Genotype Distribution of Dihydrofolatereductase Variants and their Role in Disease Susceptibility to Acute Lymphoblastic Leukemia in Indian Population: An Experimental and Computational Analysis

Objectives: To establish the allele and genotype frequencies of dihydrofolate reductase (DHFR) -317A>G and -680C>A variants in Indian population, to find the association of these variants with the risk of acute lymphoblastic leukemia (ALL) and to analyze the effects of non-synonymous SNPs (nsSNPs) and 3’untranslated region (3’UTR)variants of DHFR gene on its structure and function using in-silico tools. Methods: A total of 235 unrelated healthy volunteers (controls) and 127 ALL patients (cases) were recruited for the study. DNA was extracted from peripheral leucocytes. Genotyping of DHFR polymorphisms was done by realtime PCR. We investigated the deleterious effect of nsSNPs and variants in 3’UTR of DHFR gene through computational platforms. Results: In the present study, the frequency of DHFR -317G and -680 A alleles was found to be 33.3% and 59.8% respectively. The studied DHFR variants (rs408626 and rs442767) did not confer a significant risk for ALL. Insilico analysis revealed that three nsSNPs potentially affect the structure, function and activity of the DHFR protein. Four microRNA binding sites were found to be highly affected due to 3’UTR SNPs. Further, docking simulation suggested that the order of binding affinity of methotrexate towards native and all three mutant forms of DHFR is D153V (rs121913223)>native>G18R (rs61736208)>D187Y (rs200904105). Conclusion: This is the first study to report the normative genotype distribution of DHFR variants in Indian population and also to report that DHFR (-317A>G and -680C>A) variants do not confer a potential risk for development of ALL. Inter-ethnic differences exist in the distribution of DHFR variant genotypes, and this can lead to variability in therapeutic response to DHFR substrates. Protein sequence analysis revealed rs200904105 influences the phosphorylation process (post-translational modification) of DHFR and docking simulation suggested methotrexate to have a higher affinity towards rs121913223 mutant form. Therefore, studies are warranted to explore the clinical impact of these variants in the Indian population.