Direct Bisulfite Sequencing and Methylation Specific PCR to Detect Methylation of p15INK4b and F7 genes in Coronary Artery Disease Patients

Genome-Wide Association Studies (GWAS) have identified genetic variants contributing to the risk of cardiovascular disease (CVD) at the chromosome 9p21 locus. The chromosome 9p21 is an important susceptibility locus for several multifactorial diseases like ischemic stroke, aortic aneurysm, type 2 diabetes mellitus and coronary artery disease (CAD). F7 gene because of its role in activating the extrinsic pathway by the exposure of tissue factor after plaque disruption is related to atherothrombosis. The aim of the present study was to evaluate methylation status of two CAD related genes, p15INK4b and F7, in Iranian patients with coronary artery disease (CAD). Thirty samples from 15 male and 15 female with diagnosed 3 vessels disease CAD and 60 Samples from 60 non-CAD controls who underwent coronary angiography was analyzed by MSP and direct sequencing. DNA methylation levels at p15INK4b gene increased significantly in CAD patients in comparison with control group (p-value<0.001). To quantitative analysis of methylation, direct bisulfite sequencing method revealed 6 methylated CpGs islands. No significant difference in F7 promoter methylation was observed between CAD patients and control group. Based on the findings of this study it is possible to assume, p15INK4b methylation is associated with pathophysiology of CAD.