Importance of Additional Mutation Analysis in Chorionic Villous Sample for Prenatal Diagnosis of Thalassemia

Introduction: β thalassaemia is a genetic disorder of β globiin gene synthesis. Its presentation ranges from transfusion dependent to non transfusion dependent thalassaemia. Non transfusion dependent thalassaemia can have normal life with proper care whereas only curative option for transfusion dependent thalassaemia is bone marrow transplantation which is costly as well as requiring technical expertise. Carrier screening before marriage/conception, antenatal screening and prenatal diagnosis are the only way to prevent birth of a child with thalassaemia. Objective of prenatal diagnosis is to identify homozygous or double heterozygous baby by mutation study. However clinical course after birth may get altered depending on associated other mutation. Till date these associated mutations are not considered in cases of prenatal diagnosis. However, this approach may avoid termination of the baby. This study was carried out retrospectively in the prenatal samples to detect presence of phenotype-modifying mutations. Method and Material: The study was conducted at the Institute of Haematology and Transfusion Medicine, Medical College, Kolkata. Study population consisted of 145 CVS samples and samples of carrier mother and father. Carrier status was detected by HPLC and confirmed by ARMS- PCR for β thalassaemia mutation. Co- inheritance of α- thalassaemia was assessed by GAP- PCR and polymorphism with haplotype assessment was done by RFLP-PCR. 36 parent samples were analyzed for mutation for the carrier status and also for presence of α and Xmn 1 polymorphism. Result: Out of 145 CVS, 8.96% (13 samples) were found to be homozygous, 12.41% (18 samples) double heterozygous, 51.72% (75 samples) heterozygous, 23.44% (34 samples) normal and 3.44% (5 samples) with uncharacterized for β mutation. Our results designated that IVS 1-5 was the most common beta mutation in the state of West Bengal population (Table 1). Co-inheritance of α 3.7 deletion was found in only three affected CVS. Xmn1 polymorphism was detected in 9 CVS samples. Out of these, 3 were homozygous and 6 were heterozygous. Both co-inheritance of α mutation and xmn1 polymorphism were detected in 22 parents sample and 18 affected CVS sample ( Table: 2). Out of 31 affected sample, 18 samples showed presence of Xmn 1 and 4 for α deletion. Additional disease modifying mutations were detected in 6 samples (19.35%). In cases where the 18 parents' sample were positive for Xmn1 polymorphism and 3 for α deletion in CVS sample, either homozygous or double heterozygous for parents' mutation. Table 1. Common β globin gene mutation of CVS samples Beta genotype Homozygote Heterozygote Compound Heterozygote Normal Uncommon IVS 1-5 (G→ C) 7 48 - -- -- CD26(G→A) HbE 1 13 5 -- -- CD15(G→A) 8 9 - -- -- CD30 (G→ C) 5 -- -- -- -- CD41/42(-CTTT) 2 3 -- -- CD8/9(+G) 3 -- -- -- -- CD6 (A → T) 2 2 -- -- Total 13 75 5 34 18 Table 2. Affected CVS with additional mutations: Additional mutations Homozygous CVS Double heterozygous CVS Parents α deletion 1 -- 7 Homozygous Xmn1 +/+ 1 1 3 Heterozygous Xmn 1 -/+ 9 3 12 α deletion + Homozygous Xmn1 +/+ -- 1 -- α deletion + Homozygous Xmn1 -/+ -- 2 -- Discussion: Common thalassaemia subtypes prevalent in West Bengal are HbE beta and Beta thalassaemia . In cases where both the parents are carrier, beta mutation or mutation for HbE was first analyzed and subsequent decision for termination of baby was taken on the presence of both these mutations in the CVS sample. But phenotype is determined by presence of additional mutations like co-inheritance of α mutation and Xmn 1 polymorphism. From our study it became evident that around 19.35% cases the baby was expected to have intermedia or NTDT phenotype. Complete genotype analysis of CVS samples in prenatal diagnosis can save a life and that give a baby to the parents. However, further exploration with larger number of patients and post natal follow up is necessary to reach a definite conclusion. Conclusion: Alpha deletion and Polymorphism detection should be employed for prenatal diagnosis. While developing such program in the population, we save of multiple neonates. Disclosures No relevant conflicts of interest to declare.