Ajit Gorakshakar

Global burden, distribution and prevention of β-thalassemias and hemoglobin E disorders

The β-thalassemias, including the hemoglobin E disorders, are not only common in the Mediterranean region, South-East Asia, the Indian subcontinent and the Middle East but have now become a global problem, spreading to much of Europe, the Americas and Australia owing to migration of people from these regions. Approximately 1.5% of the global population are heterozygotes or carriers of the β-thalassemias. While the overall frequencies of carriers of these disorders are known in most countries, there have been few attempts at micromapping and wherever this has been done, significant variations are seen even within small geographic regions. Thus, the figures for the estimated numbers of births each year of homozygous β-thalassemia and the severe compound states involving other hemoglobin disorders may be an underestimate. Screening strategies have varied from premarital to antenatal in different countries depending on socio–cultural and religious customs in different populations. Prenatal diagnosis programs are ongoing in many countries and the knowledge of the distribution of mutations has facilitated the establishment of successful control programs. Many of these were through North–South partnerships and networking. Yet, there are many countries in Asia where they are lacking, and South–South partnerships are now being developed in South-East Asia and the Indian subcontinent to link centers with expertise to centers where expertise needs to be developed. Although the carrier frequencies will remain unaltered, this will eventually help to bring down the burden of the birth of affected children with β-thalassemias and hemoglobin E disorders in Asia.

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.

Carrier Screening for β-Thalassemia during Pregnancy in India: A 7-Year Evaluation

AIM Premarital screening for beta-thalassemia is not widely acceptable in India; hence, we evaluated the effectiveness of antenatal screening and counseling over 7 years. METHODS 61,935 pregnant women were screened using the single-tube osmotic fragility test during their first antenatal visit. Individuals who were positive were investigated further for diagnosis of beta-thalassemia and other abnormal hemoglobins. Spouses of carrier women were tested whenever available. Couples at risk were given the option of prenatal diagnosis. RESULTS Only 19% of the women registered at the antenatal clinic in the first trimester of pregnancy, and 14% of the women were positive per the osmotic fragility test; 1020 beta-thalassemia heterozygotes and 213 women with other hemoglobinopathies were identified, majority being in the second and third trimesters. Seven hundred and thirteen (69%) of their husbands could be tested, and 37 couples at risk were identified. Only 15 couples had a prenatal diagnosis done. Four couples with affected fetuses opted for termination of pregnancy. The remaining couples either did not respond after counseling or the pregnancies were advanced for prenatal intervention. CONCLUSION This first large study shows that antenatal screening is acceptable in India; however, awareness generation is still a primary requisite to make women register early at antenatal clinics and bring their spouses for screening when required.

Clinical, hematologic and molecular variability of sickle cell-β thalassemia in western India

BACKGROUND: Sickle cell-β thalassemia (HbS-β thalassemia) is a sickling disorder of varying severity, which results from compound heterozygosity for sickle cell trait and β thalassemia trait. The present study was undertaken to determine the genetic factors responsible for the clinical variability of HbS-β thalassemia patients from western India. MATERIALS AND METHODS: Twenty-one HbS-β thalassemia cases with variable clinical manifestations were investigated. The α and β globin gene clusters were studied by molecular analysis. RESULTS: Thirteen patients showed milder clinical presentation as against eight patients who had severe clinical manifestations. Four β thalassemia mutations were identified: IVS 1-5 (G→C), codon 15 (G→A), codon 30 (G→C) and codon 8/9 (+G). α thalassemia and XmnI polymorphism in homozygous condition (+/+) were found to be common among the milder cases. The βS chromosomes were linked to the typical Arab-Indian haplotype (#31). Framework (FW) linkage studies showed that four β thalassemia mutations were associated with different β globin gene frameworks. Linkage of codon 15 (G→A) mutation to FW2 is being observed for the first time. CONCLUSION: The phenotypic expression of HbS-β thalassemia is not uniformly mild and α thalassemia and XmnI polymorphism in homozygous condition (+/+) are additional genetic factors modulating the severity of the disease in the Indian subcontinent.

Potential of denaturing gradient gel electrophoresis for scanning of β‐thalassemia mutations in India

Over the last few years, substantial progress has been made in developing strategies for the detection and characterization of various mutations causing β‐thalassemia. The Indian population comprises of numerous endogamous caste groups and β‐thalassemia is seen in almost all of them. Knowledge of the spectrum of β‐thalassemia mutations in the population is a prerequisite for successful implementation of a prevention programme. Among the different approaches available today, Denaturing Gradient Gel Electrophoresis (DGGE) offers a valid technical approach which is applicable for screening of known mutants and polymorphisms as well as in locating regions of DNA bearing unknown mutations.

Prenatal Diagnosis of β-Thalassemia Among Indians using Denaturing Gradient Gel Electrophoresis

We have offered first trimester prenatal diagnosis to 55 couples at risk for β-thalassemia, originating from various parts of India, using polymerase chain reaction and denaturing gradient gel electrophoresis. Apart from the six common mutations, codon 30 (CAG→CAA), Cap site +1 (A→C), as well as three uncharacterized mutations were seen among the parents. in the majority of cases, the diagnosis was possible by scanning only one fragment (B) where most of the Indian mutations are situated. in 18 out of 55 cases, framework analysis could also have been used to offer prenatal diagnosis without characterizing the β-thalassemia mutations. in the two cases where the mutations were uncharacterized, prenatal diagnosis was done only on the basis of the anomalous denaturing gradient gel electrophoresis patterns seen in the parents and in previously affected children. This is the first attempt of prenatal analysis using denaturing gradient gel electrophoresis in the extremely diverse Indian population where the prof...

Cascade screening for β-thalassemia: a practical approach for identifying and counseling carriers in India.

β- thalassemia is one of the most common single gene disorders in India with an overall prevalence of 3-4%.(1) In certain communities like Sindhis, Muslims, Cutchi Bhanushalis, and some tribal groups, the prevalence of β thalassemia carriers varies between 8 and 10% or more.(2,3) It has been estimated that about 10 000-12 000 children with β thalassemia major are born every year in India. These figures might be underestimated. The epidemiology of thalassemia is changing during the past few decades in some countries such as Cyprus and Greece. This is mainly due to the successful implementation of prevention programs. A reduction in the birth rate of babies with thalassemia major from 1:250 to 1:4000 over the years has been reported in Sardinia.(4) India is a vast country where over 4000 ethnic groups with diverse cultural backgrounds are residing. Several communities have not been screened so far, and what is required is micro mapping at least at the district level to get an accurate estimate of the β–thalassemia gene in the population. The individuals who are at an increased risk have some knowledge of thalassemia as compared to the general population where majority of the people are ignorant of this disorder. This is the first report on cascade screening for β-thalassemia from India.

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.