P1620Novel single nucleotide polymorphisms in exon-10 of human coagulation factor V gene in patients with pulmonary thromboembolism

Abstract

Background: The occurrence of venous thromboembolism is a culmination of environmental and genetic risk factor. Factor V Leiden (FVL) is the most common inherited thrombophilia. The annual incidence of venous thromboembolism in patients with FVL is 0.5%. Aim: To look for factor V Leiden and other novel mutations in Exon-10 of human coagulation factor V gene in patient with documented pulmonary thromboembolism. Methods: Sixty consecutive patients with acute pulmonary thromboembolism documented on computed tomography pulmonary angiography and admitted in Sri Venkateswara Institute of Medical Sciences were included in the study. Genomic DNA was isolated from blood samples using QIAamp DNA Mini spin column (Qiagen) DNA extraction kit. Extracted DNA samples were analyzed on 1% agarose gel electrophoresis. PCR amplification was done in the Eppendorf Mastercycler nexus gradient model, Hamburg, Germany and amplified PCR products were sequenced by Sanger's dideoxy chain termination method Eurofins Genomics India Pvt Ltd., India. The sequences were compared by performing multiple sequence alignment using ClustalX tool to identify the mutations. ExPASy analysis was used to translate nucleotide sequence into amino acid sequence. Results: Of 60 patients, 48 (80%) were males. The mean age was 41.2±12-9 years. We found two novel transition type mutations, c.1538 G>A and c.G>A in exon-10 of Factor V gene which is responsible for the cleavage site for activated protein C. ExPASy (Expert Protien Analysis System) analysis showed that these point mutations resulted in single amino acid change in protein sequence at p.Arg513Lys and p.Arg534Gln respectively. Conclusion: Like most Indian studies we did not find Factor V Leiden mutation which is a missense mutation in Factor V gene at G 1691 resulting in change from arginine to glutamine (R506 Q) which slows the inactivation of Factor Va by aPC thereby increasing risk for venous thrombosis. However we found 2 novel mutation in exon 10 Factor V which are responsible for cleavage site of aPC, which prevent inactivation of Factor V by aPC and predispose to thrombosis.