Development of ARMS-PCR method to detect two mutations of MTHFR gene(C677T,A1298C)in suspected cases of thrombosis

Abstract

Background: Thrombosis is the formation of a blood clot in a blood vessel that prevents blood flow in the blood circulatory system. Nowadays thrombosis is one of the causes of mortality in human societies. Thrombosis can be caused by genetic disorders or environmental factors that help the formation of clots. Two common mutations of methylene tetrahydrofolate reductase gene known as C677T and A1298C are realized as the effective factors for thrombophilia. The purpose of this study is to design a proper ARMS-PCR method for the diagnosis of these two mutations of MTHFR gene and the evaluation of its relationship with people suspected of thrombosis. Methods: first 50 samples suspected of thrombosis were collected from cases that had gone to laboratories for coagulation tests, then the genomic DNA of the samples was extracted using the proper commercial kit. Proper ARMS-PCR primers for C677T and A1298C mutations were designed and then synthesized. After the optimization of the ARMS-PCR reaction on the positive control sample, the reaction was carried out for all 50 samples. A number of mutated and non-mutated samples were studied for confirmation by the PCR-Sequencing method.Results: the results from the reaction showed that from 50 samples under study, after the optimization, 13 people had the C677T mutation (26%) from which 5 people had the homozygous genotype and 8 people had the Heterozygous genotype and for the A1298C mutation, 12 positive sample were found (24%) from which 7 people had the Heterozygous genotype and 5 people had the homozygous genotype.