Altered methylation pattern in EXOC4 is associated with stroke outcome: an Epigenome- Wide Association Study

Abstract Background and Purpose The neurological course after stroke is highly variable and is determined by demographic, clinical, and genetic factors. However, other heritable factors such as epigenetic DNA methylation could play a role in neurological changes after stroke. Methods We performed a three-stage epigenome-wide association study (EWAS) to evaluate DNA methylation associated with the difference between the National Institutes of Health Stroke Scale (NIHSS) at baseline and at discharge (ΔNIHSS) in ischemic stroke patients. DNA methylation data in the Discovery (n = 643) and Replication (n = 62) Cohorts were interrogated with the 450K and EPIC BeadChip. Nominal CpG-sites from the Discovery (p-value < 10 -06 ) were also evaluated in a meta-analysis of the Discovery and Replication cohorts, using a random-fixed effect model. Specific cell type methylation was assessed using EpiDISH. We integrated the methylation data with 1305 plasma protein expression levels measured by SOMAscan and measured RNA expression with RT-PCR in a subgroup of 13 subjects. Results The meta-analysis revealed an epigenome-wide significant association in EXOC4 (p-value = 8.4x10 -08 ) and in MERTK (p-value = 1.56x10 -07 ). Only the methylation in EXOC4 was also associated in the Discovery and in the Replication cohort (p-value = 1.14x10 -06 and p-value = 1.3x10 -02 , respectively). EXOC4 methylation negatively correlated with the long-term outcome (coefficient = -4.91) and showed a tendency towards a decrease in EXOC4 expression (rho = -0.469, p-value = 0.091). Pathway enrichment from the meta-analysis revealed significant associations related with the exocytic process. Seventy-nine plasma proteins were differentially expressed in association with EXOC4 methylation. Pathway analysis of these proteins showed an enrichment in Natural Killer cell activation. Conclusions DNA methylation of EXOC4 is associated with a worse neurological course after stroke. Pathway analysis suggests that the exocytic pathway could be a potential biological mechanism underlying this association.