Pratibha Sawant

Response to hydroxyurea in β thalassemia major and intermedia: Experience in western India

BACKGROUND The clinical and hematological response to hydroxyurea was evaluated in beta thalassemia patients in western India with variable clinical severity and correlated with genetic factors. MATERIALS AND METHODS Seventy-nine patients-[38-beta thalassemia intermedia-(group I), 41-beta thalassemia major-(group II)] on hydroxyurea therapy were followed-up for 20-24months. RESULTS Among the frequently transfused patients in group I, 58% became transfusion independent and 16% showed a 50% reduction in transfusions after therapy which correlated with a higher mean fold increase in HbF and gamma mRNA expression levels. Forty-one percent of patients in group I had associated alpha-thalassemia and 72.7% were XmnI (+/+). beta thalassemia chromosomes among the responders of group I (41%) were linked to haplotype (- + + - + + - - +) as against haplotype (+ - - - - - - - +) being more common among the non-responders. Response was not linked to the beta thalassemia mutations. Thirty-two percent of group II patients showed a 50% reduction in their transfusion requirements after therapy which also correlated with a higher mean fold increase in HbF and gamma mRNA expression levels. A significant decrease in serum ferritin was seen in both groups. 19% of patients could not tolerate the drug. CONCLUSIONS In group I, clinical response to hydroxyurea was better in patients with alpha-thalassemia, XmnI (+/+) and a higher mean fold increase in gamma mRNA expression. In group II, only one-third of patients showed a partial response.

Hydroxyurea in sickle cell disease--a study of clinico-pharmacological efficacy in the Indian haplotype.

There is clinical variability in the presentation of sickle cell disease among Indians. Vaso-occlusive crisis is common among non-tribal patients. Hydroxyurea, induces fetal hemoglobin (HbF) synthesis and reduces the clinical severity of sickle cell disease but individual patients have a variable response. This study was undertaken to investigate the efficacy and safety of hydroxyurea in Indians with severe manifestations where the beta(s) gene is linked to the Arab-Indian haplotype and is associated with higher HbF levels. Seventy-seven patients (29 adult sickle homozygous, 25 pediatric sickle homozygous, 23 adult sickle beta-thalassemia) selected for hydroxyurea therapy were evaluated for clinical, hematological, biochemical and genetic parameters and were followed for 24 months. Ninety-eight point seven percent of the sickle chromosomes were linked to the Arab-Indian haplotype, 27% of patients had associated alpha thalassemia and 65% were Xmn I +/+. Seventy-eight percent of the patients had no further crises after starting hydroxyurea. This effect was accompanied by a significant increase in HbF (p<0.001), but this increase was variable in individual cases. There was also an increase in gamma gene mRNA expression in the few cases so studied. Hemoglobin levels increased significantly (p<0.001) resulting in the cessation of blood transfusions. Leucopoenia was observed in one patient. Hydroxyurea was effective in reducing the clinical severity in Indian patients who initially had higher HbF levels and the presence of ameliorating factors, such as alpha-thalassemia and the Xmn I polymorphism. Hydroxyurea therapy with careful monitoring can thus change the quality of life of Indians with sickle cell disease.

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.

Effect of hydroxyurea on the transfusion requirements in patients with severe HbE-β-thalassaemia: a genotypic and phenotypic study

Background Haemoglobin E (HbE)-β-thalassaemia has a very variable clinical presentation. The management of severe cases that are often transfusion dependent is similar to that of cases of β-thalassaemia major; however, this is often not possible in India because of its high cost and the lack of availability of safe blood at many places. Thus there was a need for a drug such as hydroxyurea, which is known to reduce the transfusion requirements of patients with thalassaemia intermedia. This study was undertaken to evaluate the response of Indian patients with HbE-β-thalassaemia to hydroxyurea. Materials and methods 11 patients with HbE-β-thalassaemia receiving regular transfusion plus two less frequently transfused patients were selected for hydroxyurea therapy. Clinical and haematological evaluation was performed before and after treatment for 2 years. Molecular studies included β-globin genotype, β-globin gene haplotype, Xmn I polymorphism and α-genotyping. Results Four clinically severe patients became transfusion independent (responders) after hydroxyurea therapy, four patients showed a reduction in their transfusion requirements (partial responders), and three patients were non-responders. Responders showed a statistically significant increase in Hb, mean corpuscular volume, mean cell Hb, fetal Hb and F cells with a reduction in their transfusion requirements. A reduction in serum ferritin concentration was also seen in responders and non-responders. Conclusions Genetic markers such as Xmn I polymorphism and α-gene deletions were not always beneficial for the response to hydroxyurea therapy. Thus many more markers and a larger cohort need to be studied to predict the response in these patients.

