Meschia, James F.

Background: Prior studies have shown an association between mutations within the oxidative phosphorylation (OXPHOS) pathway and familial ischemic stroke syndromes. We investigated whether common autosomal and mitochondrial genetic variants within OXPHOS genes were associated with sporadic ischemic stroke in case-control. Methods: This multicenter association study was performed using a discovery cohort of 1643 individuals and a validation cohort of 2432 individuals from sites participating in the International Stroke Genetics Consortium. We employed a gene-set enrichment analysis (GSEA) using all 95 genes structurally involved in OXPHOS to determine the probability that the OXPHOS gene-set harbored more variants associated with stroke than predicted by chance. We then constructed a genetic score consisting of common variants within these genes. This genetic score was developed in the discovery cohort, and then tested for association with ischemic stroke in the validation cohort. Results: GSEA identified an aggregate association between the OXPHOS gene-set and ischemic stroke (p = 0.012). Based on this result, we constructed and validated a genetic score which demonstrated an association between OXPHOS genetic variation and ischemic stroke (odds ratio (OR) = 1.17, p = 0.008). In order to further distill the association between OXPHOS and stroke, we developed and tested additional scores for each of the five OXPHOS respiratory complexes. This sub-analysis revealed an independent association between OXPHOS Complex I and ischemic stroke (OR = 1.06, p = 0.050). Conclusions: This pathway-based study, informed by GSEA, employing independent discovery and validation cohorts, establishes an association between common genetic variants within OXPHOS genes and ischemic stroke. Analysis of variants within each OXHPOS Complex suggests that Complex I mediates this effect. Further studies are needed to identify the individual variants which influence ischemic stroke risk. Such discoveries offer the promise of new therapeutic targets for the prevention and treatment of stroke.

BACKGROUND AND PURPOSE: Ischemic stroke is a heterogeneous complex disease that results from the interaction between numerous genetic and environmental risk factors. Stroke heritability is approximately 40% in the overall population, similar to that of cancer, although there may be important differences in the etiologies of young (<49 years old) versus older onset ischemic stroke. The goal of this study was to conduct a mechanistic genome-wide association (GWA) analysis contrasting the gene pathways involved in young- and old-onset stroke. METHODS: Our analysis included 946 young subjects (448 ischemic stroke subjects and 498 age and sex matched controls, mean age of stroke onset 41 years) and 2558 older subjects (1070 ischemic stroke subjects and 1488 controls, mean age of stroke onset 66.5 years). All subjects were of European ancestry. The young onset cases and controls were genotyped by Illumina Omni1-Quad array with imputation up to 1.8 M SNPs and the old onset cases and controls by a combination of 550 v1 and v3 as well as 610 Illumina arrays with imputation up to 5.0 M SNPs. Single SNP association testing was performed within each contributing study by logistic regression adjusting for age, sex and population structure. Numbers of genes assigned with significant SNPs (P<=0.0001) were 18 and 25 with young- and old-onset groups, respectively, which were used as an input to pathway analysis by the Pathway Studio (Ariadne Genomics, Co.) RESULTS: SNP-level analyses did not reveal any significant associations with each group that met Bonferroni adjusted thresholds for statistical significance. In analyzing the ‘suggestively associated SNPs’, protein-protein interaction pathway analysis in the young-onset group revealed a strong segregation of significant hub proteins (CAT, RUNX1, and EIF4E) with more than 100 connections, while in the old-onset group the interaction networks were dispersed without much segregation. We observed that protein-protein interaction networks have a power-law connectivity distribution in both young- and old-onset groups. We identified 3 significant and focused pathways, ROS metabolism, melanogenesis, and IGF1R->CdEBPA/FOXO1A signaling, enriched for young-onset stroke (P<0.04). In the old-onset group, 10 pathways were identified involving broader signaling pathways. CONCLUSIONS: Young-onset stroke may involve genetic predisposition characterized by a few distinct and finite pathways. Our data suggests that young onset stroke may involve a few pathways that relate to a small number of key hub proteins, including ROS metabolism, melanogenesis, and IGF1R->CEBPA/FOXO1A signaling. Our analysis is consistent with the hypothesis that different pathways may be involved in young versus old onset stroke.

