In HbE carriers of West Bengal

Name: Panchali Das

Stream : Genetics

Roll No: 21008310018 3rd Year 6th Semester

Molecular Detection of Codon 26 (G-A) Mutation

Presented by-

Thalassemia  is an inherited autosomal co-dominant blood disease.

In thalassemia, the genetic defect results in reduced rate of synthesis of one of the globin chains that make up hemoglobin.

Reduced synthesis of one of the globin chains can cause the formation of abnormal hemoglobin molecules, thus causing anemia, the characteristic presenting symptom of the thalassemias.

Introduction to Thalassemia

Thalassemia

•First described by Dr Thomas Cooley in 1925.

•He observed the disorder in patients ofmediteranean ancestry, and called “Cooley's anemia”.

•Doctors at the University of Rochester coined the name Thalassemia.

•The name is derived from the Greek word “Thalasso = Sea” and “Hemia = Blood” in reference to anemia of the sea.

History of Thalassemia

•Thalassemia is found in parts of the world where malaria is common.

•It occurs most frequently in people from : Mediterranean countries North AfricaThe Middle EastIndiaCentral & Southeast Asia.

Geographical Distribution

Now before moving on to know more about thalassemia, let us gain deeper knowledge about hemoglobin.

Now before moving on to know more about Thalassemia, let us gain deeper knowledge about Hemoglobin….

Hemoglobin & Thalassemia

Thalassemia is caused by variant or missing genes

that affect how the body makes

hemoglobin.

Hemoglobin is the protein in red blood

cells that carries oxygen. People with

thalassemia make less hemoglobin and fewer circulating red blood

cells than normal. The result is mild or severe

Anemia.

Pale eyes shows symptoms of

Anemia

Hemoglobin structure & function

Description of Hemoglobin structure

Hemoglobin has a quaternary structure characteristic of many multi-subunit globular proteins.Hemoglobin's quaternary structure comes from its four subunits in roughly a tetrahedral arrangement.In most vertebrates, the hemoglobin molecule is an assembly of four globular protein subunits.Each subunit is composed of a protein chain tightly associated with a non-protein heme group. Normal adult hemoglobin consists of 2 -globin protein chains and 2 β

-globin protein chains arranged in a heterotetramer.αThe alpha globin chains are encoded by two closely linked genes on chromosome 16.The beta globin chains are encoded by single gene on chromosome 11.

He Carries oxygen from the lungs to the body tissues and take carbon dioxide from the tissues to the lungs.

Interacts with nitric oxide (NO) to regulate the blood pressure.

Hemoglobin is an iron-protein compound in red blood cells that gives blood its red color.

Function of Hemoglobin

Hemoglobin

Hemoglobin A (α2β2)

Hemoglobin A2 (α2δ2)

Hemoglobin F (α2γ2)

Hemoglobin E (α2βE

2)

Hemoglobin Types

Hemoglobin A (α2β2) - The most common with a normal amount over 95%.

It is composed of one α2β2 tetramer and 4 Heme molecules of a weight of 614 (X4).

Hemoglobin A2 (α2δ2) –Normal variant of hemoglobin A that consists of 2α and 2δ chains.

Found in small quantity in normal human blood. Hemoglobin A2 may be increased in beta thalassemia or to people

who are heterozygous to beta thalassemia gene.

Contd..

δ chain synthesis begins late in the third trimester and in adults, it has a normal range of 1.5-3.5%.

Its biological importance is not yet known.

Hemoglobin F (α2γ2) - Fetal hemoglobin (also hemoglobin F or HbF or α2γ2) is the main oxygen transport protein in the human fetus during the last seven months of development in the uterus and in the newborn until roughly 6 months old.

In adults Hemoglobin F is restricted to a limited population of red cells called F-cells.

Contd..

However, the level of Hb F can be elevated in persons with sickle-cell disease and beta-thalassemia.

Hemoglobin E (α2βE2) - Another variant due to a variation in the

β-chain gene. This variant causes a mild chronic hemolytic anemia.

Thalassemia classification

α Thalassemia

thalassemia β

Thalassemia major

Thalassemia intermedia

Thalessemia minor

α Thalassemia - Here production of the α globin chain is affected. β Thalassemia - Here production of the β globin chain is affected. Alpha (α) thalassemia - is a form of thalassemia involving the

genes HBA1 and HBA2. Four genes located on chromosome 16 control the production of

alpha globin. Alpha thalassemia results when one or more of these genes, or parts

of them, are missing (a deletion) or carry a point mutation (a change in a single base pair of DNA). Deletions impair the production of alpha chains.

Thalassemia classification

Contd..

A point mutation may also impair the production or slightly alter the protein structure, resulting in a dysfunctional alpha chain. All affected individuals have a variable degree of anemia (low Hb) which can cause pale skin, weakness, fatigue, and more serious complications. The most severe types are usually incompatible with life, the foetus dies in the uterus or affected babies die soon after birth. Beta (β) thalassemia- Beta thalassemia’s are due to mutations in the HBB gene on chromosome 11,also inherited in an autosomal -recessive fashion. It involves decreased production of normal adult hemoglobin (Hb A).

