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BackgroundIn this genome wide association study (GWAS) we aimed to discover single nucleotide polymorphisms (SNPs) associated with motor recovery post-stroke.MethodsWe used the Vitamin Intervention for Stroke Prevention (VISP) dataset of 2,100 genotyped patients with non-disabling stroke. Of these, 488 patients had motor impairment at enrollment. Genotyped data underwent strict quality control and imputation. The GWAS utilized logistic regression models with generalized estimating equations (GEE) to leverage the repeated NIH Stroke Scale (NIHSS) motor score measurements spanning 6 time points over 24 months. The primary outcome was a decrease in the motor drift score of ≥ 1 vs.<1 at each timepoint. Our model estimated the odds ratio of motor improvement for each SNP after adjusting for age, sex, race, days from stroke to visit, initial motor score, VISP treatment arm, and principal components.ResultsAlthough no associations reached genome-wide significance (p<5 × 10−8), our analysis detected 115 suggestive associations (p<5 × 10−6). Notably, we found multiple SNP clusters near genes with plausible neuronal repair biology mechanisms. The CLDN23 gene had the most convincing association which affects blood-brain barrier integrity, neurodevelopment, and immune cell transmigration.ConclusionWe identified novel suggestive genetic associations with the first ever motor-specific post stroke recovery GWAS. The results seem to describe a distinct stroke recovery phenotype compared to prior genetic stroke outcome studies that use outcome measures, like the mRS. Replication and further mechanistic investigation are warranted. Additionally, this study demonstrated a proof-of-principle approach to optimize statistical efficiency with longitudinal datasets for genetic discovery.

Objective: To perform a genome wide association study (GWAS) of stroke recovery phenotypes as defined by the trajectories of patients’ NIH stroke scale scores over time. Background: Stroke recovery research entails tracking multiple domains of neurologic impairment over time that are measured on differing scales. This presents challenges for analytic approaches commonly used to study stroke genetics. Here, we present a novel approach using a network science-based method, Trajectory Profile Clustering (TPC), to define stroke recovery phenotypes for GWAS. Design/Methods: We analyzed data from 3,679 patients in the VISP dataset (Vitamin Intervention for Stroke Prevention) on 15 NIHSS impairment measures at 6 time points spanning 24 months post-stroke. The 4 identified TPC profiles (Figure, Panel A), were used as phenotypes for a subsequent GWAS on the 2,099 genotyped VISP patients. We used a multinomial generalized regression with TPC profiles as the response variable. The model adjusted for age, sex, treatment group, and the first ten principal components. Results: TOPMed imputation and strict quality control resulted in 6,392,745 SNPs. Although none of our loci reached genome wide significance, the GWAS identified several suggestive loci (p Conclusion: Phenotypes defined using TPC, even in this limited sample, identified multiple suggestive genetic associations with biological relevance for both stroke risk and stroke recovery.

Introduction: Social and environment factors are associated with increased stroke risk. It is unclear if favorable cardiovascular health may attenuate this risk. We evaluated the impact of neighborhood socioeconomic characteristics on stroke incidence, and if levels of cardiovascular health modify this relationship. Methods: We conducted a longitudinal cohort study of Black and White participants using data from the Reasons for Geographic and Racial Differences in Stroke. We included all stroke-free participants and geocoded their home addresses to the census-tract level. Our primary exposure was neighborhood socioeconomic condition index (nSES; in quartiles) constructed using data from the U.S. Census on income, education, and occupation of residents by census tract. We evaluated cardiovascular health based on the American Heart Association’s Life Simple 7 (ideal, adequate, inadequate). We used a Cox proportional hazards model to regress nSES and its interaction with Life Simple 7 on stroke incidence, adjusting for individual demographics, socioeconomic status, and clinical characteristics. Results: We included 26,941 adults with a mean follow-up of 11.6 years (SD ± 5.2). During the study, 1,596 (5.9%) participants had a first incidence of stroke. Participants within the lowest nSES quartile, compared to those in the highest, were more likely to be Black, have lower household income, and an inadequate Life Simple 7 score. In multivariable models, there was a non-significant increased incidence of stroke (HR-1.13; 95% CI: 0.95-1.35) between the lowest and highest nSES quartiles. The interaction between nSES and Life Simple 7 was also non-significant for stroke incidence with worse cardiovascular health across all quartiles. Discussion: Among a national cohort of Black and White adults, we found a non-significant difference in stroke incidence between adults who live in worse neighborhood socioeconomic conditions and have inadequate cardiovascular health. Characterizing the potential contribution of social, environmental, and health behaviors could inform clinical and public health strategies to mitigate stroke risk.

Background: Multi-phenotype analysis of genetically correlated phenotypes can increase the statistical power to detect loci associated with multiple traits, leading to the discovery of novel loci. This is the first study to date to comprehensively analyze the shared genetic effects within different hemostatic traits, and between these and their associated disease outcomes. Objectives: To discover novel genetic associations by combining summary data of correlated hemostatic traits and disease events. Methods: Summary statistics from genome wide-association studies (GWAS) from seven hemostatic traits (factor VII [FVII], factor VIII [FVIII], von Willebrand factor [VWF] factor XI [FXI], fibrinogen, tissue plasminogen activator [tPA], plasminogen activator inhibitor 1 [PAI-1]) and three major cardiovascular (CV) events (venous thromboembolism [VTE], coronary artery disease [CAD], ischemic stroke [IS]), were combined in 27 multi-trait combinations using metaUSAT. Genetic correlations between phenotypes were calculated using Linkage Disequilibrium Score Regression (LDSC). Newly associated loci were investigated for colocalization. We considered a significance threshold of 1.85 × 10−9 obtained after applying Bonferroni correction for the number of multi-trait combinations performed (n = 27). Results: Across the 27 multi-trait analyses, we found 4 novel pleiotropic loci (XXYLT1, KNG1, SUGP1/MAU2, TBL2/MLXIPL) that were not significant in the original individual datasets, were not described in previous GWAS for the individual traits, and that presented a common associated variant between the studied phenotypes. Conclusions: The discovery of four novel loci contributes to the understanding of the relationship between hemostasis and CV events and elucidate common genetic factors between these traits.

