles reported in 'Nomenclature for factors of the HLA system, 1996" (10) were classified into 32 allele groups.

HLA-DRB1 allele typing

High-resolution typing of HLA-DRB1 alleles was performed follow- ing the PCR-MPH (microtiter plate hybridization) method as de- scribed previously (24, 25). A summary report of the DRBl allele typing in East Asian samples appeared in the proceedings of the 12th JHW (21).

Sequencing of the HLA-B61 allele in Spanish Gypsies

The complete coding region of the HLA-B61 allele was amplified by PCR using cDNA prepared from a B-lymphoblastoid cell line from a Spanish Gypsy (GO85 HLA-Al/AB, Cw6.2 (Cw*1502)/ Cw*12022, B52B61, DR14DR15, DQ5/DQ6). The amplified frag- ment was cloned and sequenced by the method previously described (26).

Probe

B4kornmon

840-2

840-3

840-4

840-6

840-8

840-9

Position

201-218

356-373

408-425

344-360''

352-369

263-280

407-424

Sequence 5'- -3'

Specificity

GAGGAAGGAGCCGCGGGC

TCCAGAGCATGTACGGCT

GCA TGA CCA GTC CGC CTA

CTG GAT GAT GTG AGA CCC

ACT TGG CAG ACG ATG TAT

CACAGATCTTCAAGACCA

GGT ATG ACC AGT ACG CCT

B*4002.03,04,06,08,09, 11

B*4002,03,08; 8'07; 8*2707.11; B*4801; B*8101

B*4003

8*4004

8'4006; 8'7301

8*4008

8*4009; 8'2706

Derived from a sesuence of the last base of intron 2 and the first 17 bases of Exon 3.

Table 1

358 Tissue Antlgens 1998: 5 1 356-366

Ogawa et al : HIA-BGl alleles in East Asians and Spanish Gypsies

The distribution of the HLA-B61 alleles in East Asian popuiatlons and Spanish GyDsies

Number of samples Total (861-positive)')

Allele Frequency (%)

8*4002

8*4003

B'4006

South Korean

212 (36)

6.1

0.2

2.4

Chinese

Korean

197 (36)

5.0

1.0

3.7

Man Northern Han Mongolian

171 196 187 (23) (34) (43)

3.8 2.9 10.7

0 0.5 0.3

2.9 5.6 1.6

Buryat Spanish Gypsy Japaneseb)

148 8 1 117 (36) (26) (26)

13.2 0 6.1

0 0 rarec)

0.3 18.3 4.8 ~

B61 total 8.7 9.7 6.7 9.0 12.6 13.5 18.3 10.9 ~~ ~~

Number of samples exarnlned for HL4-861 alleles. Data from our previous paper (14). 0.7% in a previous study (9).

Table 2

Statistical analysis

Allele and haplotype frequencies were estimated from the genotype data of B61, C, and DRBl and serotype data of HLA-A, -C and -B using a maximum likelihood method developed for the 11th IHW (27).

Resu I t s

Frequencies of HLA-B61 alleles

From the results of serological typing, B61-positive samples were selected from population samples. The frequency of B61 vaned from 6.7% (Man) to 18.3% (Spanish Gypsy) among these populations (Table 2). The B61-positive samples obtained from 7 populations were then examined by PCR-SOP using the seven probes listed in Table 1. Three B61 alleles, B*4002, B*4003 and B*4006, were ob- served in this study. Table 2 shows the frequencies for these alleles in each population. B*4002 and B*4006 were both detected at high frequencies in the South Korean (6.1% and 2.4%, respectively), Chi- nese Korean (5.0% and 3.7%), Man (3.8% and 2.9%) and Northern Han (2.9% and 5.6%) populations. In contrast, B*4002 was pre- dominant in the Mongolian (10.7%) and Buryat (13.2%) popula- tions, while only B*4006 was detected in Spanish Gypsies (18.3%). B*4003 was found in most East Asians including the South Korean (0.2%), Chinese Korean (1.0%), Northern Han (0.5%) and Mongolian (0.3%) populations, although the frequencies were relatively low. The alleles B"4004, B*4008 and B*4009 were not found in these East Asian populations or Spanish Gypsies.

Recently, a new allele B*4011 was reported in a Mexican individ-

ual of Nahua Amerindian descent (11). The sequence of B*4011 was identical to that of B*4002, except for a single base substitution (363 G in exon 3) which was identical to that of B*4001. Probe B40- 2 is complementary to a region of B*4002 and covers the position of the substitution, and B*4001 does not hybridize with probe B40- 2. Consequently, B*4011 is expected to hybridize with only the B40- common probe but not with the other probes including B40-2. This reactive pattern was not found in these East Asians and Spanish Gypsies, thus B*4011 was concluded to be absent in these popula- tions.