Classified into- Thalassemia Major (also known as Cooley's anemia) Thalassemia Intermedia and Thalessemia Minor.

Thalassemia Major-The child born with thalassemia major has two genes for beta thalassemia and no normal beta-chain gene. The child is homozygous for beta thalassemia. This causes a striking deficiency in beta chain production and in the production of Hb A. Treatment for patients with thalassemia major includes chronic blood

transfusion therapy, iron chelation, splenectomy, and allogeneic hematopoietic transplantation.

Contd..

Thalassemia Major

Thalassemia Intermedia is milder than thalassemia major. The signs and symptoms of thalassemia intermedia appear in early childhood or later in life. Affected individuals have mild to moderate anemia and may also have slow growth and bone abnormalities.

Thalassemia Minor Caused by heterogenous mutations that affect beta globin synthesis. Usually present as mild, asymptomatic hemolytic anemia unless patient in under stress such as pregnancy, infection, or folic acid deficiency. Have one normal beta gene and one mutated beta gene. Normally require no treatment. Make sure patients are not

diagnosed with iron deficiency anemia.

Contd..

Thalassemia Minor

Genetic Prevalence

and thalassemia are often α βinherited in an autosomal recessive fashion although this is not always the case.

For the autosomal recessive forms of the disease both parents must be carriers in order for a child to be affected. If both parents carry a hemoglobinopathy trait, there is a 25% chance with each pregnancy for an affected child. 

Genetic counselling and genetic testing is recommended for families that carry a thalassemia trait.

Pathophysiology of alpha thalassemia- α thalassemias result in decreased alpha-globin production,

therefore fewer alpha-globin chains are produced, resulting in an excess of β chains in adults and excess γ chains in newborns.

The excess β chains form unstable tetramers (called Hemoglobin H or HbH of 4 beta chains) which have abnormal oxygen dissociation curves.

The excess γ chains form tetramers which are poor carriers of O2 since their affinity for

O2 is too high so it is not dissociated in

the periphery. Homozygote α0 thalassaemias, where there is lots of γ4 but no α-globins at

all (referred to as Hb Barts), often result in still birth.

Pathophysiology

Pathophysiology of β-Thalassemia : The central mechanism underlying the pathophysiology of the β

thalassemias can be related to the deleterious effects of imbalanced globin chain synthesis on erythroid maturation and survival.

An imbalance of the α/non-α globin chains leads to an excess of unmatched α globin which precipitates out, damaging membrane structures leading to accelerated apoptosis and premature destruction of the erythroid precursors in the bone marrow.

Currently, the most promising molecular therapeutic approaches include increasing β globin gene expression by stem cell gene therapy and increasing γ globin expression using pharmacological agents or by transduction of the γ globin genes.

Contd..

Case StudiesNAME CBC PARAMETER   HPLC PARAMETER

  MCV MCH MCHC RDW Hb HcT HBF HBE HBA

CASE1 66 21.5 32.6 25.6 6.3 19.5 15.2 45.4 31

CASE2 67.3 23.2 34.5 18.3 11.3 32.7 0.6 28.2 61.6

CASE3 70.4 24.5 34.9 17 10.2 29.2 1 26.3 61.8

CASE4 72.9 23.6 32.3 20.7 12.9 40.1 0.6 24.2 65.9

CASE5 68.2 24.4 35.7 17.3 12.5 35 1 26.5 61.6

CASE6 53.1 18.2 34.4 21 8.6 25.1 0.5 24.4 65.8

CASE7 74.6 28 37.5 19.7 13.6 36.2 0.3 26.4 26.4

CASE8 80.4 28.5 35.4 18.2 9.7 27.5 0.4 25.8 64.8

CASE9 71.6 25.8 36 15.8 11.6 32.3 1.4 25.2 55.2

CASE10 78.5 24.8 31.6 18.8 11.3 35.8 0.6 26.2 64.6

MCV MCH MCHC RDW Hb Hct HBF HBE HBA

0

10

20

30

40

50

60

70

80

SUMMARY OF THE CASE STUDIES

Variation of different Hb, CBC and HPLC parameters of selected patients-

LABORATORY TECHNIQUES

DNA sample was extracted from the blood sample of thalassemia patient.

ARMS-PCR from extracted DNA to detect the mutation.

Material Required :1. Resuspension Solution2. Lysis Solution3. Prewash Solution4. Wash Solution5. Elution Buffer6. Protinase K7. HiElute Miniprep Spin Column8. Rnase A Solution9. Collection Tubes,10. Polypropylene

Isolation of DNA

Prewash Solution Concentrate

Wash Solution Concentrate

Resuspension Solution

Elution Buffer

Lysis Solution

BLOOD COLLECTION• The whole blood is collected in an anticoagulant tube under

sterile condition. The blood sample must be at the room temperature before beginning the work.