Introduction: Genetic reference panels and imputation approaches have improved greatly in the last 10 years. The development of the TOPMed reference plane has led to enhanced imputation quality and quantity of single nucleotide polymorphisms (SNPs) due to greater sample diversity among various population ancestries. We revisited our prior GWAS of recurrent stroke by utilizing the TOPMed imputation server. Methods: This GWAS used a Cox proportional hazards model of time to recurrent stroke with the Vitamin Intervention for Stroke Prevention clinical trial cohort. There were 2,100 genotyped patients (64% male) in total with an average age of 67.2 (±10.8) years and ancestry distribution of 1,725 (82%) European, 258 (12%) African, and 117 (6%) Other or Mixed ancestry. Genotyped samples underwent a strict quality control process. We utilized TOPMed for imputation which totaled 10,467,887 biallelic SNPs which was 14 times greater in number compared the original analysis. Results: Recurrent stroke was observed in 182 (8.7%) patients. We identified seven novel SNPS on chromosome 1 in addition to our previous finding, rs6664786. Interestingly all chromosome 1 SNPs were located within the LINCO1362 gene. This gene has an acute change in expression in the presence of smoking even after adjustment of relevant clinical factors. Two novel SNPs were found on chromosome 16 located in gene desert nearest the pseudo-gene RNU6-21P in an intergenic region downstream of the Cadherin-8 (CDH8) gene. Both SNPs and RNU6-21P have no previously reported clinical relevance, except for their relative position to CDH8. CDH8 is highly expressed in brain tissue. Conclusions: We identified several novel SNPs associated with recurrent stroke. Capitalizing on genetic imputation advancements allows potential new insights and discoveries with past trial cohorts. Understanding these insights may provide further mechanistic knowledge of recurrent stroke to develop potential therapeutic targets.

The scientific response to the COVID-19 pandemic has elicited commentaries on the quickening of biomedical research,1–3 contrasting with literature on prolonged time to publication for clinical research projects.4–6 We investigated research project duration for three clinical departments (emergency medicine, family medicine, and pediatrics) with centralized research leadership in a community-based US medical school. Following institutional review board (IRB) approval, we identified original research reports published or accepted in academic year 2019–2020, originating at our institution, and involving faculty from these departments. Of 39 eligible publications, we reconstructed study timelines (table 1) for 17 publications based on departmental records, and 10 publications based on a survey of corresponding authors. View this table: Table 1 Data points on project stages, by project type The median overall project duration was 18 months (IQR 10–26). Median durations of …

Supplemental Digital Content is available in the text., Background and Purpose: Metabolic traits affect ischemic stroke (IS) risk, but the degree to which this varies across different ethnic ancestries is not known. Our aim was to apply Mendelian randomization to investigate the causal effects of type 2 diabetes (T2D) liability and lipid traits on IS risk in African ancestry individuals, and to compare them to estimates obtained in European ancestry individuals. Methods: For African ancestry individuals, genetic proxies for T2D liability and circulating lipids were obtained from a meta-analysis of the African Partnership for Chronic Disease Research study, the UK Biobank, and the Million Veteran Program (total N=77 061). Genetic association estimates for IS risk were obtained from the Consortium of Minority Population Genome-Wide Association Studies of Stroke (3734 cases and 18 317 controls). For European ancestry individuals, genetic proxies for the same metabolic traits were obtained from Million Veteran Program (lipids N=297 626, T2D N=148 726 cases, and 965 732 controls), and genetic association estimates for IS risk were obtained from the MEGASTROKE study (34 217 cases and 406 111 controls). Random-effects inverse-variance weighted Mendelian randomization was used as the main method, complemented with sensitivity analyses more robust to pleiotropy. Results: Higher genetically proxied T2D liability, LDL-C (low-density lipoprotein cholesterol), total cholesterol and lower genetically proxied HDL-C (high-density lipoprotein cholesterol) were associated with increased risk of IS in African ancestry individuals (odds ratio per doubling the odds of T2D liability [95% CI], 1.09 [1.07–1.11]; per standard-deviation increase in LDL-C, 1.12 [1.04–1.21]; total cholesterol: 1.23 [1.06–1.43]; HDL-C, 0.93 [0.89–0.99]). There was no evidence for differences in these estimates when performing analyses in European ancestry individuals. Conclusions: Our analyses support a causal effect of T2D liability and lipid traits on IS risk in African ancestry individuals, with Mendelian randomization estimates similar to those obtained in European ancestry individuals.

African Americans endure a nearly two-fold greater risk of suffering a stroke and are 2–3 times more likely to die from stroke compared to those of European ancestry. African Americans also have a greater risk of recurrent stroke and vascular events, which are deadlier and more disabling than incident stroke. Stroke is a multifactorial disease with both heritable and environmental risk factors. We conducted an integrative, multi-omic study on 922 plasma metabolites, 473,864 DNA methylation loci, and 556 variants from 50 African American participants of the Vitamin Intervention for Stroke Prevention clinical trial to help elucidate biomarkers contributing to recurrent stroke rates in this high risk population. Sixteen metabolites, including cotinine, N-delta-acetylornithine, and sphingomyelin (d17:1/24:1) were identified in t-tests of recurrent stroke outcome or baseline smoking status. Serum tricosanoyl sphingomyelin (d18:1/23:0) levels were significantly associated with recurrent stroke after adjusting for covariates in Cox Proportional Hazards models. Weighted Gene Co-expression Network Analysis identified moderate correlations between sphingolipid markers and clinical traits including days to recurrent stroke. Integrative analyses between genetic variants in sphingolipid pathway genes identified 29 nominal associations with metabolite levels in a one-way analysis of variance, while epigenomic analyses identified xenobiotics, predominately smoking-associated metabolites and pharmaceutical drugs, associated with methylation profiles. Taken together, our results suggest that metabolites, specifically those associated with sphingolipid metabolism, are potential plasma biomarkers for stroke recurrence in African Americans. Furthermore, genetic variation and DNA methylation may play a role in the regulation of these metabolites.