Association of B*4002 and B*4006 with HLA-C alleles in

East Asian populations

Based on B61 genotypes and Cw serotypes, three B-C haplotypes were observed at frequencies of higher than 1.4% in the East Asian populations (Table 3). In the South Korean, Chinese Korean and Man populations, B*4002-Cw10 (Cw3) was predominantly observed (4.2%, 2.5'76, and 2.4%, respectively), and B*4006-C blank (CBL) was also commonly observed (1.8%, 2.3% and 2.0%, respectively). In the Mongolian and Buryat populations, B*4002-Cw10 (Cw3) was observed at high frequencies (10.3% and 11.3%, respectively), whereas in the Northern Han population, B*4006-CBL was predom- inantly observed (4.4%). A unique haplotype, B*4006-Cw!3, was ob- served in the Chinese Korean population.

Samples possessing B*4002 were further subtyped for Cw alleles, and the number of individuals possessing each Cw*03 allele was counted (Table 4). Cw3 antigens are known to be divided into two split antigens, Cw9 and CwlO. The Cw9 antigen has been reported to be encoded by Cw*0303, and the CwlO antigen by Cw*0302 and Cw*0304 (10, 28). In the South Korean, Chinese Korean, Northern

Tissue Antigens 1998: 51: 356-366 359

Ogawa et al : HLA-B61 alleles in East Asians and Spanish Gypsies

Twelocus haplotypes carrying HLA-861 alleles and C antigens In East Aslan populatlons

Haplotype

Population B*

South Korean 4002

4006

Chinese Korean 4002

4006

4006

Man 4002

4006

Northern Han 4006

cw HF(%)*I

10 4.2

CBL'I 1.8

10 2.5

CBL', 2.3

9 1.6

3 (9/10) 2.4

c B L" 2.0

CBL=) 4.4

L D ~ I RLD" X Z

3.2 0.63 59.5

1.2 0.67 14.6

2.0 0.45 30.6

0.9 0.39 4.3

1.1 0.34 11.4

1.8 0.56 21.3

1.0 0.52 4.8

2.3 0.67 17.3

Pvaiue

<lo-7

<lo-3

<lo-6

c0.05

d 0 - J

<10-5

<0.05

<10-4

Mongolian 4002 3 (9/10) 10.3 7.0 0.94 92.1 <lo-7

Buryat 4002 10 11.3 8.0 0.81 87.0 <lo-7

Japanese B*4002-Cw*0304 5.8 5.1 0.92 96.1 <lo-7

B*4008Cw*0801 4.9 4.3 1.00 81.0 <10-7

Haplotype frequency: haplotypes whose frequencles are higher than 1.40% are presented in the Table. bl Linkage disequilibrlum parameter X10-2. c, Relatlve llnkage disequilibrium value. ") Data from our previous paper (14). *) CBL: HIAC blank.

Table 3

Han, and Buryat populations, serological typing results of Cw9 and CwlO corresponded well to the allele typing results. We determined that: 1) all CwlO-positive samples possessed Cw*0304 or Cw*0302,2) all Cw9-positive samples possessed Cw*0303, and 3) several samples which have been serologically assigned to be Cw9/- were Cw*0303/ Cw*0304 or Cw*0302/Cw*O303 heterozygotes as mentioned above (see Serological typing in Material and methods). In the Mongolian population, out of 36 Cw3-positive samples (Cw9 and CwlO split typ- ing was not performed), 31 samples possessed Cw*0304, and 4 out of the 31 samples were Cw*0302/Cw*O304 heterozygotes and one was a

Cw*0303/Cw*O304 heterozygote. The remaining two samples pos- sessed only Cw*0302 and three possessed only Cw*0303. These re- sults indicated that B*4002 is strongly associated with Cw*0304 in the South Korean, Chinese Korean, Northern Han, Mongolian and Bu- ryat populations. In contrast, in the Man population, Cw3 was divided into two alleles; 7 out of 11 samples were found to possess Cw*0303 and the remaining 4 to possess Cw*0304. Therefore, B*4002 may be associated with both Cw*0303 and Cw*0304 in this population. Our results indicated that B*4006-CBL is a common haplotype

in the East Asian populations (Table 3). B*4006 was strongly as-

The assoelatlon of HLA-B*4002 wlth Cw3 alleles In East Aslan populations

No. with Cw*03 alleles" No. with No. with

Populations B*4002 cw3 (CwlO) Cw'O302 Cw*0303 Cw*0304

South Korean 25 23 (17) 1 6 20

Chinese Korean 18 12 ( 9) 0 3 10

Man 13 11 0 7 4

Northern Han 11 9 ( 4) 1 4 6

Mongolian 36 36 6 4 3 1

Buryat 35 35 (32) 5 3 35

The sum of the number for each Cw'O3 allele was larger than the number of Cw3positive individuals, because several individuals were Cw.03 heteroygotes.

