New thalassemia mutations from West Bengal

Sir,

Prevention of birth of a child with thalassemia is the cornerstone to reduce the burden of the disease. This is achieved by prenatal diagnosis of ‘at risk’ couple in prenatal diagnosis centers or by high throughput screening of antenatal mothers undertaken by the Sankalp Thalassemia Prevention Programme¹. During prenatal diagnosis, only 28 Indian thalassemia mutations are screened for. However, 954 DNA variants and 400 thalassemia mutations are reported worldwide² Even Colah et al³ listed 63 Human Beta globin gene (HBB) mutations that characterise thalassemia variants in India. We conducted a retrospective data analysis on 86 stored DNA samples of individuals with clinical suspicion of hemoglobinopathy at the Institute of Hematology and Transfusion Medicine (IHTM), Medical College Kolkata from January 2022 to May 2023. The study was ethically approved by the Instituite Ethical Committee. The included cases comprised of co-inherited alpha thalassemia or alpha triplication determinants, homozygosity of HbE disease for confirmation, δβ-Thalassemia and β-Thalassemia heterozygote for preconception molecular screening. The male to female ratio was 4:3 and mean age of the participants was 26±4.9 yr. Thalassemia trait status was confirmed by hemoglobin analysis performed in Biorad variant II Beta Thal short programme (Biorad, Switzerland). Then samples were further processed for mutation analysis.

First seven common Indian thalassemia mutations were investigated using ARMS-PCR⁴. Then undetected samples were analyzed by DNA sequencing. Sanger sequencing (Genetic Analyzer 3500, Applied Biosystems, USA) was performed for the whole HBB gene using two sets of primers designed in the laboratory to cover the entire HBB gene and using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, USA).

Alpha thalassemia determinants of 3.7 kb and 4.2 kb deletion (-α3.7 and –α4.2) in alpha globin cluster were investigated by Gap Polymerase Chain Reaction (GAP-PCR) method described by Liu et al⁵. Cases suspected for alpha triplication (ααα^anti3.7 or ααα^anti4.2) were analyzed for alpha triplication by protocol described by Wang et al⁶. And cases suspected for δβ-thalassemia were investigated by GAP-PCR method described by Mohanty and Colah⁴. DNA chromatogram was analyzed initially by the ABI DNA sequencing programme. DNA alignment was performed in ClustalW programme ( https://www.genome.jp/tools-bin/clustalw ) to detect DNA variant.

Out of 86 samples, alpha triplication was found in six cases and alpha deletion in five cases. Asian Indian inversion deletion was detected in two samples. In 17 DNA samples IVS1-5(G>C) or CD26(G>A) mutations was detected by ARMS PCR. Then 56 samples were further analyzed by sequencing HBB gene in two fragments from HGVS coding position -394 covering the entire HBB gene. Eight rare mutations were identified in 16 samples (supplementary table). One rare Hb variant HbD Iran in heterozygous state was found in six samples. In 18 samples, no other mutations were detected except for a common mutation or polymorphism. No mutation was found in 16 DNA samples. Of the eight rare mutations, four are not reported earlier from West Bengal. These four mutations ‘-86 (C>G), CD10(C>A), IVSII-848 (C>A), CD82(A>T)’ are rare and only two reports are present till date^7,8. The figure shows the DNA alignment and chromatogram of these four rare DNA variant.

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Figure.

The four mutations mapped in HBB reference gene sequence NG_000007.3. (A): Position of three mutations is shown in the DNA alignment map in ClustalW. (B): Position of DNA mutation CD82(A>T) shown in both forward and reverse sequence.

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Figure shows the images of the alignment of the DNA sequence with the reference HBB gene (NG_000007.3) sequence for the three DNA mutations in panel A. First mutation is found at promoter region, located at -86 (C>G) upstream to exon 1 of HBB gene. It is not reported earlier in eastern Indian thalassemia cohort. It is a β⁺ class in phenotype but if co-inherited with β⁰ mutation like IVS1-5(G>C), result in thalassemia major phenotype. Second mutation is at codon 10 referred to as CD10(C>A). It is also β⁺ class in phenotype and is a synonymous mutation. When associated with IVS1-5(G>C), it may lead to severe phenotype. Third mutation is IVSII-848(C>A) in the second intron of HBB gene. It is also a β+ mutation and causes mild phenotype in homozygous condition. Fourth mutation is CD82(A>T) leading to termination codon is found in heterozygous condition in the present study. This is not yet reported in global databases and globin gene database. CD82 (A>T) (HGVS HBB:c247A>T) was reported for the first time in 2010 in a 6 yr old child in homozygous condition⁷ and in our population we found this mutation in heterozygous condition. In this position, two variants A>C (Hb Tsurumai), A>G (Hb Gambara) were mentioned in the NCBI database. Nucleotide conversion A>T is not yet reported in globin gene database. The CD82A>T substitution result in stop codon (UAG) which cause premature termination of β-gloin chain. In homozygous condition, it presents as thalassemia major phenotype.

This retrospective data analysis was performed on unsolved cases of hemoglobinopathies. Total 81.3 per cent cases were resolved by advance analysis in our center but 18.6 per cent cases remained unsolved. In a previous report from our center we showed that 7.3 per cent of samples for prenatal diagnosis remained uncharacterised⁹. In 18.6 per cent samples, eight rare DNA mutations were found. Out of eight rare mutations, two are β⁰ and other six are β⁺ in class phenotype. Four mutations ‘-86 (C>G), CD10(C>A), IVSII-848 (C>A), CD82(A>T)’ were not reported earlier in eastern Indian cohort. These four mutations are very rare and found in only one or two reports. In the global database, -86 is reported from Thailand and CD10 is only reported in TOPMED database. IVSII-848(C>A) is found many countries like Algeria, Egypt, Greece, Jordan, Tunisia and UAE. CD82(A>T) mutation is not reported so far in the global database.

Later, Colah et al¹⁰ mentioned that more than 80 thalassemia mutations are found in Indians. So far, there is no study till date which present the comprehensive list of all the thalassemia mutations found in India. There is a need for an Indian thalassemia mutation database for such a genetically diverse vast population for improving the prenatal diagnosis for thalassemia across the country.

We may conclude that in this retrospective study, 86 undefined DNA samples of suspected haemoglobinopathies were further analysed by advance mutation analysis. Eight rare mutations were identified in sixteen β-thalassemia heterozygotes. Of these, we found four rare mutations -86 (C>G), CD10 (C>A), IVSII-848 (C>A) and CD82 (A>T) that were not reported earlier from West Bengal.