BACKGROUND: Recent genome-wide association studies (GWAS) have identified a number of novel SNPs associated with blood pressure and hypertension-related traits although studies were very large (approaching 80K participants) and effect sizes were modest with per allele effect sizes as small variants as a single millimeter of mercury difference in blood pressure. However, because hypertension is the most potent modifiable risk factor for ischemic stroke, lowering blood pressure by a few mm Hg can translate into measurable decrease in stroke risk. METHODS: We investigated 22 hypertension (HTN) associated SNPs in a case-control analysis of ischemic stroke in multiple stroke studies including the Siblings With Ischemic Stroke Study (SWISS), the Ischemic Stroke Genetics Study (ISGS), the Bio-Repository of DNA in Stroke (BRAINS), the Vitamin Intervention for Stroke Prevention (VISP) trial, and the Women's Health Initiative (WHI). SWISS/ISGS/BRAINS were analyzed together and included subtype information. Genotyping was funded through the individual studies, the Genomics and Randomized Trials Network (GARNET) consortium, and the SHARe (SNP Health Association Resource). All studies had both raw and imputed GWAS data. Analyses included the following phenotypes: all ischemic stroke, stroke subtypes, and age-at-stroke-onset and were stratified by race. Analyses were minimally adjusted for age, sex and principal components 1 & 2. Alpha was set at 0.05. RESULTS: Eleven of the SNPs from the HTN GWAS were associated with ischemic stroke in European ancestry from one or more of the study populations. Two SNPs on chromosome 12 and one on chromosome 21 were associated in multiple European ancestry cohorts. No SNP was associated in all studies. A SNP previously associated with HTN in African ancestry populations was associated with stroke in African-Americans. Analysis of subtypes found associations with small vessel disease and cardioembolic stroke but not large artery disease. The case-only age-at-stroke-onset analysis also found associations for the two SNPs on chromosome 12. CONCLUSION: These data suggest that candidate SNPs from a HTN GWAS may be associated with ischemic stroke and warrant further investigation. Racial differences were observed. Replication in populations from the other SiGN (Stroke Genetics Network), GENEVA (Gene Environment Association Studies) consortium, and the ISGC (International Stroke Genetics Consortium) studies are underway.

Background: Causative Classification of Stroke (CCS) system is a novel, web-based, and fully-computerized algorithm that integrates clinical, diagnostic, and etiologic stroke characteristics in an evidence-based manner (available at http://ccs.mgh.harvard.edu ) and provides in a given patient both phenotypic subtypes where abnormal evaluation findings are simply organized in major etiologic categories and causative subtypes where abnormal evaluation findings are filtered through a decision making process to identify the most likely cause of stroke. In this study, we sought to identify the concordance between phenotypic and causative CCS subtypes in the NINDS Stroke Genetics Network (SiGN). Methods: A total of 24 adjudicators from 15 US and European sites certified in CCS retrospectively determined CCS subtypes through chart review in 7134 patients. The CCS software provided causative subtypes in 3 confidence levels as “evident”, “probable” or “possible” based on the weight of causal evidence. We estimated how often CCS classified a given major abnormal evaluation finding as the causative stroke mechanism. Results: CCS assigned 55% of patients with the phenotype of moderate-to-severe atherosclerotic stenosis into the causative category of “evident large artery atherosclerosis”. Likewise, 50% of patients with a major cardiac source (such as atrial fibrillation) were classified into “evident cardio-aortic embolism”, and 74% of those with imaging evidence of a typical lacunar infarct into “evident small artery occlusion”. In 20% to 30% of patients, phenotypic and causative categories were the same but the causative subtype was assigned with a lower level of confidence. Six to 20% of patients with a given phenotypic feature were classified into a different causative category. Failure to investigate for other subtypes in the presence of a positive test finding and the presence of multiple competing etiologies were key contributors to the observed disconcordance between phenotypic and causative subtypes. Conclusions: Our findings show that the presence of a major abnormality in stroke evaluation does not necessarily mean that it is always the cause. The ability of CCS system to discriminate between phenotypic and causative characteristics of stroke etiology provides the opportunity to refine stroke phenotypes for use in research studies.

