Molecular Basis of β-Thalassemia in Karnataka,India

Introduction

β-Thalassemia is a common autosomal recessive inherited single gene disorder in humans caused by mutations in the β-globin (HBB) gene. It is highly prevalent in the Meditertanean region, the Asian-African continents in general, and the malaria endemic region in particular (Tuzmen and Schechter, 2001; Patrinos et al., 2005). The HBB gene encodes the beta-globin chains of hemoglobin, an oxygen-carrying protein composed of two α- and two β-chain subunits found within red blood cells. This gene has three exons and two introns involved in β-thalassemic pathogenesis. Mutations in the HBB gene lead to an altered β-globin chain resulting in structural change of the protein conformation of hemoglobin. In β-thalassemia, point mutations in the β-globin structural gene are largely responsible for either decreased or no β-globin synthesis (Giardine et al., 2007). β-Thalassemia may also arise from small deletions or insertions within the β-globin gene. Approximately 600 mutations have been identified in the β-globin (HBB) gene, of which more than 200 are associated with β-thalassemia phenotype (Huisman and Carver, 1998; Patrinos et al., 2005). Previous studies have demonstrated the presence of population-specific mutations, with different frequencies probably caused by conservation of gene pools, selection, and genetic drift (Rund et al., 1991; Agarwal et al., 2000).

The Indian subcontinent is inhabited by nearly 5000 anthropologically well-defined indigenous populations, which are from diverse ethnic, genetic, tribal, cultural, and social background (Singh, 2003). The caste endogamy and clan exogamy, which are practiced among different communities since time immemorial, might have led to a conservation of gene pools among them (Bhasin et al., 1994). This in turn might have resulted in accumulation of mutations leading to increased frequency of genetic disorders (Kivisild et al., 2003). The initial studies in the Indian population on β-thalassemia were undertaken in the individuals from the states of Gujarat and Punjab and the Sindhi community. Many of these groups who originated in Pakistan and are also living in other countries revealed five major mutations, IVSI-5(G-C), IVSI-1(G-T), 619-bp del, codon 41/42 (-TCTT), and Codon 8/9 (+G), which accounted for 90% of all mutations (Kazazian et al., 1984; Thein et al., 1988). Later studies that focused on the populations of Gujarat, Punjab, and Maharashtra are consistent with the results of the previous studies (Varawalla et al., 1992; Garewal et al., 1994). Further studies revealed wide distribution and extensive heterogeneity of β-thalassemia mutations in different subpopulations of India (Gupta et al., 2003; Bashyam et al., 2004; Sinha et al., 2009a, 2009b; Agarwal et al., 2010; Kumar and Agarwal, 2011) and found that the highest percentage of rare mutations was from the Karnataka region (Colah et al., 2009).

A large number of studies have been carried out in North Indian populations (Gupta et al., 2003; Sinha et al., 2009a, 2009b; Agarwal et al., 2010; Kumar and Agarwal, 2011). The Indian population is comprised of different ethnic groups with much genetic heterogeneity (Reich et al., 2009). The population of North India is largely Indo-European and hence different from other regions of the country. Therefore, previous studies may not be sufficient to describe the complete spectrum of HBB gene mutations in this country. Very little data about HBB gene mutations in the population of South India are available so far. Therefore, the purpose of this study was to determine the spectrum of β-thalassemia mutation in the population of the South Indian region.

Subjects and Methods

Thirty-six thalassemia patients from the Karnataka region, who regularly attend the Karnataka Institute of Medical Science, Hubli and JJM Medical College, Davanagere, during January 2008 to February 2010, were included in the present study. All the thalassemia patients were confirmed with hemoglobin electrophoresis (Interlab Genio S Electrophoresis, Via Rina Monti, Roma, Italy). All patients were also transfusion dependent. Blood samples (5.0 mL) from each individual were collected just before the transfusion, with the informed written consent of all patients who are above 18 years or parents of individuals who are <18 years old following the institutional ethical guidelines. Genomic DNA was extracted from all participants using a standard procedure described elsewhere (Thangaraj et al., 2002).

Results

Ten mutations found in the patients are shown in Table 1. Among 36 samples analyzed, 13 showed IVSII-16 G>C, 10 of IVSI-5G>C, 8 of IVSII-74 T>G, 7 of codon 3 (T>C), 3 of Poly A site (T>C), and 1 each of codon 6 (-CT), codon 16 (G>A), codon 31 (G>C), codon 31 (G>A), and IVSII-837 T>G. In the present study, we documented a novel frameshift deletion (-CT) at codon 6 in one sample [Codon 6 (-CT)] (Fig. 1). IVSII-16 G>C is the predominant mutation (36%) followed by IVSI-5G>C (28%), IVSII-74 T>G (22%), codon 3 (T>C) (19%), and Poly A site (T>C) (8%).

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FIG. 1.

