SCREENING AND MOLECULAR CHARACTERIZATION OF HAEMOGLOBINOPATHIES USING ARMS (AMPLIFICATION REFRACTORY MUTATION SYSTEM) AND DIRECT DNA SEQUENCING TECHNIQUES AMONG YOUNG IN CENTRAL GUJARAT WESTERN INDIA

Haemoglobinopathies is consider the most common inherited disorders in human and results from genetic mutation in . one or more genes The present study was aimed to characterize the β-thalassemia mutation and haemoglobin variant in youth by ARMS PCR which is an uncomplicated and convenient method for identification of five common mutation from central Gujarat, western India region. This Study included 44 randomly selected haemoglobinopathies carrier student's sample of Anand People's Medicare Society (APMS), Anand for DNA analysis by ARMS PCR from March 2021-April 2021. Identification of five common Indian β thalassemia mutations along with Hb S and HB E were carried out by ARMS PCR method and δβ- thalassemia mutation was characterized by GAP-PCR. The samples which remain uncharacterized were sent to S N gene lab, Surat for DNA sequencing. In our study the most common mutation among five common mutations characterized was IVS-1, nt5 (G→C) in 22 (50%), followed by Codon41/42 (-CTTT) in 5 (11.3%). IVS-1, nt1 (G→T), Codon8/9 (+G) and 619bp del mutation was not identified in any carrier students screened for haemoglobinopathies. Other than these five common mutation Codon -88 (C→T) (2.27%) and Codon 30 (G→A) (2.27%) are also detected. The prevalence of haemoglobinopathies with respect to communities, reflects that SC/ST/OBC are at the highest risk with 50%. Communities like Rajput (22.7%), Patel (18.1%), Brahmin (6.8%) and Muslim (2.2%) are also showing prevalence. The study has included mutation in different communities reflects characterization of mutation of central Gujarat, western India which is significant for rapid and convenient identification of mutation while conducting screening programs and prenatal diagnosis. Rare mutations which are not recognized, need further confirmation for carrier detection followed by prenatal diagnosis.

Hemoglobin Ottawa (HBA2:c.46G>C) and β+ thalassemia (HBB:c.-138C>T) detected in an Indian male by capillary zone electrophoresis

Hemoglobin (Hb) Ottawa [α15(A13)Gly>Arg], also known as Hb Siam, results from GGT>CGT mutation in codon 15 of either HBA1 or HBA2. Hb Ottawa carriers typically have normal hematology but when the variant is coinherited with either α or β thalassemia, microcytic red cell indices were observed. The percentage of variant detected using routine methodology was variable (14-33%), with a higher percentage found when co-inherited with an abnormal α-globin genotype. The case presented here involved an Indian male with microcytic red cell indices, who was heterozygous for Hb Ottawa (HBA2:c.46G>C) and β+ thalassemia (HBB:c.-138C>T). This case represents the first reported finding of Hb Ottawa in the Indian population, as well as the first time capillary zone electrophoresis (CZE) has been used to identify the variant. The abnormal red cell indices were attributed to co-inheritance of β+ thalassemia mutation (HBB:c.-138C>T), which alters binding of transcriptional factors to the HBB promoter and reduces transcription from the allele. The mild β+ thalassemia mutation has commonly been found in the Indian population.

Beta-Thalassemia Intermedia: A Single Thalassemia Center Experience from Northeastern Iraq

Objective. To determine the molecular characterization and disease-associated complications of beta-thalassemia intermedia (β-TI) patients in Sulaymaniyah province, northeastern Iraq. Methods. A total of 159 β-TI patients from 114 families were enrolled. Detection of β-thalassemia mutations was done by reverse hybridization technique and direct gene sequencing. Also, the clinical and hematological data were collected through an electronic-based medical recording system using a designed comprehensive questionnaire. Results. Nineteen different β-globin gene mutations arranged in 37 various genotypes were determined. The most frequent were IVS-II-I (G>A) (47.2%), followed by IVS-I-6 (T>C) (23.3%) and IVS-I-110 (G>A) (5%). Among disease-related morbidities documented, bone disease amounted to 53% (facial deformity and osteoporosis), followed by endocrinopathies 17.6% (growth retardation and subclinical hypothyroidism), cholelithiasis 13.8%, pulmonary hypertension 11.3%, and abnormal liver function test 7.5%, whereas venous thrombosis, extramedullary hemopoiesis, and leg ulcer were less frequently observed. Age≥35 and female sex were risk factors for cholelithiasis, while age was an independent risk for hypothyroidism and female sex was associated with increased risk for osteoporosis. Mean serum ferritin of ≥1000 μg/L was associated with an increased risk of osteoporosis, whereas chelation therapy was protective for a multitude of other complications. Transfusion, on the other hand, increased the risk of osteoporosis, yet it was protective for cholelithiasis and hypothyroidism. Moreover, splenectomy was protective for cholelithiasis, although it was an independent risk for hypothyroidism. Finally, hydroxyurea was associated with an increased risk of osteoporosis, while it was protective for cholelithiasis. Discussion and Conclusion. β+-thalassemia mutation had contributed to 41.25 of families with a less severe β-thalassemia phenotype in the northeastern part of Iraq, justifying the need to investigate the contribution of genetic modifiers in ameliorating disease severity. In addition, the substantial number of β-TI patients developed disease-related morbidities, which necessitates the need for more appropriate clinical management with earlier intervention.