ObjectivesGullah African Americans are descendants of formerly enslaved Africans living in the Sea Islands along the coast of the southeastern U.S., from North Carolina to Florida. Their relatively high numbers and geographic isolation were conducive to the development and preservation of a unique culture that retains deep African features. Although historical evidence supports a West and Central African ancestry for the Gullah, linguistic and cultural evidence of a connection to Sierra Leone has led to the suggestion of this country/region as their ancestral home. This study sought to elucidate the genetic structure and ancestry of the Gullah.Materials and MethodsWe leveraged whole-genome genotype data from Gullah, African Americans from Jackson, Mississippi, Sierra Leone Africans, and population reference panels from Africa and Europe, to infer population structure, ancestry proportions, and global estimates of admixture.ResultsRelative to southeastern non-Gullah African Americans, the Gullah exhibit higher mean African ancestry, lower European admixture, a similarly small Native American contribution, and stronger male-biased European admixture. A slightly tighter bottleneck in the Gullah 13 generations ago suggests a largely shared demographic history with non-Gullah African Americans. Despite a slightly higher relatedness to Sierra Leone, our data demonstrate that the Gullah are genetically related to many West African populations.DiscussionThis study confirms that subtle differences in African American population structure exist at finer regional levels. Such observations can help to inform medical genetics research in African Americans, and guide the interpretation of genetic data used by African Americans seeking to explore ancestral identities.Research HighlightsUsing genomic data, we show that the Gullah have lower European and higher West African genomic background compared to non-Gullah African Americans, confirming their diverse African ancestry and rejecting a model that asserts a predominant Sierra Leone origin.Our data reveal a largely shared demographic history with southeastern non-Gullah African Americans, but also subtle differences related to high African genetic ancestry due to isolation in the Sea Islands.

Background and Purpose: Stroke is a complex disease with multiple genetic and environmental risk factors. Blacks endure a nearly 2-fold greater risk of stroke and are 2× to 3× more likely to die from stroke than European Americans. Methods: The COMPASS (Consortium of Minority Population Genome-Wide Association Studies of Stroke) has conducted a genome-wide association meta-analysis of stroke in >22 000 individuals of African ancestry (3734 cases, 18 317 controls) from 13 cohorts. Results: In meta-analyses, we identified one single nucleotide polymorphism (rs55931441) near the HNF1A gene that reached genome-wide significance ( P =4.62×10 −8 ) and an additional 29 variants with suggestive evidence of association ( P −6 ), representing 24 unique loci. For validation, a look-up analysis for a 100 kb region flanking the COMPASS single nucleotide polymorphism was performed in SiGN (Stroke Genetics Network) Europeans, SiGN Hispanics, and METASTROKE (Europeans). Using a stringent Bonferroni correction P value of 2.08×10 −3 (0.05/24 unique loci), we were able to validate associations at the HNF1A locus in both SiGN ( P =8.18×10 −4 ) and METASTROKE ( P =1.72×10 −3 ) European populations. Overall, 16 of 24 loci showed evidence for validation across multiple populations. Previous studies have reported associations between variants in the HNF1A gene and lipids, C-reactive protein, and risk of coronary artery disease and stroke. Suggestive associations with variants in the SFXN4 and TMEM108 genes represent potential novel ischemic stroke loci. Conclusions: These findings represent the most thorough investigation of genetic determinants of stroke in individuals of African descent, to date.

Background and Purpose: The majority of published data in cervical artery dissection (CeAD), a common cause of stroke in young adults, derive from populations of European ancestry (EA), including a recent genome-wide study identifying an association with the rs9349379 polymorphism of the PHACTR1 gene. Little is known about CeAD in individuals of African ancestry (AA) despite robust epidemiological data showing increased risk of stroke at younger ages. We hypothesize that AA patients with CeAD have different epidemiology and clinical profiles compared to those of EA, and a different genetic architecture related to rs9349379 of the PHACTR1 gene. Methods: We searched a single-center database of CeAD to identify AA and EA patients. We compared differential prevalence of CeAD versus all young stroke between AA and EA patients. We characterized clinical profiles via electronic medical record review. Data include descriptive statistics reported as medians or percentages. We also obtained publicly available allele frequencies of rs9349379 in AA and EA populations. Results: AA patients comprise 7% of CeAD cases and 27% of young stroke cases while EA patients comprise 90% of CeAD cases and 70% of young stroke cases. Prevalence of hypertension, diabetes mellitus, and hyperlipidemia were 74, 30, and 50%, respectively, in AA patients compared to 37, 6, and 25% in EA patients. Allele frequencies for the CeAD risk allele, rs9349379(A), are higher in AA populations compared to EA populations. Conclusion: AA patients represent a smaller proportion of CeAD cases compared to young stroke cases at our center. AA patients suffering CeAD have higher prevalence of both vascular risk factors and frequency of the CeAD risk allele compared to EA patients. These findings suggest a complex interplay between traditional vascular risk factors and genetic predisposition underlying CeAD pathogenesis. Further prospective research is needed to clarify these associations and disparities.

Altres ajuts: National Institutes of Health (grant U01HG005160); National Human Genome Research Institute (Supplement U01HG005160-03S1); American Heart Association Scientist Development Award (12SDG9180012); Faith Christian Center International (Charlottesville, VA). National Institute of Neurological Disorders and Stroke (R01 NS34447). Aberrant DNA methylation profiles have been implicated in numerous cardiovascular diseases; however, few studies have investigated how these epigenetic modifications contribute to stroke recurrence. The aim of this study was to identify methylation loci associated with the time to recurrent cerebro- and cardiovascular events in individuals of European and African descent. DNA methylation profiles were generated for 180 individuals from the Vitamin Intervention for Stroke Prevention clinical trial using Illumina HumanMethylation 450K BeadChip microarrays, resulting in beta values for 470,871 autosomal CpG sites. Ethnicity-stratified survival analyses were performed using Cox Proportional Hazards regression models for associations between each methylation locus and the time to recurrent stroke or composite vascular event. Results were validated in the Vall d'Hebron University Hospital cohort from Barcelona, Spain. Network analyses of the methylation loci were generated using weighted gene coexpression network analysis. Primary analysis identified four significant loci, cg04059318, ch.2.81927627R, cg03584380, and cg24875416, associated with time to recurrent stroke. Secondary analysis identified three loci, cg00076998, cg16758041, and cg02365967, associated with time to composite vascular endpoint. Locus cg03584380, which is located in an intron of ZDHHC6, was replicated in the Vall d'Hebron University Hospital cohort. The results from this study implicate the degree of methylation at cg03584380 is associated with the time of recurrence for stroke or composite vascular events across two ethnically diverse groups. Furthermore, modules of loci were associated with clinical traits and blood biomarkers including previous number of strokes, prothrombin fragments 1 + 2, thrombomodulin, thrombin-antithrombin complex, triglyceride levels, and tissue plasminogen activator. Ultimately, these loci could serve as potential epigenetic biomarkers that could identify at-risk individuals in recurrence-prone populations.