Background The Carotid Revascularization Endarterectomy versus Stenting Trial (CREST) enrolled 2502 patients between December of 2000 and mid-July of 2008. We analyzed temporal changes in outcomes for both CEA and CAS over the course of this study. Methods Enrollment was divided into 3 consecutive epochs (5 years, 14 months, and 16 months), each with approximately 834 patients ( Table ). Rates for the primary outcome of death, stroke, and myocardial infarction (DSMI) and for death and stroke (DS) during the periprocedural period were calculated. Poisson regression was used to adjust rates for age, sex, dyslipidemia, and symptomatic status, all of which were found to influence outcomes. Results For CAS, there was a 26% decline in DSMI (6.2% → 4.6%) and a 35% decline in DS (5.5% → 3.6%). For both composite endpoints in CEA there was no consistent pattern ( Table ). As CREST progressed, it enrolled younger patients, more men, patients more likely to be dyslipidemic and enrolled more asymptomatic patients (all p < 0.05). Adjustment for these changes mediated the improvement in event rates for the CAS patients, and had no consistent effect on CEA event rates ( Table ). Conclusion Periprocedural safety for CAS improved over time in CREST. Changes were inconsistent for CEA. Improvements for CAS in part appear to reflect changes in patient selection related to age, sex, and risk factors, as adjustment for those variables attenuated the decline in rates. Linking rates to operator experience was not feasible because of the large number of new interventionalists and surgeons entering CREST over time.

Background: Intracerebral hemorrhage (ICH) is the stroke subtype with the worst prognosis and has no established acute treatment. ICH is classified as lobar and non-lobar based on the location of the hemorrhage, with genetic variation contributing to risk of both subtypes. We report the first genome wide association study (GWAS) of this condition. Methods: The discovery phase included GWAS data (Illumina 610 or Affymetrix 6.0) from 6 ICH studies that enrolled subjects of European ancestry in the US and Europe. Cases were classified as lobar or non-lobar ICH based on head CT. ICH-free controls were selected from ambulatory clinics or by random digit dialing. Standard quality controls filters were applied and principal component analysis was used to account for population structure. Unobserved genotypes were inferred by imputation to 1000 genomes references panels. Association testing was carried out using logistic regression, assuming additive effects, and including age, gender and principal components in the model. Meta-analyses were completed using the inverse normal method, weighting by sample size. Replication of signals with p<1x10E-6 was pursued in an independent multi-ethnic study utilizing direct genotyping. Results: The discovery phase included 1545 cases (664 lobar and 881 non-lobar) and 1481 controls, and tested 5,358,103 SNPs. Two loci were identified: for lobar ICH, an intergenic region located on chromosome 12q21.1 (rs11179580: minor allele frequency [MAF] 0.22, OR 1.54, 95% CI 1.33 - 1.84; p = 6.7x10E-8); and for non-lobar ICH, a region on chromosome 1q22 encompassing the PMF1 and SLC25A44 genes (rs2984613: MAF 0.31, OR 1.45, 95% CI 1.2 - 1.64; p = 8.9x10E-9). Replication included 1256 cases (329 whites, 506 blacks and 421 Hispanics) and 760 controls (237 whites, 346 blacks and 177 Hispanics) and confirmed both loci: 12q21.1 (p = 0.037, meta-analysis p = 2.3x10E-8) and 1q22 (p = 3.0x10E-4, meta-analysis p = 2.2x10E-11). Conclusions: Two new susceptibility loci for ICH were identified, both showing location-specificity. These results confirm the presence of biological heterogeneity across ICH subtypes and highlight that different genetic risk factors may contribute to the small vessel diseases that underlie each ICH subtype.

The recent 130-center, international, second ACST trial (Asymptomatic Carotid Surgery Trial) involving 3625 patients found that regardless of whether a patient underwent stenting or endarterectomy, the periprocedural risk of disabling or fatal stroke was about 1% and the 5-year estimated risk of nonprocedural disabling or fatal stroke was 2.5%. With advances in technique, technology, and patient selection, stenting done by appropriately trained and experienced operators can achieve safety and efficacy comparable to endarterectomy for asymptomatic patients. The ongoing CREST-2 trial (Carotid Revascularization and Medical Management for Asymptomatic Carotid Stenosis Trial) will clarify whether revascularization, by either stenting or endarterectomy, remains an important therapeutic goal in the setting of modern intensive medical therapy.

