Introduction:

Thalassemia is the most commonly inherited blood disorder worldwide (Garewal et al, 2003). Thalassemia is an autosomal recessive disorder inherited in families, in which the body makes an abnormal form of hemoglobin (the protein in red blood cells that carry oxygen). This disorder result in excessive destruction of red blood cells which lead to anemia. Hemoglobin is made of two proteins- alpha globins and beta globins. Thalassemia occurs when there is a defect in a gene that helps control production of one of these proteins. In the thalassemia patient, a mutation or deletion of the genes that control globin production occurs. This leads to a decreased production of the corresponding globin chains and an abnormal hemoglobin ratio. This abnormal ratio leads to decreased synthesis of hemoglobin and the expression of thalassemia. The globin that is produced in normal amounts winds up in excess and forms red cell aggregates or inclusions. These aggregates become oxidized and damage the cell membrane, leading to hemolysis, ineffective erythropoiesis, or both. The quantity and properties of these globin chain aggregates determine the characteristics and severity of the thalassemia. (Giardina et al,2008)

Genetic backgroundHemoglobin comprises four globin chains: fetal hemoglobin (Hb F) has two α and two gamma chains (α2γ2) and adult hemoglobin (Hb A) has two α and two β chains (α2β2). Genes in the α-globin and β-globin gene clusters (on chromosomes 16 and 11) control globin-chain production. Due to spontaneous mutation, hemoglobin gene variants are present at low prevalence (carriers 1–1.5/1000) in all sizeable populations. (Livingstone FB, 1985) (Modell B et al, 2007)

TYPES OF THALASSEMIA: ALPHA THALASSEMIA AND BETA THALASSEMIA

Alpha Thalassemia: It occurs when a gene or genes related to alpha globin protein are missing or mutated. Alpha (α) thalassemia involves the genes HBA1 and HBA2 (OMIM 141800 and OMIM 141859). It is also connected to the deletion of 16p chromosome. α-thalassemia results in decreased alpha globin production therefore fewer alpha globin chains are produced, resulting in excess of beta (β) chains in adults and gamma (γ) chains in newborns. The excess of β-chains form unstable tetramers (called hemoglobin H or HbH of 4 beta chains) which have abnormal dissociation curves. Alpha thalassemias occur most commonly in persons from Southeast Asia, the Middle East, China, and in those of African descent.

There are at least five main types of alpha thalassemia. These are most common in people of Southeast Asian, Indian, southern Chinese, Middle Eastern and African ancestry (Cooley’s anemia Foundation, 2007). There are four genes that control the production of alpha globin. The severity of the condition is determined by how many of these genes are missing or abnormal.

Silent carrier, the mildest form, has one alpha globin gene missing or abnormal. Affected individuals generally have no symptoms, but they can pass on the genetic abnormality to their children.

Alpha thalassemia minor (also called alpha thalassemia trait) has two missing or abnormal alpha globin genes. Affected individuals may have no symptoms or a mild anemia, but they can pass the condition on to their children.

Alpha thalassemia major, the most severe form, is caused when there are no alpha globin genes. Affected fetuses suffer from severe anemia, heart failure and fluid buildup. They usually are stillborn, but some die in the first hours after birth. In rare cases, babies diagnosed and treated before birth with blood transfusions have survived. These babies require lifelong blood transfusions (Cooley’s anemia Foundation, 2007 and Northern California Comprehensive thalassemia centre, 2008).

Hemoglobin H disease is caused by three missing or abnormal alpha globin genes (so there is one normal alpha globin gene). The condition causes abnormalities in red blood cells and rapid destruction of these cells. Most affected individuals have mild to moderate anemia and can live fairly normal lives. The anemia may temporarily worsen when individuals have a viral infection or when they are treated with certain medications (such as sulfa drugs) (Northern California Comprehensive thalassemia centre, 2008).