Background and Purpose— von Willebrand factor (vWF) plays an important role in thrombus formation during cerebrovascular damage. We sought to investigate the potential role of circulating vWF in recurrent cerebrovascular events and identify genetic contributors to variation in vWF level in an ischemic stroke population. Methods— We analyzed the effect of circulating vWF on risk of recurrent stroke using survival models in the VISP trial (Vitamin Intervention for Stroke Prevention) and the use of vWF in reclassification over traditional factors. We conducted a genome-wide association study) with imputation, based on 1000 Genomes Project data, for circulating vWF levels and then interrogated loci previously associated with vWF levels. We performed expression quantitative trait locus analysis for vWF across different tissues. Results— Elevated vWF levels were associated with increased risk for recurrent stroke in VISP. Adding vWF to traditional clinical parameters also improved recurrent stroke risk prediction. We identified single-nucleotide polymorphisms significantly associated with circulating vWF at the ABO locus ( P −8 ) and replicated findings from previous genetic associations of vWF levels in humans. Expression quantitative trait locus analyses demonstrate that most associated ABO single-nucleotide polymorphisms were also associated with vWF gene expression. Conclusions— Elevated vWF levels are associated with recurrent stroke in VISP. In the VISP population, genetic determinants of vWF levels that impact vWF gene expression were identified. These data add to our knowledge of the pathophysiologic and genetic basis for recurrent stroke risk and may have implications for clinical care decision making.

Variation in the phosphatase and actin regulator-1 (PHACTR1) gene, a downstream regulator of the endothelin-1 (EDN1) gene, has been implicated in the pathogenesis of several related vascular phenotypes including atherosclerotic coronary artery disease, non-atherosclerotic coronary artery dissection, and carotid artery dissection, though it has not been studied in carotid atherosclerosis. We analyzed differential expression of PHACTR1 and EDN1 between atheromatous and non-atheromatous carotid artery tissue within the same individual and found lower levels of PHACTR1 expression in the atheromatous carotid tissue.

Introduction: Several related neurovascular phenotypes, including cervical artery dissection, fibromuscular dysplasia, large artery stroke, hypertension, and migraine headache are associated with a single nucleotide polymorphism (rs9349378) in the phosphatase and actin regulator-1 ( PHACTR1 ) gene recently identified as a distal regulator of the endothelin-1 ( EDN1 ) gene. Vascular endothelial cells release EDN1, a potent vasoconstrictor. We hypothesize that atheromatous and normal carotid tissue will differentially express PHACTR1 and EDN1 . Methods: We obtained gene expression data from the publically available Gene Expression Omnibus Carotid Atheroma Dataset (GSE43292). This resource contains 32 paired samples of carotid atheroma and distant macroscopically intact carotid tissue from the same individual and gene expression data from the Affymetrix Human Gene 1.0 ST Array. We compared log transformed count of signal intensity of PHACTR1 and EDN1 expression in the 32 paired samples using a two-sample t-test assuming equal variance with SPSS (IBM, Armonk, NY). Results: PHACTR1 expression was lower in atheromatous compared to normal carotid tissue (7.152 vs. 7.399, delta 0.248, 95%CI 0.180 - 0.315, p=1.9 E -8 ). EDN1 expression did not differ (6.514 and 6.339, delta 0.176, 95%CI -0.004 - 0.356, p = 0.055). with genetic data associating the rs9349378[G] allele with lower levels of expression and atherosclerotic vascular diseases. The lack of differential EDN1 expression may reflect the small sample size or may challenge the assertion that EDNI is the mediator of the PHACTR1 association. Our data support the hypothesis that this pathway may play an important and potentially variable role in neurovascular phenotypes including large artery atherosclerotic stroke and non-atherosclerotic arteriopathies.

Comment inThe yin and yang of magnesium and calcium: New genetic insights for stroke? [Neurology. 2019]; International audience; Objective To determine whether serum magnesium and calcium concentrations are causally associated with ischemic stroke or any of its subtypes using the mendelian randomization approach. Methods Analyses were conducted using summary statistics data for 13 single-nucleotide polymorphisms robustly associated with serum magnesium (n = 6) or serum calcium (n = 7) concentrations. The corresponding data for ischemic stroke were obtained from the MEGASTROKE consortium (34,217 cases and 404,630 noncases). Results In standard mendelian randomization analysis, the odds ratios for each 0.1 mmol/L (about 1 SD) increase in genetically predicted serum magnesium concentrations were 0.78 (95% confidence interval [CI] 0.69-0.89; p = 1.3 × 10 −4) for all ischemic stroke, 0.63 (95% CI 0.50-0.80; p = 1.6 × 10 −4) for cardioembolic stroke, and 0.60 (95% CI 0.44-0.82; p = 0.001) for large artery stroke; there was no association with small vessel stroke (odds ratio 0.90, 95% CI 0.67-1.20; p = 0.46). Only the association with cardioembolic stroke was robust in sensitivity analyses. There was no association of genetically predicted serum calcium concentrations with all ischemic stroke (per 0.5 mg/dL [about 1 SD] increase in serum calcium: odds ratio 1.03, 95% CI 0.88-1.21) or with any subtype. Conclusions This study found that genetically higher serum magnesium concentrations are associated with a reduced risk of cardioembolic stroke but found no significant association of genetically higher serum calcium concentrations with any ischemic stroke subtype.