Table 4

360 Tissue Antigens 1998: 51: 356-366

Ogawa e t al : HLA-961 al le les in East Asians a n d Spanish Gypsies

The assoclatlon of HLA-B*4006 wlth Cw'0801 In East Aslan papuiatlons

No. with C blank alleles" No. with No with

Populations B*4006 C blank Cw*0801 Cw'l.502 Other alleles (occurrence)

South Korean 11 9 9 0 0802 ( l ) , 14 (2)

Chinese Korean 16 12 11 2 (0) Man 10 9 8 1 14 (3) Northern Han 21 18 17 0 0802 (1). 1202 (1). 1203 (2), 14 (2) Mongolian 6 5 4 1 1202 (l), 1203 (1) Buryat 1 1 0 0 1203 (1)

")The sum of the numbers for each C blank allele was larger than the number of individuals with Cw blank antigen because several individuals were heterozygotes of

Table 5

blank alleles.

sociated with Cw*0801 in the Japanese population (14), whereas, in Spanish Gypsies, B*4006 was exclusively associated with Cw*1502 as described below. Both Cw alleles were serologically typed as blank with our antisera. Then the distribution of Cw alleles in the B*4006-positive East Asian samples was determined in this study, and the numbers of individuals possessing C blank alleles were counted (Table 5). Cw*0801 was detected predominantly in the East Asians except for the Buryat population, and other alleles, including Cw*1502, were only found in a few samples from each population. The results indicated that B*4006 is strongly associated with Cw*0801 in the East Asian populations.

In the Chinese Korean population, the association of B*4006 with Cw9 was observed (Table 3). All of the seven samples possessing both B*4006 and Cw9 were found to possess Cw*0303.

Association of B*4003 with Cw*0304 in East Asian populations

In the six East Asian populations, a total of 8 individuals possessed HLA-B*4003. The HLA types of these individuals are listed in Table

The assoclatlon of HLA-B*4003 with Cw*0304 In East Asian populatlons

6. All of them possessed Cw*0304. A South Korean individual (sample no. 1) carried the haplotype Cw*0304-B*4003 determined by a family study (18). Thus, B*4003 is likely to be carried by a haplotype B*4003-Cw*0304, although statistical analysis was not applicable because of the low frequency of the B*4003 allele.

Association of B*4002 and B*4006 with DRBl alleles in East Asian populations

Six kinds of B-DRB1 haplotypes were observed at frequencies of higher than 1.4% in East Asian populations (Table 7). B*4002 was strongly associated with DRB1*1501 in the Man population (1.8%), whereas B*4002 was associated with DIU31*0401 (2.5%) and DRB1*0802 (2.1%) in the Mongolian population. In the Buryat population, B*4002-DRB1*0401 was predominant (4.1 YO), followed by B*4002-DRB1*0901 and B*4002-DRB1*1401 (2.2% and 2.0%, re- spectively). Among these B*4002-DRBl haplotypes, only B*4002- DRB1*0401 was shared by both the Mongolian and Buyat popula- tions.

A common haplotype observed in the Northern Han population

H M type No A c w CW*" c w CW*=) B B") B DRBI**' Population

1

2 24 0304 9 0303

3 2.24 0304 9 0303 6 1 4003 35 1405 1407 Chinese Korean

4 2,31 10 0304 1502 6 1 4003 5102 1405 1101

6 1 4003 5 1 0101 0406 5 2, 3 10 0304 1502

6 2, 24

7 2 10 0304 1203 61 4003 35 0401 0405

8 30, 31 10 0304 2 02

24, 33 10 0304 7 0701 6 1 4003 44 1405 0701 South Korean

6 1 4003 35 1201 1501

10 0304 14 6 1 4003 51 1405 1501 Northern Han

6 1 4003 27 1403 0801 Mongolian

Alleles determined by DNA typing methods.

