Reema Surve

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.

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.

Feasibility of Antenatal Screening of β-Thalassemia in Mumbai, India

Accessible online at: www.karger.com/journals/aha We read with interest the success of premarital screening of ß-thalassemia trait done in the Province of Denizli, Turkey [1] where 9,902 couples were screened over a period of 4 years. ß-Thalassemia is a major public health problem in India, and about 10,000 homozygous children are born each year in this vast country [2]. Our earlier study on screening of 5,700 high school children in Mumbai showed that the overall prevalence of ß-thalassemia trait was 2.7%; however, it varied from 1 to 7% in different communities [3]. Premarital screening is generally not yet acceptable in India due to cultural values and social stigma. We therefore attempted antenatal screening of women during their first visit for registration to a maternity hospital. We screened 14,086 women over a period of 18 months. Preliminary screening was done by the Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT) followed by measurement of RBC indices, hemoglobin electrophoresis on cellulose acetate and quantitation of Hb A2 and other abnormal hemoglobins by elution. We have found this approach to be simple, reliable and cost effective [4]. The findings are shown in table 1. Only 15.4% of the women registered in the first trimester for an antenatal check-up while 37.6% came late in the third trimester. 270 ß-thalassemia carriers (1.92%) were identified along with 22 cases who were heterozygous for other hemoglobinopathies (Hb S, D, E, Q and ‰ß thalassemia). After counselling these 292 women, only 86 (29.5%) came for a follow-up along with their husbands. Two of the husbands were also found to have ß thalassemia trait. Both couples opted for prenatal diagnosis. In one case, chorionic villus sampling and DNA analysis were done and the second underwent cordocentesis and fetal blood analysis as her gestation was already 18 weeks. Both fetuses were found to be normal. While in Turkey premarital screening has been shown to be an effective way of controlling thalassemia, in India our experience with screening in a large maternity hospital in Mumbai catering mainly to a lower socioeconomic group has shown that even antenatal screening is not the solution as the majority of women do not come to the hospital till the second or third trimester of pregnancy. In spite of counselling, the husbands of carriers do not readily come for screening. These are some of the difficulties in controlling thalassemia in India. Table 1. Antenatal screening for ß-thalassemia trait

Clinical Diversity of Sickle Cell Disease in Western India – Influence of Genetic Factors

Accessible online at: www.karger.com/journals/aha We read with interest the report entitled ‘Sickle cell disease in Brazzaville, Congo: genetical, hematological, biochemical and clinical aspects’ by Mouele et al. [1]. We have been studying the natural history, clinical and molecular mechanism of sickle cell disease in western India for 1 decade. We have studied 143 sickle cell disease patients aged 6–36 years, with varying clinical manifestations from tribal and nontribal populations. The severity grading was used according to the NIH (USA protocol). DNA samples of these patients were analyzed for ßS haplotypes and ·-genotypes by PCR [2] and Southern blot hybridization [3] to understand the clinical diversity among these groups. A summary of our findings is presented in table 1. Part of these data have been published elsewhere [4, 5]. Mouele et al. [1] have not correlated the influence of ·-thalassemia with the severity of sickle cell disease, whereas our study showed an ameliorating effect of the –·3.7 deletion on the severity of sickle cell disease. The incidence of splenomegaly in Congolese patients was similar to our patients with the –·3.7 deletion. Similar observations have been made by Kar et al. [6] from Orissa populations in the eastern part of India. Unlike their series, none of our patients had leg ulcers. Contrary to this, other studies have shown that the rate of painful crisis, acute chest syndrome and aseptic necrosis is more common among the patients with ·-thalassemia [7, 8]. Hence, the influence of ·thalassemia on the clinical severity of sickle cell disease is controversial. In the majority of Congolese patients, the ßS gene was linked with the severe Bantu haplotype while all our patients had the milder Arab-Indian haplotype with much higher HbF levels (14.5 vs. 8.8%). This could offer an added advantage to our patients. Further, the majority of our patients were identified during population surveys, where cases with a milder presentation could be picked up in contrast to the hospital-based study of Mouele et al. [1] which is more biased as more severely affected patients may be included. Thus, our study indicates that the clinical manifestation of sickle cell disease was influenced by ·-thalassemia rather than the ßS haplotype or higher HbF levels in Indians.

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.

Variable haematological and clinical presentation of β‐thalassaemia carriers and homozygotes with the Poly A (T→C) mutation in the Indian population

To study the varied clinical and haematological profile of β‐thalassaemia homozygotes, compound heterozygotes and heterozygotes with the Poly A (T→C) mutation and its implication in prenatal diagnosis.

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.

Hb M Hyde Park and Hb M Boston in two Indian families - a rare cause of methaemoglobinemia.

Sir, The M haemoglobins include a group of haemoglobin variants which display an autosomal dominant inheritance and in most cases result from amino acid substitutions of tyrosine for histidine in the haem-binding pocket of either the alpha, beta or the gamma chain of haemoglobin. These substitutions lead to structural alterations in the haem-binding pocket resulting in the oxidized Fe (ferric state) from the normal Fe (ferrous state) of haem iron to produce methaemoglobinemia. Methaemoglobin does not function as an oxygen transporter and consequently leads to cyanosis. We describe two cases of Hb M with cyanosis that remained undiagnosed into adulthood.