Hemoglobin H-Constant Spring is a more severe form of hemoglobin H disease. Affected individuals have one normal alpha globin gene, plus a specific mutation (change) called Constant Spring on one of their three abnormal genes. People with this condition generally have moderate to severe anemia and often develop complications, such as an enlarged spleen. Some need blood transfusions from time to time, such as when they develop an illness with a fever, while others need more frequent transfusions (Northern California Comprehensive thalassemia centre, 2008) and (Cohen, A.R., et al. 2004)

BetaThalassemia: occurs when similar gene defects affect production of beta globin protein (Giardina et al, 2008 and DeBaun MR et al, 2007). Β-thalassemia are due to mutation in HBB gene on chromosome 11 (OMIM 141900) also inherited in autosomal recessive fashion. The severity of disease depends on the nature of the mutation. Mutations are characterized as (βo or β thalassemia major or Cooley’s anemia) if they prevent any formation of β chains (which is the most severe form of β thalassemia); they are characterized as (β+ or β thalassemia intermediate) if they allow some β chain formation to occur. In either case there is a relative excess of α chains, but these do not form tetramers: rather, they bind to the red blood cell membranes, producing membrane damage, and at high concentrations they form toxic aggregates. Beta thalassemias occur in persons of Mediterranean origin, and to a lesser extent, Chinese, other Asians, and AfricanAmericans.There are three main forms of beta thalassemia. These are most likely to affect people of Greek, Italian, Middle Eastern, Southeast Asian, southern Chinese and African descent (Cooley’s anemia Foundation, 2007). Two genes control the production of beta globin. Mutations on one or both of them can cause the disorder. The severity of the condition is determined by whether one or both beta globin genes carry a mutation and by the severity of the mutation.

Thalassemia minor (also called thalassemia trait) is caused by a mutation on one beta globin gene. Most affected individuals have no symptoms, though some have mild anemia. Affected individuals can pass the abnormal gene on to their offspring.

Thalassemia intermedia results from abnormalities in both beta globin genes. These gene abnormalities are generally less severe than those that cause thalassemia major. Affected children usually have mild to moderate anemia, and they may develop some of the complications seen in thalassemia major, including enlarged spleen and bone abnormalities. Many affected individuals require occasional or more frequent blood transfusions to reduce complications (Rund D et al, 2005).

Thalassemia major, the most severe form, results from severe mutations on both beta globin genes. It also is called Cooley's anemia, named after the doctor who first described it in 1925. Most affected children appear healthy at birth. However, during the first year or two of life, they become pale and fussy and have a poor appetite. They grow slowly and often develop jaundice (yellowing of the eyes and skin). Without treatment, they develop an enlarged spleen and liver, thinning bones that break easily, abnormal facial bones, frequent infections and heart problems, and they die in the first decade of life. Affected children require regular blood transfusions beginning in infancy.

Other forms of thalassemia include:

E-beta thalassemia results from one beta globin gene carrying a thalassemia mutation (thalassemia minor) and one beta globin gene carrying a mutation that produces a variant form of hemoglobin called hemoglobin E. This condition is most common in people from Southeast Asia, including Cambodia, Vietnam and Thailand (Cooley’s anemia Foundation, 2007). Individuals who produce hemoglobin E generally are healthy or have only a mild anemia. However, those with E-beta thalassemia have mild to severe anemia, resembling beta thalassemia intermedia or beta thalassemia major (Cooley’s anemia Foundation, 2007).

Hb S/beta thalassemia results from one beta globin gene carrying a thalassemia mutation (thalassemia minor) and one gene for sickle cell disease, another inherited anemia. It is most common in those of African or Mediterranean ancestry (Cooley’s anemia Foundation, 2007)). Symptoms generally resemble those of sickle cell disease, including varying degrees of anemia, serious infections, pain and damage to vital organs.

Delta-thalassemia: It is associated with HbD (Drakoulakou O et al, 1997). As well as alpha and beta chains being present in hemoglobin about 3% of adult hemoglobin is made of alpha and delta chains. Just as with beta thalassemia, mutations can occur which affect the ability of this gene to produce delta chains.