Table 6

Tissue Antigens 1998: 51: 356366 361

Ogawa et al : HLA-561 alleles in East Asians and Spanish Gypsies

TwrAocus haplotypes carrylng HLA-B61 and D R B l alleles In East Asian populatlons

HaDlotvDe ~

Population B* DRB1* HF(%)" L D ~ ] RLD" X2 P-value

Man 4002 1501 1.8 1.4 0.41 18.6 <lo-5

Northern Han 4006 0901 2.2 1.5 0.31 14.4 c10-3

Mongolian 4002 0401 2.5 1.7 0.27 16.9 ~ 1 0 - 5

4002 0802 2.1 1.6 0.34 19.8 -40-5

Buryat 4002 0401 4.1 2.4 0.22 13.7 <lo-3

4002 0901 2.2 1.2 0.19 5.7 <0.02

4002 1401 2.0 1.7 0.71 27.4 <10-e

Japanese") 4006 0901 4.0 3.2 0.78 38.6 c10-7

'1, n', and a); see Table 3.

Table 7

(2.2%), B*4006-DRB1*0901, has already been reported in the Ja- panese population (14). This haplotype was also observed in the South Korean and Chinese Korean populations, although its fre- quency was low (1.1% in both populations). In these two popula- tions, no haplotypes were found at frequencies higher than 1.1%. Consequently, the common B-DRB1 haplotypes were found to differ among various East Asian populations.

confirmed the nucleotide sequence of the complete coding region of the allele cloned from a Spanish Gypsy with a characteristic haplo- type, Al-Cw*1502-B61-DR14-DQ5. Out of 26 samples used for SSOP typing, 25 samples possessed Cfl1502, and 19 samples shared Al, Cw*1502, DR14 and DQ5. Consequently, B*4006 is strongly associ- ated with Cfl1502 and mainly carried by the haplotype, Al- Cw*1502-B*4006-DR14-DQ5 in Spanish Gypsies.

Three- and four-locus haplotypes in East Asian populations

Common three- and four-locus haplotypes carrying B61 alleles in East Asian populations are shown in Table 8. A2-Cw*0304-B*4002 Discussion

was shared by the East Asian populations except for the Northern Han population. In addition, A24-Cw*0304-B*4002 was predominant in both the Mongolian (5.7%) and the Buryat populations (4.7%), al- though four-locus haplotypes were associated with different DRB l alleles in these populations: A24-Cw*0304-B*4002-DRBI*O802 was common in the Mongolian population (2.1 %), while A24-Cw*0304- B*4002-DRB1*1401 was common in the Buryat population (2.0%).

As for haplotypes including B*4006, A2-Cw*0801-B*4006 was observed in the South Korean (1.4%) and Northern Han populations (2.9%). Moreover, A24-Cw*0801-B*4006 (1.7%) and A24Cfl0801- B*4006-DRl31*0901 (1.4%) were observed in the Northern Han population. These haplotypes have also been reported in the Ja- panese population (14).

Association of B*4006 In Spanish Gypsies

In Spanish Gypsies possessing the B61 antigen, all the alleles were typed as B*4006 by PCR-SOP as shown in Table 2. Moreover, we

The distribution of HLA-B61 alleles in six East Asian populations and Spanish Gypsies was analyzed. We previously reported the existence of three B61 alleles, B*4002, B*4003 and B*4006, as well as three common haplotypes, B*4002-Cw*0304, B*4006-Cw*0801 and B*4006-DRB1*0901, in the Japanese population (9, 14). This study confirmed that B*4002 and B*4006 commonly exist in East Asian populations, although the allele frequencies differ among the populations (Table 2). B*4002 was predominant in the Mongolian and Buryat populations, while B*4006 was predominant in the Northern Han population. The B-C haplotypes carrying the B61 alleles were, however, shared by these East Asian populations. B*4002-Cw*0304 was observed in the South Korean, Chinese Kore- an, Mongolian, Buryat, and Japanese populations, and B * W Cw*0801 was observed in the South Korean, Chinese Korean, Man, Northern Han, and Japanese populations (Tables 3-5). In Spanish Gypsies, the most common haplotype was Al-Cw6.2-B61-DR14-DQl @IF, 0.130; LD, 0.127) (16). The allele for Cw6.2 was previously iden-

362 Tissue Antigens 1998: 5 1 356-366

Ogawa et al : HLA-B61 alleles in East Asians and Spanish Gypsies

Table 8 Three. and four-locus haplotypes carrying the HLA-B61 alleles In East Aslan populatlons

Haplotype

Population A

South Korean 2

2

Chinese Korean 2

Man 2

2

Northern Han 2

24

Mongolian

Buryt

2

24

24

2

24

24

24

2

3 1

24

24

24

Cw (alleie)"

