ANGPT1 methylation and delayed cerebral ischemia in aneurysmal subarachnoid hemorrhage patients

Abstract

Background: Delayed cerebral ischemia (DCI) is a common secondary complication and an important cause of disability and mortality among patients who survive aneurysmal subarachnoid hemorrhage (aSAH). Knowledge on DCI pathogenesis, risk factors, and biomarkers are essential for early detection and improved prognosis. To investigate the role of DNA methylation in DCI risk, we conducted an epigenome-wide association study (EWAS) in 68 patients followed up to 1 year after the initial aneurysm rupture. Blood samples were collected within 48 h post hemorrhage and used for DNA methylation profiling at ~ 450k CpG sites. A separate cohort of 175 patients was sequenced for the top CpG sites from the discovery analysis for a replication of the EWAS findings.

Results: EWAS did not identify any epigenome-wide significant CpGs. The top signal, cg18031596, was annotated to ANGPT1, a gene with critical functions in angiogenesis after vascular injury. Post hoc power calculations indicated a well-powered discovery analysis for cg18031596. Analysis of the replication cohort showed that four out of the five CpG sites sequenced at the ANGPT1 locus passed a Bonferroni-adjusted significance threshold. In a pooled analysis of the entire sample, three out of five yielded a significant p-value, and the top association signal (p-value = 0.004) was seen for a CpG that was not originally measured in the discovery EWAS. However, four ANGPT1 CpG sites had an opposite effect direction in the replication analysis compared to the discovery EWAS, marking a failure of replication. We carefully examined this observed flip in directions and propose several possible explanations in addition to that it was a random chance that ANGPT1 ranked at the top in the discovery EWAS.

Conclusions: We failed to demonstrate a significant and consistent effect of ANGPT1 methylation in DCI risk in two cohorts. Though the replication attempt to weaken the overall support of this gene, given its relevant function and top rank of significance in the EWAS, our results call for future studies of larger aSAH cohorts to determine its relevance for the occurrence of DCI.

Keywords: ANGPT1; Aneurysmal subarachnoid hemorrhage; Delayed cerebral ischemia; Epigenome-wide association study; Methylation.

Fig. 1

EWAS of DCI. A Quantile-Quantile plot showing expected (x-axis) and observed (y-axis) -log10-transformed p-values. B Manhattan plot showing the -log10-transformed p-values (y-axis) for each CpG by its genomic position (x-axis) organized by chromosome. C coMET plot for the ANGPT1 locus. For the coMET plot, the upper panel shows the EWAS p-values; the middle yellow track represents the gene position with exons indicated by boxes; the lower panel shows the correlation between selected CpGs in this genomic region

Fig. 2

Correlations between the 450k array M values and the MethylSeq M values at four CpG sites among 58 discovery samples with both measurements available. The Spearman correlation coefficients are shown in the upper left of each sub-figure. Colored by DCI status

Fig. 3

Opposite effect directions of cg18031596 in the discovery and replication analysis. The horizontal line segment represents the median. DCI cases on average had a higher M value than controls in the discovery stage, yet in the replication samples the opposite was observed

Fig. 4

Regression coefficients in simulated discovery (x-axis) and replication (y-axis) samples. Results from 10,000 simulation experiments are plotted. Black and red dots mark the 1229 samplings which yielded a discovery p-value < 0.05; the background gray dots represent the remaining non-significant ones (p-value > 0.05). Coefficients were derived from a model where M value at cg18031596 was regressed on DCI and covariates. Marginal distributions are indicated by the rug plots along the inner side of the axes. The horizontal and vertical dashed line identify the observed replication coefficient (−0.2) and observed discovery coefficient (0.1; can be found in Table S3), respectively. Red dots represent a flip of association direction with the simulated replication coefficient more extreme than the observed replication coefficient