PROTEINASE TREATMENT• 20µl of the reconstituted Proteinase k solution is added into a

collection tube containing 200µl of the whole blood. Vortex is performed for 10-15 seconds to ensure thorough mixing.

LYSIS REACTION• 200µl of the lysis solution is added to the sample. The vortex

is then performed for a few second to homogenise the solution. It is incubated for 10 minutes at 550C.

DNA Isolation- Procedure

PREPARE FOR BINDING• 200µl of ethanol is added to the lysate obtained from step 3

for preparation of lysate for binding to the spin column. It is mixed thoroughly by gentle pipetting.

LOAD LYSATE IN HIULUTE IN MINI PREP SPIN COLUMN• The lysate obtained from step 4 is transferred into the spin

column provided. It is centrifuged at 10,000 rpm for 1 min. The flow –through liquid is discarded & the column is placed in a new 2.0 ml collection tube.

PRE WASH• 500µl of diluted prewash solution is added to the column. It is

centrifuged at ≥6,500×g(≈10,000rpm) for 1 minute. The flow-through liquid is discarded & the same collection tube with the column is re-used.

WASH• 500µl of diluted wash solution is added to the column &

centrifuge at 12,000-16,000×g (≈13,000-16,000 rpm) for 3 minutes to dry the column. The collection tube containing the flow through liquid is discarded & the column is placed in a new 2 ml collection tube.

DNA Isolation• Pipette 100µl of the elution buffer directly on to the column.

It is incubated for 1 minute at R.T. Centrifuge at ≥6,500×g (≈10,000 rpm) for 1 minute to elute the DNA. The step is repeated again with another 100µl of Elution buffer for high yield of DNA.

Material Required:1. dNTPs2. ARMS-PCR buffer 3. Taq polymerases

4. Tris-borate-EDTA(TBE) buffer

ARMS-PCR to detect the mutation

Principle of ARMS-PCR

The technique of ARMS is based on the principle of allele specific

priming of the PCR process, a specific primer will only permit

amplification to take place, when it’s 3’ terminal nucleotide

matches with it’s target sequence.

Thus to detect the HbE mutation Cod 26 (G->A), the 3’ nucleotide of the ARMS primer is T in order to base-pair with A in the mutant DNA. The primer forms the G-T mismatch with the normal DNA ,but this is a weak mismatch and will not prohibit the extension of the primer itself. Only

strong mismatches (C-C , G-A and A-A) were found to reduce priming efficiency to zero or below 5%.

A reaction mixture prepared for 200 reactions(4 ml) comprising of 0.5 ml of 10 X ARMS-PCR buffer , 1.25 ml of 1.35 mM dNTP mixture ; 2.65ml of sterile distilled water.

20 μl of PCR reaction mixture is pipetted out into a 1.5 ml tube.

1 μl of each primer (1 OD unit/ml) is added.

0.05 μl of Taq polymerase (5 u/μl) is added.

Mutation Detection - PROCEDURE

More than one test is performed , the primer and the enzyme are mixed together in a separate tube before addition to the reaction mix. This decreases pipetting errors in as larger quantities are used.

1 μl of genomic DNA (100mg/μl) & overlay with 25μl of mineral oil is added and mixed.

Then the mixture is centrifuged and placed in thermal cycler.

Contd..

Amplify for 25 cycles as follows: 1 min at 94 C / 1 min at 65 C / 1.5 min at 72 C with a final extension period of 3 min at 72 C following the 25 cycle.

Tubes are removed from thermal cycler and 5 ml of blue dye is mixed and centrifuged.

A 20 ml aliquot is loaded onto a 3% agarose gel and run at 100 V for approx. 45 min in TBE.

Gel is stained in ethidium bromide solution (0.5 mg/ml)for 15 to 30 minutes and bands can visualize on a UV light box (312 nm) and photograph with an electronic camera system or a Polaroid CU-5 fitted with an orange filter

Contd..

Mol. wt. MarkerSample with Codon 26 (G-C) mutation Sample with Codon 26 (G-C) mutationSample with Codon 26 (G-C) mutationNegative control

OBSERVATION & RESULT

236 bp

Codon 26 (G-A)

751 bpM1

2

3

4

M 1 2 3 4

Lane Content

HbE Mutation

CONCLUSION

Thalassemia is highly prevalent and is a major public health problem along the parts of Indian Subcontinent including West Bengal and Middle East . Cross-sectional surveys indicate that in many regions of the Indian Subcontinent and Middle East, only a few prevalent Hb mutations comprise the majority of patients with beta and HbE beta thalassemia.

So, HbE carriers are having major risk of developing HbE-beta offspring in their next generation, if they marry beta carriers/ patients.

So, molecular detection of HbE mutation [Cod 26 (G-A)] is a very important study in the context of the disease.

REFERENCES

www.wikipedia.com

pmoi@unica.it and sadelain@ski.mskcc.org.

www.urmc.rochester.edu/geneticsHemoglobin E-beta Thalassemia: Factors AffectingPhenotype I. Panigrahi, S.Agarwal, T. Gupta, P. Singhal and M. Pradhan

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