Introduction: Cervical artery dissection (CeAD) of the carotid and vertebral arteries is a prevalent cause of stroke in young adults. While the pathogenesis of incident CeAD remains poorly understood, clinical and genetic associations suggest a systemic arteriopathy at time of event. Hypothesis: Individuals suffering CeAD express a distinct gene expression profile at time of event compared to (1) age and sex-matched controls and (2) convalescent profiles within the same individual at a later time point. Methods: We enrolled 37 consecutive patients with carotid or vertebral artery dissection (with and without ischemic stroke) from 2013-2016, excluding concurrence of major trauma. Cases were age and sex matched to non-CeAD ischemic stroke controls (n=16) and healthy controls (n=11). Whole blood samples were collected within four weeks from time of event, and again at 3 and 6-month follow-up. We used IlluminaHT-12 microarrays to assess differential gene expression at time of CeAD relative to controls, and relative to gene expression at >3 month follow-up within CeAD cases. Mixed effects regression models included relevant covariates of age, sex, race/ethnicity, time of enrollment, and occurrence of stroke. We used a False Discovery Rate (FDR) cutoff of 5% to account for multiple testing as well as a 1.5 fold change cutoff to identify robust, differential gene expression. Results: We identified 538 differentially expressed genes between CeAD patients and healthy controls with 30 of these genes reaching our predetermined 1.5x fold change limit. Within CeAD cases, 1,238 genes showed differential expression at a 5% FDR compared to time points at 3-6 month follow-up, with 31 genes showing at least a 1.5 fold change. Compared to controls and convalescent profiles, 12 genes were significant using the 5% FDR and 1.5 fold change cutoffs. Preliminary gene-ontology analysis indicate the largest differential expression for genes associated with oxygen and gas transport. Conclusion: In this consecutive cohort of individuals with CeAD, we identified a distinct gene expression profile associated with incident dissection. These data are limited by small sample size and results are hypothesis generating. Replication in larger, independent cohorts is warranted.

Introduction: Recent success identifying genetic variation in ischemic stroke (IS) has yet to translate into actionable targets or pathways. Weighted Gene Co-Expression Network Analysis (WGCNA) uses an unsupervised approach to cluster genes into modules based on co-expression profiles. Modules likely contain biologically related genes. The correlation between a gene’s expression and the module’s overall expression pattern (eigengene) defines connectivity. Genes with high connectivity may inform and supplement genome-wide association studies (GWAS). Methods: Gene Expression in Carotid Artery GSE43292 (GEO) resource contains publically available Affymetrix Human GeneChip Gene 1.0 ST data from 34 carotid atherosclerotic plaques and paired normal carotid tissue from the same individual. WGCNA resulted in 16 co-expression modules tested against the atherosclerosis phenotype. Significant modules had strong correlation between module eigengene and atherosclerosis. We took the top genes from significantly associated modules and looked them up in two existing IS genetic datasets: Stroke Genetic Network (SiGN) (17,000 IS cases & 32,000 controls) and METASTROKE (10,307 cases & 19,326 controls). We conducted a secondary GWAS informed by WGCNA for only known functional variants in METASTROKE with replication in SiGN. Results: Five of 16 modules showed evidence of association with two standouts (“Turquoise” and “Brown”) both enriched for genes associated with vascular disease related traits (carotid IMT, coronary artery disease, etc.) and contain genes associated with IS, especially large artery atherosclerotic stroke (including HDAC9). We took the top 20 genes from these modules, not currently known to associate with IS, and looking them up in SiGN and METASTROKE, we found association with variants in three genomic regions (SLC7A7, ATXN2/SH2B3, ALDH2). The informed GWAS approach identified ADAMTSL3 as a potential novel translational target. Conclusion: Leveraging expression data in a convergent genomics approach holds promise to supplement on-going GWAS and accelerate identification of potential translational targets. Gene ontology analyses of genes from the top modules are underway.

Background and Purpose— The majority of genome-wide association studies (GWAS) of stroke have focused on European-ancestry populations; however, none has been conducted in African Americans, despite the disproportionately high burden of stroke in this population. The Consortium of Minority Population Genome-Wide Association Studies of Stroke (COMPASS) was established to identify stroke susceptibility loci in minority populations. Methods— Using METAL, we conducted meta-analyses of GWAS in 14 746 African Americans (1365 ischemic and 1592 total stroke cases) from COMPASS, and tested genetic variants with P −6 for validation in METASTROKE, a consortium of ischemic stroke genetic studies in European-ancestry populations. We also evaluated stroke loci previously identified in European-ancestry populations. Results— The 15q21.3 locus linked with lipid levels and hypertension was associated with total stroke (rs4471613; P =3.9×10 −8 ) in African Americans. Nominal associations ( P −6 ) for total or ischemic stroke were observed for 18 variants in or near genes implicated in cell cycle/mRNA presplicing ( PTPRG , CDC5L ), platelet function ( HPS4 ), blood–brain barrier permeability ( CLDN17 ), immune response ( ELTD1, WDFY4 , and IL1F10-IL1RN ), and histone modification ( HDAC9 ). Two of these loci achieved nominal significance in METASTROKE: 5q35.2 ( P =0.03), and 1p31.1 ( P =0.018). Four of 7 previously reported ischemic stroke loci ( PITX2, HDAC9, CDKN2A/CDKN2B , and ZFHX3 ) were nominally associated ( P Conclusions— We identified a novel genetic variant associated with total stroke in African Americans and found that ischemic stroke loci identified in European-ancestry populations may also be relevant for African Americans. Our findings support investigation of diverse populations to identify and characterize genetic risk factors, and the importance of shared genetic risk across populations.

