HbD Punjab/HbQ India Compound Heterozygosity: An Unusual Association.

BACKGROUND: Haemoglobinopathies are the commonest hereditary disorders in India and pose a major health problem. Both beta thalassaemia and structural haemoglobin variants are relatively common in northwestern India. Here we report a 29-year-old Sindhi female who was referred to us for a haemoglobinopathy work up and genetic counseling since her spouse was a classical beta thalassaemia carrier. METHOD: A complete blood count was done on an automated cell counter. Haemoglobin analysis was carried out using HPLC Variant Haemoglobin Testing System. The cellulose acetate electrophoresis was carried out [pH 8.9]. Confirmation of mutations was done by automated DNA sequencing. RESULTS: HPLC analysis showed four major peaks, HbA0, a peak in the HbD window, an unknown peak [retention time 4.74 minutes] and a peak in the HbC window. The HbA2 level was 2.2%, and the HbF level was 0.7%. Cellulose acetate electrophoresis at alkaline pH, a slow moving band was seen at the HbS/D position along with a prominent band at the HbA2 position. DNA sequencing of the ß and α genes showed presence of the two hemoglobin variants: Hb D [ß 121GAA → CAA] and Hb Q [α 64 AAG → GAG]. The δ globin gene was normal. The additional peak in the HbC window was due to the formation of a heterodimer hybrid. CONCLUSION: Both HbD Punjab and HbQ India are relatively common in India, but their co-inheritance has not been described in the country. This case is the third report of compound heterozygosity for HbQ India/HbD Punjab haemoglobinopathy globally and the second one from India.

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

OBJECTIVES: To study the varied clinical and haematological profile of ß-thalassaemia homozygotes, compound heterozygotes and heterozygotes with the Poly A (T→C) mutation and its implication in prenatal diagnosis. MATERIALS AND METHODS: Forty individuals were included in the study. Peripheral smear examination, complete blood count and haemoglobin analysis were carried out. ß-thalassaemia mutation analysis was carried out by reverse-dot-blot hybridization, amplification refractory mutation system and DNA sequencing of the ß-globin gene. RESULTS: Five of the six ß-thalassaemia homozygotes with the Poly A (T→C) mutation and five individuals who were compound heterozygous for the Poly A (T→C) mutation along with another common Indian ß-thalassaemia mutation showed a severe ß-thalassaemia major phenotype, while one individual presented as a thalassaemia intermedia. Majority of the 28 heterozygous individuals with this mutation showed borderline HbA2 (mean HbA2 = 3.7 ± 0.4%) levels as compared to individuals with common ß-thalassaemia mutations (mean HbA2 = 5.2 ± 1.4%). The Mean Corpuscular Volume (MCV) levels in individuals heterozygous for the Poly A (T→C) mutation (mean MCV 70.0 ± 5.2 fl) were significantly higher than in individuals with other common ß-thalassaemia mutations (mean MCV 60.7 ± 7.7 fl) (P < 0.001). CONCLUSION: It is important to identify these often silent carriers of ß-thalassaemia for prenatal diagnosis as homozygotes have a severe disease.

Epidemiology of beta-thalassaemia in Western India: mapping the frequencies and mutations in sub-regions of Maharashtra and Gujarat.

Although the average frequency of beta-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 beta-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 beta-thalassaemia heterozygotes. There was an uneven distribution of the frequencies of beta-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 beta-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.

Regional heterogeneity of beta-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.

Hematological and molecular analysis of novel and rare beta-thalassemia mutations in the Indian population.

A variety of mutations causing beta-thalassemia (beta-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 beta-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.

Carrier screening for beta-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.

Prenatal diagnosis of sickle syndromes in India: dilemmas in counselling.

