ROCK2 Polymorphism in Patients with Vasospastic Angina

Abstract

Backgrounds and Objectives: Recent studies indicate that in response to vasoconstrictor stimuli the small GTPase RhoA and its downstream effector, ROCK/Rho-kinase, are associated with hypercontraction of the vascular smooth muscle of coronary arteries through augmentation of myosin light chain phosphorylation and Ca2+ sensitization. Expression of ROCK/Rho-kinase mRNA was significantly increased and up-regulated at the spastic coronary artery in a porcine model, and a specific inhibitor of ROCK/Rho-kinase inhibited coronary artery spasm in humans. We, therefore, explored the role of ROCK2 polymorphisms in the pathogenesis of vasospastic angina (VA).

Materials and Methods: We studied 106 patients with VA who exhibited spontaneous or provoked coronary spasm during coronary angiography and compared the prevalence of ROCK2 polymorphisms between this group of patients with VA and controls whose angiograms were normal and in whom the ergonovine test did not cause spasm (n=107). Genomic DNA was isolated from peripheral blood leukocytes by using the QuickGene SP kit (FUJIFILM Corporation, Tokyo, Japan) according to the instructions of the manufacturer Five single nucleotide polymorphisms (SNPs) of the ROCK2 gene (rs978906, rs2271621, rs2230774, rs1515219, rs3771106) were selected based on the following criteria: the functionality with the priority order of picking SNPs being based on their potential functions [nonsynonymous SNPs > splicing-site SNPs > synonymous SNPs > 5’ untranslated-region (UTR) SNPs > 3’ UTR SNPs > intronic SNPs], the frequency of minor alleles ≥ 20% in Asians population, the spacing of SNPs should be relatively even across the gene region, and then genotyped by high-resolution melting. Linkage disequilibrium and haplotype analyses were performed by using SHEsis program. The incidence of coronary risk factors, including age, sex, dyslipidemia, hypertension, diabetes, cigarette smoking and the polymorphisms of the ROCK2 were compared in control and VA groups by forward stepwise selection.

Results: The prevalence of genotypes of the five interesting SNPs in patients with VA was not different from that in the control group. Among the coronary risk factors, including age, sex, dyslipidemia, hypertension, diabetes and cigarette smoking, and the polymorphisms of the ROCK2, male gender was the only independent risk factor for VA by multiple logistic regression analysis (odd ratio 7.745, CI 3.977 - 15.080). In haplotype analysis, the haplotype G-T-C-T-G (in order of rs978906, rs2271621, rs2230774, rs1515210 and rs3771106) was significantly associated with a decreased risk of VA (p = 0.007).

Conclusion: The haplotype G-T-C-T-G in ROCK2 gene had a protective effect against VA, suggesting the involvement of ROCK2 in VA pathogenesis. However, we cannot exclude the possibility of having missed non-coding functional variants or rare coding variants.