BETA THALASSEMIA MINOR:Thalassemia minor is most common form of beta-thalassemia, and is also known as the ‘thalassemia trait’, in which affected individuals are asymptomatic (CaO A GR, 2005 and Rund D et al 2005) .These subjects are typically heterozygous for beta-thalassemia since they carry one normal HBB allele and one thalassemia allele - either B0 or B+ (Thein SL,2004) . Thalassemia minor occurs if you receive the defective gene from only one parent. Persons with this form of the disorder are carriers of the disease and usually do not have symptoms. Asymptomatic patients are usually detected through routine hematologic testing, but in retrospect some newly diagnosed patients are observed to have mild anemia and small RBCs (Bunn HF FB, 1984). The primary caution for individuals with thalassemia minor is a potential risk of having

children affected with more serious thalassemia if their partner is also a carrier of thalassemia minor (Thein SL, 2004).

THE BETA-GLOBIN LOCUS The beta-globin chain is encoded by the HBB gene, which spans 1.6 kb on the short-arm of chromosome 11 (11p15.4; MIM: 141900) [Cao A GR.cited 2007 and OMIM. Hemoglobin-Beta Locus; HBB. John Hopkins University1986.]. The genomic sequence of HBB contains three exons, two intervening sequences (IVS1 and IVS2) and the 5' and 3' untranslated regions (UTRs). The HBB gene is regulated by a 5' promoter region that contains the classical TATA, CAAT and duplicated CACCC boxes. Upstream of the beta-globin cluster is another regulatory element for HBB, namely the locus control region (βLCR). The expression of individual globin genes is governed by direct physical interaction between the βLCR and globin promoters, mediated by the binding of tissue- restricted and ubiquitous transcription factors [Grosveld F, de Boer E, Dillon N, et al 1998]. The most important transcription factor for HBB is the erythroid Kruppel-like factor (EKLF), which binds the proximal CACCC box [Cao A GR.cited 2007]

Schematic representation of beta-globin locus. (a) Schematic structure of the beta-globin gene, with 3 exons (grey boxes) and the two intervening sequences (IVS). The gene is flanked by the 3’ and 5’ UTR regions (white boxes). The promoter region upstream of the 5’UTR (stripped box) contains the following regulatory sequences: TATA, CAAT, and duplicated CACCC boxes. (Adapted from Thein 2005); (b) Schematic structure of the beta-globin locus that resides on chromosome 11p15.4. From 5’ the first gene, HBE (ε), is expressed at the embryonic stage to produce embryonic-hemoglobin (Hb-E). The next two genes, HBG-G (γ-G) and HBG-A (γ-A), are expressed during fetal development (12 weeks post-conception) to form fetal-hemoglobin (Hb-F). After 7 or 8 months after birth, adult-hemoglobin (Hb-A) is produced due to initiation of

expression of the HBB (β) gene. 3% of adult-hemoglobin is adult-hemoglobin-type 2 (Hb-A2), composed of the δ-globin chain.Prevalence of Beta Thalassemia in India: β-thalassaemia is detectable in almost every Indian population; however, it is seen with highest frequency in north-west and Far East. Sindhis, Guajarati’s, Bengalis, Punjabis and Muslims account for most of β- thalassaemia (Agarwal MB et al, 1982).Carrier state for β-thalassaemia in India varies from 1-17% with an average of 3.2 %.( Agarwal MB et al, 1982). Based on this mean carrier frequency and the demographic profile of India, estimated number of beta-thalassaemia carriers is about 29.7 million, while the birth of b-thalassaemics are 7000 per year (Verma IC,1994)

Associated Gene Studies Internationally and Nationally

The molecular studies done on β-thalassemia are reviewed for the current study. A study from Canada by Cornut G et al in 2007 revealed new mutations causing a beta (+)-thal minor phenotype was found in a patient of Arabic origin. The insertion frameshift mutation (+A) between codons 45 and 46 [codons 45/46 (+A)] results in a premature termination signal at codon 52. No truncated beta-globin or abnormal hemoglobin (Hb) was identified.

Another study in Czech in 1999 the authors describe a newly identified beta0-thalassaemic mutation found in two subjects from two generations of a Slovak family. The beta0-thalassaemic allele developed by insertion of one nucleotide (+G, CD 7/8) into the first exon of the beta-globin gene. The mutation causes a shift of the open globin reading frame which leads to the development of a terminal codon in codon 22. The thalassaemic allele is associated with the mediterranean haplotype IX. The mutation has in both heterozygotes the phenotype of beta0-thalassaemia minor with a slightly elevated level of HbF.

