Supriya Phanasgaonkar

Regional heterogeneity of β-thalassemia mutations in the multi ethnic Indian population

To determine the frequencies of beta-thalassemia mutations in different states of India and to compare this with the available data in Asian Indians for a comprehensive catalogue of molecular defects in the Indian population. beta-thalassemia mutations were characterized in 2456 heterozygotes using reverse dot blot hybridization, ARMS and DNA sequencing. 36 beta-thalassemia mutations were characterized from 18 different states in India. Seven mutations were common, accounting for 95.8% of mutated alleles. Marked regional diversity was seen in different parts of the country. Among the tribal populations, only 2 mutations (IVS I-5 (G-->C) and CD15 (G-->A) accounted for over 90% of mutant alleles. A compilation of all the studies in Asian Indians reported so far showed the presence of 63 mutations in the Indian population. This large study adds to the existing data to give a detailed account of the molecular basis of beta-thalassemia in India. This information is important for establishing prenatal diagnosis programmes in different states in India as well as other countries in which there is a major influx of Indian immigrants.

Epidemiology of β‐thalassaemia in Western India: mapping the frequencies and mutations in sub‐regions of Maharashtra and Gujarat

Although the average frequency of β‐thalassaemia carriers in India is 3–4% and the prevalent mutations have been studied, no micromapping has been done. This is the first attempt to provide an accurate estimate of the frequencies of β‐thalassaemia and the expected annual births of homozygous children in different districts of Maharashtra and Gujarat in Western India as well as to determine the molecular heterogeneity in different sub‐regions in these states. A total of 18 651 individuals were screened for haemo‐globinopathies and mutations were characterized in 1334 β‐thalassaemia heterozygotes. There was an uneven distribution of the frequencies of β‐thalassaemia, varying from 1·0% to 6·0% and 0% to 9·5% in different districts of Maharasthra and Gujarat. The rate of homozygosity per 1000 births annually was 0·28 in Maharashtra and 0·39 in Gujarat. The three most prevalent β‐thalassaemia mutations in Maharashtra were IVS 1‐5(G→C), Codon 15(G→A) and Codon 30(G→C) (87·9%) while in Gujarat they were IVS 1‐5(G→C), 619 bp deletion and Codon 5(‐CT) (68·5%). These data will help to develop adequate programmes for control and care where they are most needed. They also emphasize the importance of subgroup micromapping for determining the health burden of a common genetic disease.

Prevalence and Molecular Characterization of α-Thalassemia Syndromes among Indians

OBJECTIVE This study was undertaken to determine the prevalence and molecular basis of alpha-thalassemia in the Indian population and its implications in genetic counseling and prenatal diagnosis. METHODS 1253 random samples were screened for hemoglobinopathies. Red cell indices were measured on the Sysmex K 1000 cell counter; HbA2 and HbF levels were quantitated using high performance liquid chromotography (HPLC). Cellulose acetate electrophoresis and isoelectric focusing (IEF) was done to detect the presence of Hb Barts in cord blood samples. alpha-Globin gene mapping was done using Southern blot hybridization of BamHI and BglII digests. RESULTS Of the 1253 subjects, 132 had a single alpha-gene deletion (10.5%) while 29 had two alpha-gene deletions (2.31%). Fifteen cases showed the presence of alpha-gene triplication (1.1%). A single case showed the presence of one alpha-gene deletion as well as alpha-gene triplication (-alpha/alphaalphaalpha). Overall, the prevalence of alpha-thalassemia was 12.9%. Region-wise and caste-wise analysis showed the highest prevalence of alpha-thalassemia among the Punjabi population originating from the northern region of India. CONCLUSION alpha-Thalassemia is by far the commonest hemoglobinopathy in India, but it is not a cause of serious genetic risk is the milder form (-alpha/alphaalpha) of alpha-thalassemia, which is predominant. Knowing the alpha-genotype is useful for genetic counseling for prenatal diagnosis in couples where one of the parents may have reduced indices coupled with a raised RBC count and normal HbA2 levels.

A NOVEL β0-THALASSEMIA MUTATION AT CODON 55 (−A) AND A RARE 17 bp DELETION AT CODONS 126–131 IN THE INDIAN POPULATION

A new mutation at codon 55 (−A) and a rare mutation, a 17 bp deletion at codons 126–131, that gives rise to β0-thalassemia, were found in the Indian population by means of direct sequencing of two polymerase chain reaction products generated from a 2.3 kb DNA fragment containing the whole β-globin gene. Each polymerase chain reaction product was sequenced on both strands in a mutation-loading format, showing all nucleotide substitutions or deletions/insertions, including mutations and polymorphisms, in the product. The entire protocol requires four sequencing reactions/gel loadings after two successive polymerase chain reactions, which simplifies the mutation search process and reduces the reading error rate.

