In this study our aim was to analyze whether the neurological course could be associated with epigenetic modifications. With this objective, we studied the genome-wide DNA methylation pattern associated with ΔNIHSS at discharge. DNA methylation is probably the most studied epigenetic variation, consisting of the addition of a methyl group to a cytosine, mainly in the context of cytosines and guanines (CpG sites). We selected ΔNIHSS at discharge as the exposure variable because it was independently associated with functional long-term outcome. The results from our EWAS suggest that the neurological course of stroke patients measured as the difference between NIHSS at baseline and NIHSS at discharge has an impact on DNA methylation in specific CpG sites.
From the 44 candidate CpG sites identified in the Discovery Analysis, two CpG sites, located in genes bodies, were epigenome-wide significance (p-value < 2.4x10− 7) in the meta-analysis and only one CpG site (cg00039070) located in the body of the EXOC4 gene was validated.
EXOC4, also known as SEC8, encodes for a subunit in the exocyst complex, a protein complex involved in the tethering of secretory vesicles to the plasma membrane (34). Different functions are attributed to the exocyst complex, including but not limited to, exocytosis, cell growth cytokinesis, and neuronal development (37,38). It is highly expressed in the brain and is enriched in axon growth cones and dendritic branches (37).
The Sec8 protein, encoded by EXOC4, has been seen to control the synaptic targeting and the insertion of glutamate receptors in the synapsis, controlling the directional movement of glutamate receptors to the post-synaptic membrane (38). In stroke, the release of the glutamate neurotransmitter is associated with ischemic cell death in a process known as excitotoxicity. Briefly, the glutamate neurotransmitter is increased because of the ischemic insult (39) and over-activates two kind of glutamate receptors: the N-methyl-D-aspartate receptor (NMDAR) and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) (39,40). Activation of the synaptic NMDAR (containing the GluN2A subunit) leads to pro-survival signaling (41), while activation of extra-synaptic NMDAR induces a downstream neurotoxic cascade (42) that finally causes delayed neuronal death. Both NMDAR and AMPAR have been reported to be associated with the Sec8 subunit of the exocyst complex (38,43). Sec8 is involved in the targeting of these receptors to the post-synaptic membrane (38,43). Our results indicated that higher expression of EXOC4 (Sec8) is associated with a better neurological course. Given that Sec8 is controlling trafficking of synaptic glutamate receptors, it could be related to survival signaling (41).
Moreover, apart from the differential methylation identified in EXOC4, we found another gene, GRM3, nominally associated with ΔNIHSS in the discovery, that encodes glutamate metabotropic receptor 3, also involved in excitotoxicity processes. This gene was associated with memory impairment in a genetic study in Alzheimer disease patients (44).
Our pathway analysis has shown an enrichment of genes belonging to exocytic process and vesicle-mediated transport to the cell membrane. These results seem to be strengthening the effect that this process related with excitotoxicity, regulated by methylation, could have on stroke outcome.
A recently published GWAS has identified seven loci associated with stroke outcome measured through the NIHSS scale (calculating the difference between NIHSS at baseline and at 24h) (10). Their functional annotation strongly suggested GRIA1 and ADAM23 associated with ΔNIHSS. Both genes are also involved in excitotoxicity processes. Both results support a role of excitotoxicity in processes related to stroke neurological outcomes modulated by genetic and epigenetic variations. Despite clinical trials using drugs to modulate excitotoxicity processes having failed, progress has been made in clarifying the mechanisms that explain this failure (45).
One in vivo study in EXOC4 mutant’s drosophila showed that apart from this gene being involved in glutamate receptor trafficking, it is also required for regulating synaptic microtubule formation and synaptic growth, thus suggesting that EXOC4 methylation could be altering different processes in the synapsis (46).
The results from the pathway analysis based on the proteomic results also suggest that the inflammatory pathway, regulated by NK cells, could be involved in the regulation of stroke outcome by methylation. The results from the differential methylation studied by cell type also supported the involvement of this pathway. It showed some associations for the CpG sites identified in our study, especially in NK cells, although not for EXOC4. Both the excitotoxicity and neuroinflammatory pathways have been suggested to be pathological mediators of ischemic brain damage (47).
Limitations
The first limitation is the difference in sample size and clinical features between the Discovery and Replication Cohorts. However, we looked for which clinical variables were associated with the methylation of EXOC4 and we did not find any. Therefore, there is no reason to believe that they would affect EXOC4 methylation in the Replication Cohort. Despite the differences between both cohorts, we have been able to replicate the results, which reinforces the plausible implication of EXOC4 methylation in stroke outcome.
Another limitation is the use of whole blood to study DNA methylation in association with stroke outcome. However, other epigenomic, transcriptomic, and proteomic studies on stroke (13,48) have also used blood samples as it is also a relevant tissue in stroke outcome. Additionally, the blood and brain tissues have been found to have a 0.86 correlation in global methylation (49). Finally, we were not able to find a significant correlation between EXOC4 mRNA levels and EXOC4 methylation, despite a trend being observed. Probably, the sample size of for the transcriptomic analysis was not large enough to obtain significant results.