Introduction: Patients with carotid stenosis may have silent cerebrovascular disease due to chronic hypoperfusion and atheroembolism. If so, progression of silent disease may be preventable through revascularization. We aim to evaluate the association between carotid stenosis/occlusion and cerebral small vessel disease (SVD) burden ipsilateral to the cervical carotid stenosis. Methods: Patients age ≥40 years with carotid stenosis or occlusion on carotid ultrasound who underwent MRI brain and intracranial angiogram with gradable SVD on MRI from 2011-2015 were included. Severity of carotid stenosis was defined using NASCET criteria as 50-69% (moderate), 70-99% (high-grade), or occluded. WMH volume was quantified using an automated artificial intelligence algorithm applied to axial T2 FLAIR images. Images were also scored for presence of chronic lacune, cerebral microbleeds (CMB), and silent infarcts using STRIVE criteria. Differences in WMD volume between hemispheres ipsilateral and contralateral to the carotid stenosis were calculated, and prevalence of SVD and silent infarct were compared across severities of carotid stenosis. Results: Of the 183 patients, mean age was 71±10 years and 38% were female. Moderate and severe stenosis was present in 36% and 46.5%, respectively, and 18% had carotid occlusion. Mean WMH volume was 7271±522mm 3 . Patients with carotid stenosis had greater WMH volume ipsilateral to the side of carotid stenosis than the contralateral side (mean difference 224±206mm 3 ), and higher severity of stenosis was associated with greater hemispheric difference in WMH volume (moderate vs high; 164±274mm 3 vs 743±309 mm 3 , p=0.009). Silent infarcts were more prevalent in high-grade carotid stenosis than moderate grade (moderate vs high, 10.8% vs 36.5%; p=0.002). There were no differences in either lacunae or CMBs across severities. Conclusions: Greater than one third of patients with high-grade carotid stenosis had ipsilateral silent brain infarction, and the volume of WMH was greater on the side of carotid stenosis. Future analyses should consider the potential yield of screening for carotid atherosclerosis in patients with varying degrees of hemispheric WMH asymmetry.

Introduction: Genetic studies of early-onset disease have been an effective strategy to identify novel pathways and drug targets generalizable also to later-onset disease. Few studies have investigated the sex-specific genetic associations with early-onset ischemic stroke even though several features of ischemic stroke differ between males and females. We hypothesized that stratifying the GWAS by sex would reveal novel stroke loci. Methods: We performed a transethnic ischemic stroke GWAS of 3,056 female cases and 4,462 male cases < 60 years-old and 16,192 and 16,048 sex-matched controls, respectively, from the Early Onset Stroke Genetics Consortium. Results: We identified a significant association in women with a locus in close proximity to TMX1 , a transmembrane platelet protein that inhibits platelet function. Additionally, we identified 2 other suggestive (P < 5 x 10 -6 ) loci in females (see Table), i.e., at APOH , which encodes beta2-glycoprotein I, an established GWAS locus for lipoprotein(a), and LRFN2 which has been previously reported to associate with obesity-related measures and type II diabetes. We observed suggestive evidence for association in males with MMP3/MMP12 , a known stroke susceptibility locus. Conclusions: Despite a very modest sample size, sex-specific analyses identified suggestive associations at biologically important novel loci in females and a known stroke locus in males. Further studies of sex-specific associations in both early- and later-onset ischemic stroke are needed.

Abstract Stroke represents a considerable burden of disease for both men and women. However, a growing body of literature suggests clinically relevant sex differences in the underlying causes, presentations and outcomes of acute ischaemic stroke. In a recent study, we reported sex divergences in lesion topographies: specific to women, acute stroke severity was linked to lesions in the left-hemispheric posterior circulation. We here determined whether these sex-specific brain manifestations also affect long-term outcomes. We relied on 822 acute ischaemic patients [age: 64.7 (15.0) years, 39% women] originating from the multi-centre MRI-GENIE study to model unfavourable outcomes (modified Rankin Scale &gt;2) based on acute neuroimaging data in a Bayesian hierarchical framework. Lesions encompassing bilateral subcortical nuclei and left-lateralized regions in proximity to the insula explained outcomes across men and women (area under the curve = 0.81). A pattern of left-hemispheric posterior circulation brain regions, combining left hippocampus, precuneus, fusiform and lingual gyrus, occipital pole and latero-occipital cortex, showed a substantially higher relevance in explaining functional outcomes in women compared to men [mean difference of Bayesian posterior distributions (men – women) = −0.295 (90% highest posterior density interval = −0.556 to −0.068)]. Once validated in prospective studies, our findings may motivate a sex-specific approach to clinical stroke management and hold the promise of enhancing outcomes on a population level.