Single nucleotide polymorphisms (SNPs) located in the chromosomal region 16p13.13 have been previously associated with risk for several autoimmune diseases, including type 1 diabetes. To identify and localize specific risk variants for type 1 diabetes in this region and understand the mechanism of their action, we resequenced a 455-kb region in type 1 diabetic patients and unaffected control subjects, identifying 93 novel variants. A panel of 939 SNPs that included 46 of these novel variants was genotyped in 3,070 multiplex families with type 1 diabetes. Forty-eight SNPs, all located in CLEC16A, provided a statistically significant association (P < 5.32 × 10−5) with disease, with rs34306440 being most significantly associated (P = 5.74 × 10−6). The panel of SNPs used for fine mapping was also tested for association with transcript levels for each of the four genes in the region in B lymphoblastoid cell lines. Significant associations were observed only for transcript levels of DEXI, a gene with unknown function. We examined the relationship between the odds ratio for type 1 diabetes and the magnitude of the effect of DEXI transcript levels for each SNP in the region. Among SNPs significantly associated with type 1 diabetes, the common allele conferred an increased risk for disease and corresponded to lower DEXI expression. Our results suggest that the primary mechanism by which genetic variation at CLEC16A contributes to the risk for type 1 diabetes is through reduced expression of DEXI.

Type 2 diabetes (T2D) affects Latinos at twice the rate seen in populations of European descent. We recently identified a risk haplotype spanning SLC16A11 that explains ∼20% of the increased T2D prevalence in Mexico. Here, through genetic fine-mapping, we define a set of tightly linked variants likely to contain the causal allele(s). We show that variants on the T2D-associated haplotype have two distinct effects: (1) decreasing SLC16A11 expression in liver and (2) disrupting a key interaction with basigin, thereby reducing cell-surface localization. Both independent mechanisms reduce SLC16A11 function and suggest SLC16A11 is the causal gene at this locus. To gain insight into how SLC16A11 disruption impacts T2D risk, we demonstrate that SLC16A11 is a proton-coupled monocarboxylate transporter and that genetic perturbation of SLC16A11 induces changes in fatty acid and lipid metabolism that are associated with increased T2D risk. Our findings suggest that increasing SLC16A11 function could be therapeutically beneficial for T2D. Video Abstract [Figure presented] Keywords: type 2 diabetes (T2D); genetics; disease mechanism; SLC16A11; MCT11; solute carrier (SLC); monocarboxylates; fatty acid metabolism; lipid metabolism; precision medicine

Reduced glomerular filtration rate defines chronic kidney disease and is associated with cardiovascular and all-cause mortality. We conducted a meta-analysis of genome-wide association studies for estimated glomerular filtration rate (eGFR), combining data across 133,413 individuals with replication in up to 42,166 individuals. We identify 24 new and confirm 29 previously identified loci. Of these 53 loci, 19 associate with eGFR among individuals with diabetes. Using bioinformatics, we show that identified genes at eGFR loci are enriched for expression in kidney tissues and in pathways relevant for kidney development and transmembrane transporter activity, kidney structure, and regulation of glucose metabolism. Chromatin state mapping and DNase I hypersensitivity analyses across adult tissues demonstrate preferential mapping of associated variants to regulatory regions in kidney but not extra-renal tissues. These findings suggest that genetic determinants of eGFR are mediated largely through direct effects within the kidney and highlight important cell types and biological pathways. J.T. and P.H. are consultants for Servier. J.C. received research grants and honoraria from Servier. K.S. obtained research support from Boehringer Ingelheim. The remaining authors declared no competing financial interests.

Genome-wide association studies have identified associations between type 1 diabetes and single nucleotide polymorphisms (SNPs) at chromosome 12q13, surrounding the gene ERBB3. Our objective was to fine map this region to further localize causative variants. Re-sequencing identified more than 100 putative SNPs in an 80 kb region at 12q13. By genotyping 42 SNPs, spanning approximately 214 kb, in 382 affected sibling pair type 1 diabetes families, we were able to genotype or tag 67 common SNPs (MAF ≥ 0.05) identified from HapMap CEU data and CEU data from the 1000 Genomes Project, plus additional rare coding variants identified from our re-sequencing efforts. Fifteen SNPs provided nominal evidence for association (P≤ 0.05) with type 1 diabetes. The most significant associations were observed with rs2271189 (P = 4.22×10−5), located in exon 27 of the ERBB3 gene, and an intergenic SNP rs11171747 (P= 1.70×10−4). Follow-up genotyping of these SNPs in 2 740 multiplex type 1 diabetes families validated these findings. After analyzing variants spanning more than 200 kb, we have replicated associations from previous GWAS and provide evidence for novel associations with type 1 diabetes. The associations across this region could be entirely accounted for by two common SNPs, rs2271189 and rs11171747.

Summary Variants in the engulfment and cell motility 1 (ELMO1) gene are associated with nephropathy due to type 2 diabetes mellitus (T2DM) in a Japanese cohort. We comprehensively evaluated this gene in African American (AA) T2DM patients with end-stage renal disease (ESRD). Three hundred and nine HapMap tagging SNPs and 9 reportedly associated SNPs were genotyped in 577 AA T2DM-ESRD patients and 596 AA non-diabetic controls, plus 43 non-diabetic European American controls and 45 Yoruba Nigerian samples for admixture adjustment. Replication analyses were conducted in 558 AA with T2DM-ESRD and 564 controls without diabetes. Extension analyses included 328 AA with T2DM lacking nephropathy and 326 with non-diabetic ESRD. The original and replication analyses confirmed association with four SNPs in intron 13 (permutation p-values for combined analyses = 0.001–0.003), one in intron 1 (P = 0.004) and one in intron 5 (P = 0.002) with T2DM-associated ESRD. In a subsequent combined analysis of all 1,135 T2DM-ESRD cases and 1,160 controls, an additional 7 intron 13 SNPs produced evidence of association (P = 3.5 × 10−5– P = 0.05). No associations were seen with these SNPs in those with T2DM lacking nephropathy or with ESRD due to non-diabetic causes. Variants in intron 13 of the ELMO1 gene appear to confer risk for diabetic nephropathy in AA.