In 2003, a study by Jazayeri M et al in Iran Ninety-three (56 females) documented BTM cases and 104 (54 females) controls were enrolled in the study. All three C282Y mutations were found in BMT patients with one of them being a compound heterozygote. Results suggest that HFE mutations C282Y and H63D are more frequent in Iranian BTM patients than in the normal population, causing no significant changes in serum ferritin level.

A study from China by Zheng MQ et al in 2008 indicated that indicated that the C-->T heterozygous mutation occurred at the IVS-2 -654 site in 4 cases; the TTCT deficiency appeared at CD41/42 site in 1 case; in 2 sites existed single nucleotide polymorphisms occurring at the 59th site of exon 1 (T/C, CAT/CAC, His) and IVS-2 nt 665 (T/C). It is concluded that singte nucleotide polymorphism of minor beta-thalassemia patients born in Wenzhou had specificity; this study found too kinds of gene mutations which are IVS-2 -654 C-->T heterozygous mulation and CD41/CD42 site-TTCT deficiency.

A study from Italy in 2009 by Piras IS et al showed the presence of a correlation between a (CA) n repeat located in exon 29 of NOS1 gene and the beta-thalassemia trait in Corsica Island (France). They genotyped a sample of individuals with beta-thalassemia minor (N=110) and an ethnically matched control (N=113) from Balagna, a region of Corsica Island (France). Results highlighted the high frequencies of allele with 16 and 17 repeats in the thalassemic sample.

Maria Antonietta Melis et al in 2002 studied H63D mutation in the HFE gene increases iron overload in β-thalassemia carriers. β-thalassemia carriers have higher ferritin levels than β-

thalassemia carriers with who are homozygotes for the H63D mutation the H/H genotype , suggesting that the H63D mutation may have a modulating effect on iron absorption.

T.M. Oliveira et al in 2004 studied 102 patients with alpha-thalassemia (69 women and 33 men), 168 patients with the beta-thalassemia trait (96 women and 72 men), and 173 patients without hemoglobinopathies (16 women and 157 HFE mutations in thalassemic patients men). Allelic frequencies in the alpha-thalassemic group were 13.72% for the H63D mutation, 0% for the S65C mutation and 0.98% for the C282Y mutation. In the beta-thalassemic group, mutation frequencies were 13.70% for H63D, 0.60% for S65C and 2.38% for C282Y.

Rute Martins et al in. 2004 evaluate the effect of genetic markers (HFE mutations C282Y, H63D, and S65C) on the iron status of beta-thalassemia carriers. A total of 101 individuals heterozygous for beta-thalassemia and 101 normal control individuals were studied. The allelic frequencies of C282Y (1.5 versus 3.5%), H63D (15.3 versus 18.3%), and S65C (1.0 versus 1.5%) did not differ significantly between beta-thalassemia carriers and normal controls. These results suggest that the beta-thalassemia trait tends to be aggravated with the coinheritance of H63D mutation, even when present in heterozygosity.

In 2005 A study was conducted by Garewal G et al in Chandigarh shows that H63D is prevalent and C282Y is rare in north Indians and the presence of H63D mutation does not increase body iron as measured by serum ferritin in beta thalassemia traits. Haplotype of H63D gene mutation is of European haplotype, indicating a common origin.

Another study from Lucknow in 2007 by Agarwal S et al analyzed 729 north Indian samples for C282Y and H63D mutations. Of these, no allele of the C282Y mutation was seen, while 3 homozygous and 43 heterozygous for the H63D mutation were seen in the patients of thalassemia group. However, 47 cases were found heterozygous for the H63D mutation among the normal groups (11.16%).

Methodology: Blood samples will be gathered from the subjects (cases and controls) and DNA will be extracted out. Then amplified by using PCR technique and SNP will be studied.

Objectives:

1. To study single nucleotide gene polymorphism in beta thalassemia minor subjects in north-west population of Punjab.

2. Screening the mutations in beta globin HBB gene in patients (diagnosed) and controls.

3. To evaluate the prevalence of thalassemia carriers in north-west population of Punjab.

4. To identify the relation of these mutations with other diseases.

5. To make people aware about the risk factors in further generations especially in pregnant women’s having thalassemia minor trait.

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