Evaluation of the Use of Monoclonal Antibodies and Nested PCR for Noninvasive Prenatal Diagnosis of Hemoglobinopathies in India

Our purpose was to develop and evaluate isolation and enrichment of fetal erythroblasts and a nested polymerase chain reaction (PCR) approach using fetal erythroblasts for detecting the beta-globin gene mutations for a noninvasive prenatal diagnosis of hemoglobinopathies. Maternal blood at different periods of gestation was layered on a Percoll density gradient for enrichment of fetal nucleated RBCs (NRBCs). A combination of 3 monoclonal antibodies (CD45-peridinin chlorophyll protein, glycophorin A-phycoerythrin, and anti-hemoglobin F-fluorescein isothiocyanate) was used for flow cytometric sorting of fetal NRBCs from enriched cells. Different nested PCR-based approaches were used for identification of fetal mutations. Owing to heterogeneity of beta-thalassemia mutations in the population of India, we had to screen for 12 mutations and were able to give an accurate diagnosis in 84 (84.0%) of 100 cases when compared with chorionic villus sampling or cordocentesis and DNA analysis.This nested PCR approach enabled amplification of small quantities of DNA from fetal erythroblasts, providing a cost-effective method for noninvasive diagnosis.

Clinical and hematological presentation among Indian patients with common hemoglobin variants

BACKGROUND Co-inheritance of structural hemoglobin variants like HbS, HbD(Punjab) and HbE can lead to a variable clinical presentation and only few cases have been described so far in the Indian population. METHODS We present the varied clinical and hematological presentation of 22 cases (HbSD(Punjab) disease-15, HbSE disease-4, HbD(Punjab)E disease-3) referred to us for diagnosis. RESULTS Two of the 15 HbSD(Punjab) disease patients had moderate crisis, one presented with mild hemolytic anemia; however, the other 12 patients had a severe clinical presentation with frequent blood transfusion requirements, vaso occlusive crisis, avascular necrosis of the femur and febrile illness. The 4 HbSE disease patients had a mild to moderate presentation. Two of the 3 HbD(Punjab)E patients were asymptomatic with one patients sibling having a mild presentation. The hemoglobin levels of the HbSD(Punjab) disease patients ranged from 2.3 to 8.5 g/dl and MCV from 76.3 to 111.6 fl. The hemoglobin levels of the HbD(Punjab)E and HbSE patients ranged from 10.8 to 11.9 and 9.8 to 10.0 g/dl whereas MCV ranged from 67.1 to 78.2 and 74.5 to 76.0 fl respectively. CONCLUSIONS HbSD(Punjab) disease patients should be identified during newborn screening programmes and managed in a way similar to sickle cell disease. Couple at risk of having HbSD(Punjab) disease children may be given the option of prenatal diagnosis in subsequent pregnancies.

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.

Challenges in prenatal diagnosis of beta thalassaemia: couples with normal HbA2 in one partner

To undertake β‐genotyping in couples having normal/borderline HbA2 levels in one partner to offer the possibility of prenatal diagnosis of thalassaemia.

Hemoglobin Fontainebleau [a21(B2)Ala>Pro]: The second report from India.

Structural hemoglobin (Hb) variants are mainly due to point mutations in the globin genes resulting in single amino acid substitutions. Until date, about 200 alpha chain variants have been identified and they are usually detected during the hemoglobinopathy screening programs. Under a community control program for hemoglobinopathies, which involved screening of antenatal cases followed by prenatal diagnosis if indicated. Here, we report a rare alpha globin gene variant Hb Fontainebleau [a21(B2)Ala>Pro] detected in the heterozygous condition in a 35-year-old pregnant lady screened during this program. This is the second report of this alpha globin variant from India. Unlike the earlier case from India where Hb Fontainebleau was reported in a neonate who was also a carrier of Hb Sickle and had no clinical problems, this case presented with a bad obstetric history associated with the secondary infertility. However, the presence of the variant and the obstetric complications may be unrelated.