Background and Aims 35 loci have been associated with stroke in Genome-Wide association studies (GWAS). We previously found an altered DNA methylation pattern in one gene identified in GWAS to be associated with stroke risk (ZFHX3). DNA methylation in this gene was causally associated with atherothrombotic stroke. Our aim is to determine whether genes associated with stroke risk in GWAS are also linked with stroke susceptibility through epigenetic regulation. Moreover, to study the implication of these genes in the atherosclerotic process using an in vitro model.

Methods DNA methylation was assessed in 253 ischemic stroke patients and 43 controls using the Infinium 450KBeadChip and EPICBeadChip. We selected all the CpG-sites located in the 35 loci previously associated with stroke in the Megastroke + Uk Biobank cohorts. The significant associations were evaluated in an in vitro model of human coronary artery endothelial cells exposed to normal laminar flow (LSS) and atherogenic flow environments: oscillatory (OSS) and elevated laminar shear stress (ESS).

Results 134 CpG-sites located in 27 different loci were associated with stroke (p<0.05) and 8 CpG-sites remained significant after Bonferroni adjustment. These CpG-sites corresponded to 6 different genes: ZFHX3, SH2B3, SMARCA4, TSPAN2, ILF3 and CDK6. All of them presented hypomethylation in stroke patients compared with controls. We found a significant increased expression of ZFHX3 and CDK6 in ESS compared to LSS. Expression of ILF3 was 60% higher in OSS and 40% lower in ESS compared to LSS. The phosphorylation status of ZFHX3 and SMARCA4 was found to be increased in the OSS environment.

Conclusions Our findings indicate that epigenetic regulation of genes that are risk factors for stroke is associated with stroke susceptibility.

Conflict of Interest No