Association of DNA Methylation in Blood Pressure-Related Genes With Ischemic Stroke Risk and Prognosis

Abstract

Background: A genome-wide association study identified 12 genetic loci influencing blood pressure and implicated a role of DNA methylation. However, the relationship between methylation and ischemic stroke has not yet been clarified. We conducted a large-sample sequencing study to identify blood leukocyte DNA methylations as novel biomarkers for ischemic stroke risk and prognosis based on previously identified genetic loci.

Methods: Methylation levels of 17 genes were measured by sequencing in 271 ischemic stroke cases and 323 controls, and the significant associations were validated in another independent sample of 852 cases and 925 controls. The associations between methylation levels and ischemic stroke risk and prognosis were evaluated.

Results: Methylation of AMH, C17orf82, HDAC9, IGFBP3, LRRC10B, PDE3A, PRDM6, SYT7 and TBX2 was significantly associated with ischemic stroke. Compared to participants without any hypomethylated targets, the odds ratio (OR) (95% confidence interval, CI) for those with 9 hypomethylated genes was 1.41 (1.33-1.51) for ischemic stroke. Adding methylation levels of the 9 genes to the basic model of traditional risk factors significantly improved the risk stratification for ischemic stroke. Associations between AMH, HDAC9, IGFBP3, PDE3A and PRDM6 gene methylation and modified Rankin Scale scores were significant after adjustment for covariates. Lower methylation levels of AMH, C17orf82, PRDM6 and TBX2 were significantly associated with increased 3-month mortality. Compared to patients without any hypomethylated targets, the OR (95% CI) for those with 4 hypomethylated targets was 1.12 (1.08-1.15) for 3-month mortality (P = 2.28 × 10^-10).

Conclusion: The present study identified blood leukocyte DNA methylations as potential factors affecting ischemic stroke risk and prognosis among Han Chinese individuals.

Keywords: blood pressure; methylation; mortality; prognosis; stroke.

Figure 1

Results for the associations between DNA methylation and ischemic stroke. The crude P-values for the associations between DNA methylation and ischemic stroke. The x-axis represents the chromosome positions. The y-axis shows the –log₁₀ P-values for the associations between the levels of DNA methylation sites and ischemic stroke. A total of 866 methylation sites were analyzed in stage one (black circles), and 224 of them passed the significance threshold of 5.75 × 10⁻⁵ (red line). A total of 377 methylation sites were analyzed in stage two (green triangles), and 296 of them passed the significance threshold of 5.75 × 10⁻⁵. The associated methylation sites tend to cluster in targets.

Figure 2

Ischemic stroke risk according to the number of hypomethylated genes. ROC curves were used to obtain the optimal cutoff points for the representative targets of 9 genes, including AMH_3 (<3.21%), C17orf82_3 (<3.35%), HDAC9_1 (<1.88%), IGFBP3_2 (<3.90%), LRRC10B_1 (<1.09%), PDE3A_3 (<2.47%), PRDM6_3 (<2.01%), SYT7_1 (<1.98%) and TBX2_3 (<3.00%), to define hypomethylation. CI, Confidence interval; OR, Odds ratio.

Figure 3

Association between DNA methylation and 3-month mortality. The upper panel shows the adjusted ORs and corresponding 95% CIs for the associations between hypomethylation of each target and 3-month mortality. The lower panel shows the adjusted ORs and corresponding 95% CIs for 3-month mortality according to the number of hypomethylated targets among acute ischemic stroke patients.

Figure 4

Hazard ratios for the association between DNA methylation and 3-month mortality. The four panels show the hazard ratios for each of the CpG sites in the four genes assessed by Cox proportional hazard models. The associations were adjusted for covariates.

Figure 5

Association between DNA methylations and survival. The patients were separated into three groups by tertiles of methylation levels of each of the four targets. The survival rates between the three groups were compared. Patients who have higher methylation levels of these genes have higher survival rates than patients with lower methylation levels.

Figure 6

Interactions between methylations and disease severity on mortality. The upper panel shows the interaction effects between hypomethylation of each of the four targets and NIHSS score on 3-month mortality. The increased risk of death per 1-point increase in NIHSS score was higher in patients with hypomethylation of each of these 4 targets than in those without hypomethylation of the corresponding target. The middle panel shows the adjusted ORs and corresponding 95% CIs for the associations between NIHSS score and 3-month mortality among subgroups of acute ischemic stroke patients according to the number of hypomethylated targets. The increased risks of death per 1-point increase in NIHSS score were higher in patients with 3 or 4 hypomethylated targets than in those without hypomethylated targets. The lower panel shows the combined effect of the number of hypomethylated targets and disease severity on 3-month mortality. Patients were separated by the number of hypomethylated targets (separated by≤2 hypomethylated targets and ≥3 hypomethylated targets) and disease severity (minor stroke: NIHSS score <5, moderate stroke: NIHSS score 5–14, severe stroke: NIHSS score ≥15). Approximately 25.4% of ischemic stroke patients may have 3 or 4 hypomethylated genes, and nearly 10% of them would die within 3 months of onset, while the 3-month mortality for the overall patients was ~3%. Notably, for severe ischemic stroke patients with 3 or 4 hypomethylated genes, half of them died within 3 months of onset.