Previously, we performed a genome scan for type 2 diabetes (T2DM) using 638 African-American (AA) affected sibling pairs from 247 families; non-parametric linkage analysis suggested evidence of linkage at 6q24-27 (LOD 2.26). To comprehensively evaluate this region, we performed a two-stage association study by first constructing a SNP map of 754 SNPs selected from HapMap on the basis of linkage disequilibrium (LD) in 300 AAT2DM end-stage renal disease (ESRD) subjects, 311 AA controls, 43 European American controls and 45 Yoruba Nigerian samples (Set 1). Replication analyses were conducted in an independent population of 283 AA T2DM-ESRD subjects and 282 AA controls (Set 2). In addition, we adjusted for the impact of admixture on association results by using ancestry informative markers (AIMs). In Stage 1, 137 (18.2%) SNPs showed nominal evidence of association (P < 0.05) in one or more of tests of association: allelic (n = 33), dominant (n = 36), additive (n = 29), or recessive (n = 34) genotypic models, and 2- (n = 47) and 3-SNP (n = 43) haplotypic analyses. These SNPs were selected for follow-up genotyping. Stage 2 analyses confirmed association with a predicted 2-SNP "risk" haplotype in the PARK2 gene. Also, two intergenic SNPs showed consistent genotypic association with T2DM-ESRD: rs12197043 and rs4897081. Combined analysis of all subjects from both stages revealed nominal associations with 17 SNPs within genes, including suggestive associations in ESR1 and PARK2. This study confirms known diabetic nephropathy loci and identifies potentially novel susceptibility variants located within 6q24-27 in AA.

OBJECTIVE—Variants in the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene have shown positive associations with diabetes and related phenotypes, including insulin resistance, metabolic syndrome, and type 1 diabetic nephropathy. Additionally, evidence for linkage for type 2 diabetes in African Americans was observed at 6q24-27, with the proximal edge of the peak encompassing the ENPP1 gene. Our objective was to comprehensively evaluate variants in ENPP1 for association with type 2 diabetic end-stage renal disease (ESRD). RESEARCH DESIGN AND METHODS—Forty-nine single nucleotide polymorphisms (SNPs) located in the coding and flanking regions of ENPP1 were genotyped in 577 African-American individuals with type 2 diabetic ESRD and 596 African-American control subjects. Haplotypic association and genotypic association for the dominant, additive, and recessive models were tested by calculating a χ2 statistic and corresponding P value. RESULTS—Nine SNPs showed nominal evidence for association (P < 0.05) with type 2 diabetic ESRD in one or more genotypic model. The most significant associations were observed with rs7754586 (P = 0.003 dominant model, P = 0.0005 additive, and P = 0.007 recessive), located in the 3′ untranslated region, and an intron 24 SNP (rs1974201: P = 0.004 dominant, P = 0.0005 additive, and P = 0.005 recessive). However, the extensively studied K121Q variant (rs1044498) did not reveal evidence for association with type 2 diabetic ESRD in this African-American population. CONCLUSIONS—This study was the first to comprehensively evaluate variants of the ENPP1 gene for association in an African-American population with type 2 diabetes and ESRD and suggests that variants in the distal region of the ENPP1 gene may contribute to diabetes or diabetic nephropathy susceptibility in African Americans.

We previously investigated the estrogen receptor alpha gene (ESR1) as a positional candidate for type 2 diabetes (T2DM), and found evidence for association between the intron 1-intron 2 region of this gene and T2DM and/or nephropathy in an African American (AA) population. Our objective was to comprehensively evaluate variants across the entire ESR1 gene for association in AA with T2DM and end stage renal disease (T2DM-ESRD). One hundred fifty SNPs in ESR1, spanning 476 kb, were genotyped in 577 AA individuals with T2DM-ESRD and 596 AA controls. Genotypic association tests for dominant, additive, and recessive models, and haplotypic association, were calculated using a chi(2) statistic and corresponding P value. Thirty-one SNPs showed nominal evidence for association (P < 0.05) with T2DM-ESRD in one or more genotypic model. After correcting for multiple tests, promoter SNP rs11964281 (nominal P = 0.000291, adjusted P = 0.0289), and intron 4 SNPs rs1569788 (nominal P = 0.000754, adjusted P = 0.0278) and rs9340969 (nominal P = 0.00109, adjusted P = 0.0467) remained significant at experimentwise error rate (EER) P

Genome scans in African-Americans with end-stage renal disease (ESRD) identified linkage on chromosome 13q33 in the region containing the ephrin-B2 ligand (EFNB2) genes. Interactions between the ephrin-B2 receptor and ephrin-B2 ligand play essential roles in renal angiogenesis, blood vessel maturation, and kidney disease.The EFNB2 gene was evaluated as a positional candidate for non-diabetic and diabetic ESRD susceptibility in 1,071 unrelated African-American subjects; 316 with non-diabetic etiologies of ESRD, 394 with type 2 diabetes-associated ESRD and 361 healthy controls. Single nucleotide polymorphism (SNP) genotyping was performed on the Sequenom Mass Array System. Statistical analyses were computed using Dandelion version 1.26, Snpaddmix version 1.4 and Haploview version 3.32.Twenty-eight HapMap tag SNPs were genotyped spanning the 39 kilobases (kb) of the EFNB2 coding region, with average spacing of 1.43 kb. Analysis of 710 ESRD patient samples and 361 controls provided no evidence of single SNP associations in either diabetic or non-diabetic ESRD; although nominal evidence of association with all-cause ESRD was observed with a two SNP (p = 0.022) and three SNP (p = 0.023) haplotype, both containing SNPs rs7490924 and rs2391335 in intron 1.Although an attractive positional candidate gene, polymorphisms in the EFNB2 gene do not appear to contribute in a substantial way to non-diabetic, diabetic or all-cause ESRD susceptibility in African-Americans. Additional genes within the chromosome 13q33 linkage interval are likely contributors to African-American non-diabetic ESRD.

