Higher frequency of rs4977574 (the G Allele) on chromosome 9p21.3 in patients with myocardial infarction as revealed by PCR-RFLP analysis.

Single Nucleotide Polymorphisms (SNPs) can genetically predispose individuals for certain diseases and therefore are of clinical significance. Myocardial infarction (MI) was investigated in large genetic association studies revealing novel SNPs associated with MI. rs4977574 is a non-protein coding SNP (A>G) that is located in proximity of cyclin-dependent kinase inhibitor 2A and B genes on chromosome 9p21.3. rs4977574 has been recently found to be associated with the early-onset of MI, and rs4977574 is characterized by a guanine nucleotide (G) instead of an adenine nucleotide (A). rs4977574 has been reported to increase the risk for MI by 28%. In this study, we developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for detecting rs4977574 in Turkish population that consisted of 28 controls without previous MI record and 44 patients with MI. An intergenic genomic region containing the target SNP was amplified by PCR using patient's genomic DNA. Amplified DNA fragments were digested with a restriction enzyme, HhaI that cuts the amplified sequence if only the sequence has GCGC that carries rs4977574. After digestion with HhaI, DNA fragments were visualized in order to detect genotypes. PCR-RFLP revealed that the frequency of rs4977574, the MI-associated allele (G), was 56.8% (25/44) in patients with MI and 33.9% (9.5/28) in controls; the frequency of rs4977574 in patients with MI was significantly higher compared to controls (P = 0.027). Importantly, for the first time in this study, we have developed a novel PCR-RFLP method to detect the presence of rs4977574.