Genetic and Epigenetic Signatures in Cerebrovascular Disease

Abstract

Cerebrovascular disease is one of the predominant causes of morbidity and mortality worldwide. This thesis encompasses three studies aimed at understanding the genetic underpinnings of the rate of cell death in ischemic stroke as well as vasospasm and neurological dysfunction after aneurysmal subarachnoid hemorrhage in mice and humans. Part 1: Following ischemic stroke, the penumbra, at-risk neural tissue surrounding the core infarct, survives for a variable period of time before progressing to infarction. In part 1 of this thesis, we investigated genetic determinants of the size of penumbra in mice subjected to middle cerebral artery occlusion (MCAO) using a genome-wide approach. 449 male mice from 33 inbred strains underwent MCAO for 6 hours (215 mice) or 24 hours (234 mice). A genome-wide association study using genetic data from the Mouse HapMap project was performed to examine the effects of genetic variants on the penumbra ratio, defined as the ratio of the infarct volume after 6 hours to the infarct volume after 24 hours of MCAO. Efficient mixed model analysis was used to account for strain interrelatedness. Penumbra ratio differed significantly by strain (F = 2.7, P < 0.001) and was associated with 18 significant SNPs, including 6 protein coding genes. We have identified 6 candidate genes for penumbra ratio: Clint1, Nbea, Smtnl2, Rin3, Dclk1, and Slc24a4. Part 2: Delayed ischemic neurological deficits and infarction (DINDI) after subarachnoid hemorrhage (SAH) are a manifestation of multiple processes including cerebral vasospasm and neuronal dysfunction. This constellation of findings is responsible for significant morbidity and mortality after SAH. In part 2 of this thesis, we employed an intra-cisterna magna blood injection model of subarachnoid hemorrhage and determined the degree of cerebral vasospasm and change in neurological score after 24 hours in 31 inbred strains of mice. Genetic determinants of vasospasm and neurological score were analyzed using genome-wide association analyses with efficient mixed models to account for strain interrelatedness. 403 mice were included in the study, 268 of which were in the SAH group. Cerebral vasospasm (F = 1.97, P = 0.003) and neurological outcome (F = 3.85, P < 0.001) varied across strains. Vasospasm and neurological outcome were not correlated (ρ = 0.08, P = 0.22). There were 6 genome-wide significant SNPs all in chromosome 1 associated with cerebral vasospasm. Neurological score was associated with 46 genome-wide significant SNPs across 11 chromosomes in 23 protein coding genes. No SNPs were significant for both outcomes. Several candidate genes associated with neurological outcome may function in Wnt/ beta-catenin signaling or were GABA receptor subunits and Ptgs2, the gene encoding COX-2, was within 500kbp of a SNP associated with vasospasm. Part 3: Delayed ischemic neurologic deficits and infarction (DINDI) are a significant cause of morbidity and mortality in ~25% of patients with aneurysmal subarachnoid hemorrhage (SAH). The drivers of DINDI are diverse and not well characterized. In part 3 of this thesis, we investigated dynamic epigenetic variation in peripheral blood after SAH to both identify pathways involved and to define CpG signatures for prognostic purposes. Peripheral blood samples from 59 patients with SAH were collected at the time of presentation and at subsequent time points during their hospital stay and at follow up clinic visits. Linegaard ratios (mean velocity in middle cerebral artery / mean velocity in ipsilateral extracranial internal carotid artery) were obtained for each patient at each time point. Glasgow coma scores (GCS) were obtained for each time point. Reduced representation bisulfite sequencing (RRBS), sequencing of CpG enriched areas associated with the majority of promoters genome-wide, has been performed on samples from 10 patients to date. A mixed model was utilized to analyze sequencing data including age, race, smoking status, and percentage of lymphocytes, neutrophils, and monocytes as covariates, and post-bleed day as a random effect. Lindegaard ratio, dichotomized into either above or below 3, was used as the outcome. The Reactome tool in Cytoscape was used to cluster significant proteins and identify relevant pathways. 1,237 significant CpG islands (Benjamini and Hochberg adjusted P value < 0.05) in 995 unique associated features were associated with Linegaard ratio over 3, including 352 unique genomic features. 61 proteins encoded by genes containing significant CpG sites were identified that had interactions with at least one other protein encoded by a gene within in the set. Reactome identified 32 statistically significant pathways enriched within those 61 nodes, the most significant of which was PI3K/Akt signaling. CpG methylation in many unique protein coding genes varies between patients who go on to develop ultrasonographic vasospasm over the first several days following SAH. Akt and Wnt signaling have previously been implicated in phenotypic switching, in the case of Akt in the acute setting following SAH. Further analysis is required, and is ongoing, to identify the temporal nature of Akt methylation and to identify CpG signatures for prognostic purposes.