10 (0304)

CBL (0801)

10 (0304)

3 (0303)

3 (0303)

CBL (0801)

CBL (0801)

CBL (0801)

CBL (0801)

3 (0304)

3 (0304)

3 (0304)

3 (0304)

3 (0304)

10 (0304)

10 (0304)

10 (0304)

10 (0304)

10 (0304)

10 (0304)

B*

4002

4006

4002

4002

4002

4002

4006

4006

4006

4006

4006

4002

4002

4002

4002

4002

4002

4002

4002

4002

4002

4002

4002

4002

4002

4002

DRBl*

1501

1501

0901

0901

0901

0401

0802

0802

0802

0401

1401

0901

0401

1401

HF(%)'' LDb'

3.1

1.4

1.6

2.2

1.7

2.3

2.9

1.7

2.0

1.5

1.4

5.1

2.4

2.4

2.1

2.1

2.1

4.1

3.6

1.5

3.7

2.0

1.8

2.4

2.0

2.0

2.8

1.3

1.4

1.9

1.7

2.2

2.2

1.3

1.1

1.3

1.3

4.9

1.7

2.1

2.0

2.0

2.1

3.9

2.5

1.3

3.2

1.9

1.5

2.0

1.9

2.0 ~ 10 (0304) 1401

Japanesed' A*3101-C~*0304-B*4002 2.7 2.6

A*2402Cw*0801-B*4006 1.8 1.6

Cw*0801 -B*4006DRB1*0901 4.0 3.9

'I, t.' and 'I; see Table 3. Corresponding allele IS shown in parentheses.

tified as Cw*1502 (17) and the allele for B61 was identified as B*4006 in this study. This haplotype B*4006-Cw*1502 was con- sidered to be infrequent in the East Asian populations (Table 5). In European populations, including German, Italian, Sardinian, Span- ish and Hungarian populations, an association of B61 with Cw2 has been reported (12). Several reports have shown that the B61 antigen is encoded by B*4002 (29) and the Cw2 antigen by Cw*02022 (30, 31). Consequently, B*4002-Cw*0304 and B*4006-Cw*0801 are

characteristic of the East Asian populations studied here. The Man population exhibited unique association of B*4002: the allele was associated with both Cw*0303 and Cw*0304 (Table 4). B*4002-Cw9 (Cw*0303) was also observed in the Northern Han population at a low frequency (l.l%), and three samples from the South Korean population (0.7%) were shown to possess the haplotype in a family study (18). These results suggest that B*4002-Cw*0303 is also shared by several East Asian populations although its frequency is

Tissue Antigens 1998: 51: 356-366 363

Ogawa et al : HLA-BGI alleles in East Asians and Spanish Gypsies

relatively low. According to the results of the serological analysis reported in the 11th IHW (12), B61-CwlO is widely distributed in many ‘Mongoloid’ groups including Tibetan, Orochon, Inuit, Yakut, and Amerindians in addition to the East Asian populations de- scribed in this study (Japanese, Korean, Mongolian, and Buryat). Therefore, the haplotype B*4002-Cw*0304 is likely to be widely dis- tributed among many Mongoloid populations.

In contrast to the relatively wide distribution of the specific B- C haplotypes, B-DRB1 haplotypes exhibited considerable diversity among the East Asian populations. This phenomenon can be ex- plained by the longer physical distance between B and DRBl loci than between B and C loci on chromosome 6 (13,14). Although five B*4002-DRBl haplotypes were observed at frequencies higher than 1.8%, only one of them (B*4002-DRB1*0401) was shared by the Mongolian and Buryat populations (Table 7). A common haplotype carrying B*4006 in the Northern Han population (B*4006- DRB1*0901) has been previously described in the Japanese popula- tion (14). In Spanish Gypsies, B*4006 was strongly associated with DR14. The DR14 is considered to be encoded by DRB1*1404, ac- cording to the previous report using the PCR-SOP method (16).

Three-locus haplotypes, A24-Cw*0304-B*4002 and Cw*0304- B*4002-DRB1*0401, were shared by. the Mongolian and Buryat populations, whereas A24-Cw*0801-B*4006 and Cw*0801-B*4006- DRB1*0901 were shared by the Northern Han and Japanese popula- tions (Table 8). Although two kinds of A-C-B three-locus haplotypes,

Fig, 1. The distribution of seven B*40 al- leles (B*4002, B*4003, B*4004, B*4005, B*4006, B.4008 and B*4011). The allele fre- quencies obtained in this study and in the Ja- panese population (14) are shown in bar charts. The other distribution data were summarized from the following reports; Caucasoid (a), North Amerindian (3, 11, 34, 36), El Salvador (5), and South Amerindian (8, 35, 37).

