A multiplex ARMS PCR approach to detection of common ß-globin gene mutations.

BACKGROUND: ß-thalassaemia is a group of inherited single-gene disorders worldwide. Each ethnic population has its own common mutations. Heterogeneity of ß-thalassaemia mutations in multi-ethnic population of Surat, makes molecular diagnosis expensive and time consuming. METHODS: Specific primers were used to differentiate four common mutations, IVS I-5 (G→C), Codon 41/42 (- TCTT), 619-bp deletion and FS 8/9 (+G), by a simple PCR involving a multiplex amplification refractory mutation system. RESULTS: Several high prevalence ß-Thalassemia trait groups constituted by Muslims, Patels, Sindhis, ModhBanias, and Mahayavanshi. Four most common mutations detected in them are IVS I-5 (G→C), Codon 41/42 (- TCTT), 619-bp deletion and FS 8/9 (+G). We identified each of these ß-thalassemia mutations in multiplexed ARMS from positive control samples. Our multiplex-ARMS-PCR system was first standardized on positive DNA samples with above known four most common ß-thalassemia mutations, and these positive samples had been diagnosed with ß-thalassemia and also all these samples belonged to Surat ethnic groups. The system was subsequently tested on 110 blood samples from different ethnic backgrounds with unknown ß-thalassemia mutations which were in all specimens. CONCLUSION: The ARMS multiplex system was found reliable, cost effective, fast and most applicable for mutation screening of Thalassemia in Surat populations.

Molecular characterization of ß-thalassemia in four communities in South Gujarat--codon 30 (G → A) a predominant mutation in the Kachhiya Patel community.

Different thalassemia mutations have been reported in various ethnic groups and geographical regions in India. In this study, we have investigated Kachhiya Patel, Dhodia Patel, Modh Bania, and Muslim communities of Surat, Gujarat to identify molecular defects causing ß-thalassemia in them. Covalent reverse dot blot hybridization technique was used to detect six common Indian ß-thalassemia mutations while the seventh mutation (619-bp deletion) was identified by PCR. The less common mutations were detected by amplification refractory mutation and the uncharacterized samples were directly sequenced. Characterization of ß-thalassemia mutations was carried out in a total of 175 unrelated ß-thalassemia trait cases. We identified IVS 1 nt 5 (G → C) in 31 out of 65 Muslims, codon (Cd) 41/42 (-CTTT) in 14 out of 16 in Modh Banias, Cd 15 (G → A) in 19 out of 24 Dhodia Patels. The most significant observation was an uncommon mutation; Cd 30 (G → A) detected in 61 out of 70 Kachhiya Patels. The 619-bp deletion was detected in 6 out of 10 Muslim-Memons. Many other rare mutations like Cd 15 (-T), Cd 8 (-AA), -88 (C → A), Capsite +1 (A → C), Cd 16(-C), and Cd 5 (-CT) were detected. To our knowledge, our study is the first to characterize ß-thalassemia mutations in the Kachhiya Patel community. This study will facilitate molecular analysis and prenatal diagnosis in these four communities.

Antenatal screening for identification of couples for prenatal diagnosis of severe hemoglobinopathies in surat, South gujarat.

PURPOSE: Our aim was to identify couples at risk of having a homozygous or compound heterozygous child with a severe hemoglobinopathy by antenatal screening and prenatal diagnosis in Surat, South Gujarat. METHOD: Pregnant women were screened for hemoglobinopathies by means of red cell indices, the solubility test, cellulose acetate electrophoresis tests, and confirmation by HPLC. Husbands of the pregnant women having hemoglobinopathies were counseled and screened for hemoglobinopathies. The couples at risk were again counseled and referred to the National Institute of Immunohematology, where mutations in parents and fetuses were identified by molecular analysis. After prenatal diagnosis, the continuing pregnancies were followed up and infants were tested at birth. RESULTS: Out of 3,009 women, 37.04, 52.6, and 10.3 % were in the first, second, and third trimester of pregnancy, respectively. Among those having hemoglobinopathies, 102 (3.38 %) had the ß-thalassemia trait, 46 (1.5 %) the Sickle cell trait, and 26 (0.86) had hemoglobin variants like Hb DPunjab, Hb E, Hb DIran, Hb QIndia, Hb JParis-I, and Hb OIndonesia. Out of the 14 couples at risk of having an affected child, 11 (78.5 %) couples opted for prenatal diagnosis. Three fetuses had homozygous ß-thalassemia and hence the pregnancies were terminated. Follow up of normal or heterozygous fetuses confirmed the diagnosis. CONCLUSION: During antenatal screening, we found many Hb variants of ß and α globin chains. Late antenatal registration, non-cooperation of the husband for investigation, and refusal for prenatal diagnosis are the main hurdles in the hemoglobinopathy prevention program and awareness is necessary.

Prevalence and hematological profile of ß-thalassemia and sickle cell anemia in four communities of Surat city.

BACKGROUND: From the data of transfusion-dependent thalassemia major cases, the 4 communities (Muslim, Dhodia Patel, Kachhiya Patel, and Modh Bania) with high prevalence but not studied methodically were selected. AIM: The aim of this study is to find prevalence of ß-thalassemia and sickle cell anemia in 4 selected communities and also to evaluate hematological profile in them. MATERIALS AND METHODS: For screening of ß-thalassemia trait (BTT) and sickle cell trait (SCT), all samples were tested for red cell indices, solubility, HbA(2) level and doubtful cases confirmed on HPLC. STATISTICAL ANALYSIS: Mean ± SD, χ(2) and 't' tests were used to evaluate the significance. RESULTS AND CONCLUSION: Among 4 selected communities, the highest prevalence of BTT was observed in Modh Bania (6.2%) and Kachhiya Patel (6.05%) and that of SCT in Dhodia Patel (14.0%). Significantly higher prevalence of BTT was observed in Memon (P < 0.0001) and of SCT in Khalifa 6.6% (P < 0.0001) compared to other Muslim sub castes. Anemia was more prevalent in BTT compared to non-BTT and non-SCT subjects. 80% of Dhodia Patel non-BTT and non-SCT subjects showed microcytic red cell morphology. Their Mean ± SD Hb concentration was 12.1 ± 1.73, hence iron deficiency cannot be a sole reason. This community needs α-thalassemia and iron studies.