Nucleotide sequence of the proband β-globin gene, codons 1-9. The upper sequence is normal. The lower sequence shows a mutation at codon 3 (ATC>ACC) and novel frameshift change, starting at codon 6 deletion of CT in codon 6 (CTG>GAG).

Discussion

Although several causative mutations have been reported to be associated with β-thalassemia syndromes, the spectrum of mutations and their frequencies in most populations usually consist of a limited number of common mutations and a slightly larger number of rare mutations (Hardison et al., 2002). Sequence analysis of the HBB gene in the populations of the Karnataka region revealed that among some common mutations, IVSII-16 G>C is the predominant mutation (36%), followed by IVSI-5G>C (28%), IVSII-74 T>G (22%), codon 3 (T>C) (19%), and Poly A site (T>C) (8%) and a few rare mutations: IVSII-16 G>C, IVSI-5G>C, IVSII-74 T>G, codon 3 (T>C), Poly A site (T>C), codon 6 (-C), codon 6 (-T), codon 16 (G>A), codon 31 (G>C), IVSII-837 T>G, and codon 31 (G>A) (3% each). Interestingly, for the first time we have found two deletions at codons 6 (-C) (c.16delC) and 6 (-T) (c.18delT) in one patient whose beta thalassemia was diagnosed in 1998 when she was 4 months old and till now she is dependent on blood transfusion. This girl presented with peculiar and hematological features such as progressive pallor, frontal and parietal bone prominence, depressed nasal bridge, prominent malar eminence, maloccluded teeth, hyperpigmentation of palm, sole, and skin due to hemochromatosis, hepatomegaly, and stunted growth and hemoglobin electrophoresis shows HbA 95.1% and HbA2 4.9%.

IVSII-16 G>C is in the alternative splicing region of mRNA, which might lead to differential expression. Chinese IVSII-16 C>G subjects have shown a greater improvement of running economy indexes after 18-week 5000-m running program than those with other genotypes (He et al., 2006). This polymorphism is quite common in Indian populations. Fifty-five populations that were studied all over India have revealed this C allele frequency to be ranging from 0.21 in Indo-Europeans to 0.66 in Dravidian linguistic groups. IVSI-5(G>C) was found in an Asian Indian (Kazazian et al., 1984) and in a Chinese (Cheng et al., 1984). Although this is the second major mutation in the present study, this accounts for 54.7% of all β-thalassemia alleles nationally, and the majority of subjects with this mutation originate from or are residents of the major states of Maharashtra, Gujarat, Uttar Pradesh, and West Bengal (Agarwal et al, 1997, 2000; Kukreti et al., 2002). IVSI-5 (G>C) frequency varied from 15% in the immigrant population from Pakistan and Goa on the west coast to 88.6% in the eastern state of Orissa (Colah et al., 2009). Codon 3 (T>C) mutation changes the third codon of the HBB gene from CAT to CAC (rs713040), both coding for histidine. This silent mutation with no apparent effect in thalassemia has been observed in patients of Bangladeshi origin (Ibn Ayub et al., 2010).

The Poly A site mutation, AATAAA→AACAAA, was reported earlier in American Blacks (Gonzalez-Redondo et al., 1991). Poly A site (T>C) is a rare mutation in Indian populations (Gorakshakar et al., 2004). The rare codon 31 (G>C) (c.92G>C) mutation found in only one patient completely prevented mRNA splicing and could not produce mRNA in carriers of this mutation as previously reported in a Turkish thalassemic patient (Tadmouri et al., 2000) and a southern Iranian patient (Rahimi et al., 2006). Another rare codon 31 (G>A) (c.92G>A) mutation found in only one patient has been previously reported in a southern Iranian patient (Karimi et al., 2002). Codon 15 (G>A) (c.47 G>A) mutation TGG>TAG, a change of one nucleotide, results in the termination of translation (Gray et al., 1995). The novel codon 6 (-CT) frameshift mutation nucleotide sequence was translated to amino acids and compared with the reference sequence. There is a mismatch of 57% in these two sequences when compared globally in EMBOSS pairwise alignment tool. This frameshift mutation leads to abnormal splicing of β-globin mRNA, resulting in the formation of a nonfunctional β-globin gene product (Fig. 1). The present study reports a novel mutation; the remainder is atypical in Indians. This indicates the increasing collection of molecular defects causing β-thalassemia in Indian populations. According to previous reports, 11 HBB mutations have been shown to be common in India. Among these 11, 6 account for about 94% of all positive cases. These include IVS1+1G>T, IVS1+5G>T, c.124_127delTTCT, c.47G>A, c.366_494del, and c.20A>T. The other two less common mutations are c.27_28insT and c.79G>A.

The spectrum of mutations that are reported in this study confirm the heterogeneity observed in the different population groups within India (Bhasin et al., 1994; Edison et al., 2008; Colah et al., 2009; Sinha et al., 2009b). Population screening associated with genetic counseling is particularly helpful by allowing families at risk to make informed decision on their reproductive choices.