A2-Cw*0304-B*4002 and A2-Cw*0801-B*4006, were observed to be shared among these East Asian populations, genotyping of the A2 alleles is necessary for the precise characterization of these haplo- types, because the A2 antigen is known to be encoded by several different alleles in these populations (32, 33).

B*4002 is widely observed throughout the world, whereas B*4003, B*4004, B*4005, B*4006, B*4008 and B*4011 exhibit limited distribution as summarized in Fig. 1. The nucleotide se- quences of these six alleles and B*4009 are identical to that of B*4002 except for short segments, the sequences of which are ident- ical to those of some other HLA-B alleles (10). Therefore, B*4002 is consider to be the ancestral allele of these seven alleles, as pre- viously discussed (13). In this study, we found that both B*4002 and B*4003 are strongly associated with a specific C allele, Cw*0304, in the East Asian populations. Thus, B*4003 is considered to be gener- ated from B*4002 on the haplotype B*4002-Cw*0304 in certain populations of East Asia.

B*4003 and B*4004 were originally found in the South Amerind- ian populations together with several other new HLA-B alleles (8, 34, 35). In these reports, the authors hypothesized that the new alleles arose in the South Amerindian populations and that a marked evolution of HLA-B had occurred since humans migrated to South America from Asia. We previously reported the existence of the B*4003 in the Japanese population (9). In this study, B*4003 was also observed in the East Asian populations, and was corn-

364 Jissue Antigens 1998: 51: 35€-366

Ogawa et al : HLA-1361 alleles in East Asians and Spanish Gypsies

monly associated with Cw*0304. The B*4003 detected in the South Amerindian population appears to be associated with the Same HLA-C allele as that in the East Asian populations, because it was isolated from a Guarani Amerindian individual possessing Cw3 (HLA-A*0201, A*0211, B*4003, B*1504 and Cw3)(8). These results indicate that B*4003 is not a unique allele in the South Amerindian population but a shared allele between East Asians and Amerindi- ans. Therefore, further studies, especially on members of East Asian populations, are required to evaluate the hypothesis of the rapid evolution of HLA-B gene in South America.

B*4006 was observed in both East Asians and Spanish Gypsies and was found to be mostly carried by the B*4006-Cw*0801 haplo- type in East Asians and by the B*4006-Cw*1502 haplotype in Span- ish Gypsies. We confirmed that the sequence of B*4006 from a Spanish Gypsy was completely identical to that from a Japanese individual (2) in the entire coding reaon. Genetic distance analysis for Spanish Gypsies based on HLA antigen frequencies showed their affinity to Asian Indians, and this result concurs with their proposed historical origin, namely that Spanish Gypsies belong to a Caucasoid group which originated from northern India (16). From our East Asian data, a few samples were considered to possess the haplotype B*4006-Cw*1502, based on the identification of a Cw*1502 homozygote and a heterozygote (Table 5). Therefore, the haplotype B*4006-Cw*1502 is probably a shared haplotype between East Asians and Spanish Gypsies. These results suggest that B*4006 arose in some population in Asia, and spread to East Asians and Spanish Gypsies after it had been included in two different haplotypes. According to the serological analysis reported at the 11th IHW (12), the B61-CBL haplotype is frequent in Asian Indians (9.1%) and Thais (3.5%). It is of much interest to determine whether

References

1. Lopez de Castro J, Bragado R, Strong DM, Strominger JL. Primary structure of papain- solubilized human histocompatibility antigen HLA-B40 (-Bw60). An outline of alloantigenic determinants. Biochemistry

2. Kawaguchi G, Kato N, Kashiwase K et a]. 1983 22: 3961-9.

Structural analysis of HLA-B40 epitopes. Hum Immunol1993: 36: 193-8.

3. Hildebrand WH, Madrigal JA, Belich MI' et al. Serologic cross-reactivities poorly reflect allelic relationships in the HLA-B12 and HLA-B21 groups. Dominant epitopes of the alpha 2 helixc./lminuno11992: 149: 3563-5.

the B61 antigen is coded by B*4006 and whether C blank is coded by Cw*1502 or Cw*0801 in these populations, in order to elucidate the evolution of B61 alleles.