Red Cell Genetic Abnormalities, [Beta]-Globin Gene Haplotypes, and APOB Polymorphisms in the Great Andamanese, A Primitive Negrito Tribe of Andaman and Nicobar Islands, India

The Great Andamanese are a primitive Negrito tribe of the Andaman and Nicobar Islands, India, with a total population of 37. We studied 29 individuals from eight families from this population for abnormal hemoglobins, G6PD deficiency, DNA haplotypes, and apolipoprotein B (APOB, gene) polymorphism. Hb E was detected in five individuals, the prevalence of Hb E heterozygotes being 14.3%. One individual had b-thalassemia trait. One female was G6PD deficient and showed the G6PD Orissa mutation. Haplotype analysis of the b-globin gene cluster showed that the bE chromosomes were linked to two haplotypes (- - - - - + + and + + - + + + +) representing the framework 1 gene, whereas the bA chromosomes showed eight different haplotypic patterns corresponding to framework 1 and 3 genes. APOB polymorphism analysis showed that the 631-base-pair (bp) allele was the predominant one with a high homozygosity rate, which could be due to the higher rate of inbreeding in this isolated group. The presence of Hb E and our findings on haplotype analysis supports the hypothesis that the Great Andamanese are reasonably believed to be the surviving representatives of the Negrito race that once flourished in the entire Southeast Asian region in ancient times.

Hb QIndia and its interaction with β-thalassaemia : a study of 64 cases from India

Abstract Haemoglobin Q (Hb Q), a relatively uncommon α-chain structural Hb variant, has been reported either in the heterozygous state or interacting with β-thalassaemia. Individuals inheriting Hb Q generally are asymptomatic and are diagnosed by chance during population screening or as a part of a family study. This paper represents the first large study from India of 64 cases of Hb Q, documenting the haematological and molecular findings on 36 cases of Hb Q trait, 22 of Hb Q β-thalassaemia trait and three of Hb Q β-thalassaemia major, as well as a family of Hb Q homozygous cases. Hb Q is detected by Hb electrophoresis and chromatography. Hb Q levels in homozygous cases ranged from 32% to 35%, while in Hb Q heterozygotes the level was 20%. When there was an interaction of β-thalassaemia heterozygotes the level was 14%, and in interacting β-thalassaemia homozygotes the levels range from 7% to 9%. β-thalassaemia mutations were characterised in cases showing elevated Hb A2 levels, which were markedly reduced in the majority of cases in which β-thalassaemia was absent. Hb Q is rare and not a single homozygous case has been reported. However, Hb Q disease showed wide variation in clinical and haematological presentation in the same family.

Hematological and Molecular Analysis of Novel and Rare β-Thalassemia Mutations in the Indian Population

A variety of mutations causing β-thalassemia (β-thal) have been seen in the Indian subcontinent. We report eight families in whom two novel mutations [codon 16 (C>T), IVS-II-613 (C>T)] and three rare mutations [codons 22/23/24 (−7 bp) (−AAGTTGG), −87 (C>A), codon 15 (−T)] were encountered among 375 β-thal heterozygotes. They were referred to us for molecular characterization or prenatal diagnosis during a period of 2 years. Haplotyping was also done for linkage analysis.

Detection of two rare β -thalassemia mutations [-90 (C ® T) and CD 26 (C ®T)] among Indians

BACKGROUND : β -Thalassemia (β -thal) is present in practically every caste group in Indians. Molecular characterization of β -thal in these groups has revealed an extremely heterogeneous picture. AIM : To identify all the mutations and to detect the novel mutations using a versatile mutation detection technique. MATERIALS AND METHODS : Denaturing gradient gel electrophoresis (DGGE) has been established to scan the entire β -globin gene to localize the mutation followed by DNA sequencing for characterization. The DNA samples from two families referred to us either for prenatal diagnosis or for DNA studies were studied. RESULTS : Atypical DGGE patterns in fragments B & A indicating the presence of the mutation, have been detected in both the families. DNA sequencing revealed two rare patterns fragments with patterns in fragments β -thal mutations [CD 26 (C®T) and -90 (C®T)]. CONCLUSION : DGGE is a useful mutation detection technique to identify β -thal mutations among the heterogeneous Indian population.

Detection of Rare β‐Thalassemia Mutations by Denaturing Gradient Gel Electrophoresis Among Indians

We report four rare β‐thalassemia (thal) mutations, viz. AATAAA→AACAAA [polyadenylation (poly A) site mutation], IVS‐II‐745 (C→G), codon 121 (G→T) and IVS‐II‐1 (G→A), detected by denaturing gradient gel electrophoresis (DGGE) among Indians. Of these, the poly A site mutation has been found in combination with deletional δβ‐thal in one case, and with the IVS‐1‐5 (G→C) mutation in another. Two DGGE patterns, corresponding to the same IVS‐II‐1 (G→A) mutation, were seen in one family. Framework (FW) analyses in family studies have shown that the poly A site mutation is associated with FW‐1, while both the codon 121 (G→T) and IVS‐II‐1 (G→A) mutations are associated with FW‐2. Denaturing gradient gel electrophoresis facilitates the screening of rare β‐thal mutations in the diverse Indian population with its many ethnic groups, covering a vast geographic territory.

Prenatal Diagnosis in a Family at Risk for β‐Thalassemia and Hemophilia A: An Uncommon Association

β‐Thalassemia (thal) is an autosomal recessive disorder with a prevalence of 2–3% in Indians, while hemophilia A is X‐linked with a prevalence of 1 in 5,000–10,000 male births. The chances of both these disorders being present together is extremely rare (1 in 250,000). We report an interesting consanguineous family from Western India with a combination of these two disorders, which was referred to us for prenatal diagnosis.