Investigation of Methyl Tetrahydro-Folate Reductase C677t Polymorphism in a Sample of Patients in a Hospital Based Diagnostic Setting in Sri Lanka

Abstract

The present study was conducted to identify the prevalence of MTHFR gene mutation in a group of Sri Lankan patients seeking routine diagnostic testing for the C677T mutation through PCR followed by RFLP diagnostic methods. Human MTHFR gene codes for the production of MTHFR enzyme in humans which plays major role in homocysteine and folate metabolism in humans. The mutation in MTHFR gene at nucleotide position 677 has two possibilities: C (cytosine, which occurs in the wild type gene) or T (thymine, which occurs in the mutated gene).In C677T variant, modification from C to T gives rise to the replacement of alanine by valine residue in protein at 222 aa position. Eight serum samples were collected from the MTHFR mutation suspected patients. Genomic DNA extraction was done and extracted DNA was subjected to PCR amplification. PCR products were subjected to RFLP assay with overnight digestion. The substitution of "C" to "T" at 677 position of MTHFR gene creates a restriction site for the enzyme Hind111. RFLP products were subjected to gel electrophoresis and further analysis. The results obtained shows that, of the 8 samples tested, 2 had 677CT genotype, 1 had 677TT genotype whereas the remaining 5 had 677CC wild type. Homozygous 677TT genotype leads to lower MTHFR activity and hyperhomocysteinaemia, which can cause defects in DNA repair. However, as this is a small sample of the population, it is recommended to carry out studies with a large sample volume to ascertain the prevalence of this mutation in the community.