In a joint study on unrelated bone marrow transplantation (UBMT) in the Japanese population, retrospective high-resolution genotyping of HLA-A and -B was carried out in donors and recipi- ents who had been matched for HLA-A and -B antigens serologi- cally and for DRBl alleles by DNA typing ( S a m k i , T et al., manu- script in preparation). The results showed that the matching of HLA-A and -B alleles at the sequence level was important for pro- ducing high survival rates and a low incidence of severe graft-ver- sus-host disease (GVHD). Moreover, 80% of the allele mismatches at the HLA-A locus were caused by HLA-A2 group alleles (A*0201, A*0206, A*0207 and A*0210) and 70% at HLA-B were caused by B61 group alleles (B*4002, B*4003, and B*4006) in Japanese UBMT. Our present study indicated that other East Asian populations also show heterogeneity in B61 alleles; thus allele mismatch should occur between serologically matched UBMT donors and recipients.

Amino acid differences of the antigens encoded by B*4002,4003 and 4006 are observed in a P-sheet of the 02 domain, which is located in the bottom of the peptide binding groove of the HLA molecule. Thus, the unique sequence of each B61 antigen is usually masked by a binding peptide, which prevents the access of antibodies. This is probably the reason why we could not identify any antiserum to distinguish these subtypes. However, the differ- ences in the B61 antigens certainly affected the outcome of UBMT, as mentioned above. Further studies including disease association analyses are necessary to elucidate the biomedical significance of the diversity of B61 alleles.

4. Lin L, Tokunaga K, Ishikawa Y et al. A new member of the HLA-B40 family of alleles, B*4007, coding for B'FU' serological specificity. Tissue Anfzgens 1995: 45 276-9.

5. A d a m EJ, Little AM, Arnett KL, Leushner J, Parham P. Identification of a novel HLA- B40 allele (B*4008) in a patient with leukemia. Tissue Antigens 1995: 46: 204-5.

6. Domena JD, Arnett KL, Marsh SG, Bodmer JG, Parham P. Alloantibodies can discriminate three populations of HLA-B40 molecules encoded by B*4002. Tissue Atlti&ils 1994: 44: 57-8.

7. Lin L, Watanabe Y, Tokunaga K et al. A common Japanese haplotype HLA-A26-Cw3- B61-DR9-DQ3 carries HLA-B*4002. Tissue Antigens 1992 40 257-60.

8. Belich W, Madrigal JA, Hildebrand WH et al. Unusual HLA-B alleles in two tribes of Brazilian Indians. Nature 1992: 357 326-9.

9. Bannai M, Tokunaga K, Lin L, Ogawa A, Fujisawa K, Juji T. HLA-B40, B18, B27, and B37 allele discrimination using group- specific amplification and SSCP method. Hum lmmunol1996. 46: 107-13.

Tissue Anligens 1998: 51: 356-366 365

Ogawa et al : HIA-B61 alleles in East Asians and Spanish Gypsies

10. Bodmer JG, Marsh SG, Albert ED et al. Nomenclature for factors of the HLA system, 1996. Tissue Antigens 1997 49: 297-321.

11. VargasAlarcon G, Gbmez-Casado E, Martinez-Leso J et al. Description of a new HLA-W allele (B*4011) found in a Mexican individual of Nahua (aztec) descent. Immunogenetics 1997: 4 6 359-360.

12. Imanishi T, Akaza T, Kimura A, Tokunaga K, Gojobori T. Allele and haplotype frequencies for HLA and complement loci in various ethnic groups In: Tsuji K, Aizawa M, Sasazuki T, eds HLA 1991. Vol 1. New York Oxford University Press, 1992 1065-1220.

Genotyping and association analysis of HLA-B61 in Japanese. Hum Immunoll993 37 95-100.

14. Tokunaga K, Ishikawa Y, Ogawa A et al. Sequence-based association analysis of HLA class I and II alleles in Japanese supports conservation of common haplotypes Imrnunogenetics 1997 4 6 199- 205.

15. Lee KW, Kim YS. Serologic ambiguity and allelic frequency of the HLA-B40 family in the Korean population. Tissue Antigens 1997: 49 383-8.

16. de Pablo R, Vilches C, Moreno ME, Rementeria M, Solis R, Kreisler M. Distribution of HLA antigens in Spanish Gypsies: a comparative study. Tissue Antigens 1992 4 0 187-96.

17. Vilches C, de Pablo R, Herrero MJ, Moreno ME, Kreisler M. Molecular cloning and polymerase chain reaction-sequence- specific oligonucleotide detection of the allele encoding the novel allospecificity HLA-Cw6.2 (Cw*1502) in Spanish Gypsies. Hum Irnmunoll993 37 259-63.