OBJECTIVES: The sickle gene is prevalent in the scheduled caste and tribal populations in India. The clinical presentation of sickle cell disease is extremely variable, and there are no neonatal screening programmes. This is the first report on prenatal diagnosis of sickle syndromes in 85 couples at risk (sickle cell anemia-69; sickle thalassemia-16) from different regions in India. Most of the couples were from a low socioeconomic group and their decisions were entirely dependent on the local counselling given. We have evaluated the acceptability of prenatal diagnosis and the dilemmas faced in counselling these families. METHODS: Chorion villus sampling was done in the first trimester and DNA analysis using reverse dot blot hybridization or restriction enzyme digestion with Dde1 in 65 cases. Cordocentesis was done in the second trimester and fetal blood analyses by automated HPLC in 20 cases who came late. RESULTS: 32.9% of couples came prospectively for diagnosis. 23.5% of fetuses were affected (sickle cell anemia-18, sickle thalassemia-2). The beta-thalassemia mutation in both cases was IVS 1-5(G->C). All the couples with an unfavourable diagnosis opted for termination of pregnancy. CONCLUSION: Sickle cell anemia has a relatively benign clinical course in some tribal groups in India. This raises a dilemma whether we are justified in advising prenatal diagnosis in all such cases.

Impact of beta globin gene mutations on the clinical phenotype of beta thalassemia in India.

The beta thalassemias are one of the commonest group of autosomal recessive disorders in India. Although majority of patients are severe and transfusion-dependent, about 10-15% of cases have a milder phenotype. We evaluated the role of beta gene mutations in modulating the clinical presentation of 342 beta thalassemia patients which included 278 severe thalassemia major (TM) and 64 thalassemia intermedia (TI) cases (severe TI: 27; mild TI: 37) from this region. Thirteen beta thalassemia mutations were characterized by reverse dot blot hybridization or amplification refractory mutation system (ARMS); denaturing gradient gel electrophoresis (DGGE) analysis and DNA sequencing helped to characterize the remaining nine mutations. Majority of the patients in the thalassemia major and thalassemia intermedia groups had severe beta+ or beta0 mutations. IVS 1-5 (G-->C) was the commonest mutation in the three groups. The six severe and common Indian mutations [(IVS 1-5 (G-->C), 619 bp deletion, IVS 1-1 (G-->T), codons 8/9 (+G), codon 15 (G-->A), codons 41/42 (-CTTT)] accounted for 92.0% of molecular lesions in the thalassemia major group, 86.8% in the severe TI group, and 72.9% in the mild TI group. IVS 1-1 (G-->T) and codon 30 (G-->C) were significantly more common in thalassemia intermedia cases. The mild capsite +1 (A-->C) mutation was present in both severe and mild cases. Three other mild beta+ mutations, poly A (T-->C), -28 (A-->G), and -88 (C-->T), were seen only in the thalassemia intermedia cases. These four mild mutations in combination with other severe beta+ or beta0 mutations resulted in a very variable clinical presentation. This study reveals that, in majority of Indian patients, the beta genotype cannot predict the phenotype.

Beta-globin gene cluster haplotypes linked to the betaS gene in western India.

The present survey, which involves 70 sickle cell anemia patients from Gujarat and Maharashtra revealed a high prevalence of the typical Arab-Indian haplotype (91.5 %). Six atypical haplotypes, including a Cameroon one were also found. Correlation of these various haplotypes with HbF expression was studied.

Prenatal diagnosis in a family at risk for beta-thalassemia and hemophilia A: an uncommon association.

beta-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.

Usefulness of automated chromatography for rapid fetal blood analysis for second trimester prenatal diagnosis of beta-thalassemia.

Prenatal diagnosis of beta-thalassemia is now ideally done in the first trimester of pregnancy by chorionic villus tissue DNA analysis. Nevertheless, fetal blood analysis in the second trimester is required either when the mutation in both parents cannot be characterised or when the couple comes late for investigations. We evaluated the usefulness of analysis of fetal blood on the Biorad Variant Hemoglobin Testing System using the beta-thalassemia short programme in comparison with the conventional globin biosynthesis in 58 pregnancies. The beta/alpha biosynthesis ratios in 13 homozygous fetuses ranged from 0 to 0.03 and the adult hemoglobin (HbA) levels by automated chromatography varied from 0% to 0.4%. The normal or heterozygous fetuses had beta/alpha ratios of >0.04 and HbA levels ranging from 2.1% to 10.6%. In 17 fetuses we also correlated the beta gene mutations with the predicted genotypes using automated high-performance liquid chromatography (HPLC). Follow-up of 18 unaffected fetuses using the Variant System at birth showed a significant increase in HbA levels.