Identification of 20 novel loci associated with ischaemic stroke. Epigenome-wide association study

Abstract

DNA methylation is dynamic, varies throughout the life course, and its levels are influenced by lifestyle and environmental factors, as well as by genetic variation. The leading genetic variants at stroke risk loci identified to date explain roughly 1-2% of stroke heritability. Most of these single nucleotide polymorphisms are situated within a regulatory sequence marked by DNase I hypersensitivity sites, which would indicate involvement of an epigenetic mechanism. To detect epigenetic variants associated with stroke occurrence and stroke subtypes. A two-stage case-control epigenome-wide association study was designed. The discovery sample with 401 samples included 218 ischaemic stroke (IS) patients, assessed at Hospital del Mar (Barcelona, Spain) and 183 controls from the REGICOR cohort. In two independent samples (N = 226 and N = 166), we replicated 22 CpG sites differentially methylated in IS in 21 loci, including 2 CpGs in locus ZFHX3, which includes known genetic variants associated with stroke. The pathways associated with these loci are inflammation and angiogenesis. The meta-analysis identified 384 differentially methylated CpGs, including loci of known stroke and vascular risk genetic variants, enriched by loci involved in lipid metabolism, adipogenesis, circadian clock, and glycolysis pathways. We identified a set of 22 CpGs in 21 loci associated with IS. Our analysis suggests that DNA methylation changes may contribute to orchestrating gene expression that contributes to IS.

Keywords: DNA methylation; EWAS; Ischaemic stroke.

Figure 1.

Manhattan and QQ-plot in the discovery sample. (a) Manhattan plot (red line and blue line for statistically and nominally significance threshold) and (b) QQ-plot.

Figure 2.

Summary of loci harbouring significant CpGs in meta-analysis, overlapping known genome-wide associations to phenotypes and diseases. (a) 115 CpG (30%) of the 384 joint CpGs are associated with known variants. Bars represent the number of loci associated with each trait; 37 loci are associated with more than 1 trait. (b) List of loci with known associations. In bold, loci validated in discovery.

Figure 3.

Venn Diagrams showing the overlap of methylation-variable positions between meta-analysis and the ischaemic stroke subtypes. LAA, large-artery atherosclerosis: CE, cardiembolic; SVD, small vessel diseases; UND, undetermined IS subtypes.

Figure 4.

Summary of loci harbouring nominally significant CpGs, by TOAST, associated with phenotypes and diseases in genome-wide association studies. One locus can be associated with more than one trait. LAA, large-artery atherosclerosis: CE, cardiembolic; SVD, small vessel diseases; UND, undetermined IS subtypes; BMI, body mass index; BP, blood pressure; DM, diabetes mellitus; CHD, coronary heart disease.