18. Park MH, Hwang Y-S, Park KS et al. KLA haplotypes in Koreans based on 107 families. Tissue Anrigens. (In press).

19. Chimge N, Kashiwase K, Tanaka H et al. The HLA system in the population of Mongolia. Tissue Antigens 1996: 4 9 477- 483.

13. Lm L, Tokunaga K, Ogawa A et al.

20. Tokunaga K, Sideltseva EW, Tanaka H et al. Distribution of HLA antigens and haplotypes in the Buryat population of Siberia. Tissue Antigens 1995 4 5 98-102.

21. Tanaka H, Kashiwase K, Ishikawa Y et al. Distribution of HLA-A-B-DRB1 haplotypes in five ethnic groups in North-East Asia. In: Charron D, ed. Genetic diversify of HLA: functional and medical impricntions Vol II. Paris: EDK Publishers, 1997 183-184.

22. Cereb N, Maye P, Lee S, Kong Y, Yang SY. Locus-specific amplification of HLA class I genes from genomic DNA locus-specific sequences in the first and thiid introns of HLA-A,-B, and -C alleles. Tissue Antigens 1995: 4 5 1-11.

WE, Thomson W. Definition of HLA-C alleles using sequence-specific oligonucleotide probes (F'CR-SOP). Tissue Antigens 1995: 4 6 187-95.

24. Kawai S, Maekawajiri S, Tokunaga K, Juji T, Yamane A A simple method of HLA DRB typing using enzymaticallyamplified DNA and immobilized probes on microtiter plate. Hum Immunoll994 41: 121-6.

25. Kawai S, Maekawajiri S, Tokunaga K et al. Routine low and high resolution typing of the HLA-DRB gene using the PCR-MF'H (microtiter plate hybridization) method. Eur J Immunogenet 1996 23 471436.

26. Vilches C, Sam L, de Pablo R Moreno ME, Puente S, Kreisler M. Molecular characterization of the new alleles HLA- Bf8101 and B*4407. Tissue Antigens 1996: 47: 13942.

27. Imanishi T, Akaza T, Kimura A, Tokunaga K, Gojobori T. Estimation of allele and haplotype frequencies for KLA and complement loci. In: Tsuji K, Aizawa M, Sasazuki T, eds. HLA 1991. Vol 1. New York Oxford University Press, 1992 76-79.

28. Zemmour J, Gumperz JE, Hildebrand WH. The molecular basis for reactivity of anti- Cwl and anti-Cw3 alloantisera with HLA- B46 haplotypes. Tissue Antigens 1992: 39 249-57.

23. Kennedy LJ, Poulton W, Dyer PA, Ollier

29. Domena JD, Johnston-Dow L, Parham P The B*4002 allele encodes the B61 antigen: B40* is identical to B61. Tissue Anfigens 1992 4 0 254-6.

30. Prasad VK, Yang SY. Allele assignment for HLA-A, -B, and -C genes to the Tenth International Histocompatibility Workshop cell lines. Tissue Antigens 1996: 47: 538- 46.

31. Bunce M, Barnardo MC, Procter J, Marsh SG, Vilches C, Welsh KI. High resolution HLA-C typing by PCR-SSP identification of allelic frequencies and linkage disequilibria in 604 unrelated random UK Caucasoids and a comparison with serology. Tissue Antigens 1996: 48: 680-91.

32. Gao X, Jakobsen E3, Serjeantson SW. Characterization of the HLA-A polymorphism by locus-specific polymerase chain reaction amplification and oligonucleotide hybridization. Hum Immunoll994: 41: 267-79.

33. Ishikawa Y, Tanaka H, Semana G et al. Alleles and haplotypes Society No2 (A2, A28, A9). In: Charron D, ed. Genetic diversity of HLA: functional and medical implications Yo1 I. Paris: EDK Publishers, 1997: m1.

34. Watkins DI, McAdam SN, Liu X et al. New recombinant HLA-B alleles in a hibe of South American Amerindians indicate rapid evolution of MHC class I loci. Nature 1992 357 329-33.

35. Garber TL, Butler LM, Trachtenberg EA et al. HLA-B alleles of the Cayapa of Ecuador: new B39 and B15 alleles [published erratum appears in hunogenet ics 1995 42: 3081. Immunogenetics 1995 42: 19-27.

36. Garber TL, McAdam SN, Butler LM et al. HLA-B alleles of the Navajo: no evidence for rapid evolution in the Nadene. Tissue Antigens 1996 47: 143-6.

37. Parham P, Ohta T. Population biology of antigen presentation by MHC class I molecules. Science 1996 272 67-74.

366 Tksue Antigens 1998: 51: 356366