Objective: To investigate the genetic contributors to cerebrovascular disease and variation in biomarkers of ischemic stroke. Methods: The Vitamin Intervention for Stroke Prevention Trial (VISP) was a randomized, controlled clinical trial of B vitamin supplementation to prevent recurrent stroke, myocardial infarction, or death. VISP collected baseline measures of C-reactive protein (CRP), fibrinogen, creatinine, prothrombin fragments F 1+2 , thrombin-antithrombin complex, and thrombomodulin prior to treatment initiation. Genome-wide association scans were conducted for these traits and follow-up replication analyses were performed. Results: We detected an association between CRP single nucleotide polymorphisms (SNPs) and circulating CRP levels (most associated SNP, rs2592902, p = 1.14 × 10 −9 ) in 2,100 VISP participants. We discovered a novel association for CRP level in the AKR1D1 locus (rs2589998, p = 7.3 × 10 −8 , approaching genome-wide significance) that also is an expression quantitative trait locus for CRP gene expression. We replicated previously identified associations of fibrinogen with SNPs in the FGB and LEPR loci. CRP-associated SNPs and CRP levels were significantly associated with risk of ischemic stroke and recurrent stroke in VISP as well as specific stroke subtypes in METASTROKE. Fibrinogen levels but not fibrinogen-associated SNPs were also found to be associated with recurrent stroke in VISP. Conclusions: Our data identify a genetic contribution to inflammatory and hemostatic biomarkers in a stroke population. Additionally, our results suggest shared genetic contributions to circulating CRP levels measured poststroke and risk for incident and recurrent ischemic stroke. These data broaden our understanding of genetic contributors to biomarker variation and ischemic stroke risk, which should be useful in clinical risk evaluation.

Background: B vitamins play an important role in homocysteine metabolism, with vitamin deficiencies resulting in increased levels of homocysteine and increased risk for stroke. We performed a genome-wide association study (GWAS) in 2,100 stroke patients from the Vitamin Intervention for Stroke Prevention (VISP) trial, a clinical trial designed to determine whether the daily intake of high-dose folic acid, vitamins B6, and B12 reduce recurrent cerebral infarction. Methods: Extensive quality control (QC) measures resulted in a total of 737,081 SNPs for analysis. Genome-wide association analyses for baseline quantitative measures of folate, Vitamins B12, and B6 were completed using linear regression approaches, implemented in PLINK. Results: Six associations met or exceeded genome-wide significance (P ≤ 5 × 10−08). For baseline Vitamin B12, the strongest association was observed with a non-synonymous SNP (nsSNP) located in the CUBN gene (P = 1.76 × 10−13). Two additional CUBN intronic SNPs demonstrated strong associations with B12 (P = 2.92 × 10−10 and 4.11 × 10−10), while a second nsSNP, located in the TCN1 gene, also reached genome-wide significance (P = 5.14 × 10−11). For baseline measures of Vitamin B6, we identified genome-wide significant associations for SNPs at the ALPL locus (rs1697421; P = 7.06 × 10−10 and rs1780316; P = 2.25 × 10−08). In addition to the six genome-wide significant associations, nine SNPs (two for Vitamin B6, six for Vitamin B12, and one for folate measures) provided suggestive evidence for association (P ≤ 10−07). Conclusion: Our GWAS study has identified six genome-wide significant associations, nine suggestive associations, and successfully replicated 5 of 16 SNPs previously reported to be associated with measures of B vitamins. The six genome-wide significant associations are located in gene regions that have shown previous associations with measures of B vitamins; however, four of the nine suggestive associations represent novel finding and warrant further investigation in additional populations.

Objective— Tissue plasminogen activator (tPA), a serine protease, catalyzes the conversion of plasminogen to plasmin, the major enzyme responsible for endogenous fibrinolysis. In some populations, elevated plasma levels of tPA have been associated with myocardial infarction and other cardiovascular diseases. We conducted a meta-analysis of genome-wide association studies to identify novel correlates of circulating levels of tPA. Approach and Results— Fourteen cohort studies with tPA measures (N=26 929) contributed to the meta-analysis. Three loci were significantly associated with circulating tPA levels ( P −8 ). The first locus is on 6q24.3, with the lead single nucleotide polymorphism (SNP; rs9399599; P =2.9×10 −14 ) within STXBP5 . The second locus is on 8p11.21. The lead SNP (rs3136739; P =1.3×10 −9 ) is intronic to POLB and PLAT . We identified a nonsynonymous SNP (rs2020921) in modest linkage disequilibrium with rs3136739 ( r 2 =0.50) within exon 5 of PLAT ( P =2.0×10 −8 ). The third locus is on 12q24.33, with the lead SNP (rs7301826; P =1.0×10 −9 ) within intron 7 of STX2 . We further found evidence for the association of lead SNPs in STXBP5 and STX2 with expression levels of the respective transcripts. In in vitro cell studies, silencing STXBP5 decreased the release of tPA from vascular endothelial cells, whereas silencing STX2 increased the tPA release. Through an in silico lookup, we found no associations of the 3 lead SNPs with coronary artery disease or stroke. Conclusions— We identified 3 loci associated with circulating tPA levels, the PLAT region, STXBP5 , and STX2. Our functional studies implicate a novel role for STXBP5 and STX2 in regulating tPA release.

High blood pressure (BP) is more prevalent and contributes to more severe manifestations of cardiovascular disease (CVD) in African Americans than in any other United States ethnic group. Several small African-ancestry (AA) BP genome-wide association studies (GWASs) have been published, but their findings have failed to replicate to date. We report on a large AA BP GWAS meta-analysis that includes 29,378 individuals from 19 discovery cohorts and subsequent replication in additional samples of AA (n = 10,386), European ancestry (EA) (n = 69,395), and East Asian ancestry (n = 19,601). Five loci (EVX1-HOXA, ULK4, RSPO3, PLEKHG1, and SOX6) reached genome-wide significance (p < 1.0 × 10(-8)) for either systolic or diastolic BP in a transethnic meta-analysis after correction for multiple testing. Three of these BP loci (EVX1-HOXA, RSPO3, and PLEKHG1) lack previous associations with BP. We also identified one independent signal in a known BP locus (SOX6) and provide evidence for fine mapping in four additional validated BP loci. We also demonstrate that validated EA BP GWAS loci, considered jointly, show significant effects in AA samples. Consequently, these findings suggest that BP loci might have universal effects across studied populations, demonstrating that multiethnic samples are an essential component in identifying, fine mapping, and understanding their trait variability.

Summary Background The discovery of disease-associated loci through genome-wide association studies (GWAS) is the leading genetic approach to the identification of novel biological pathways underlying diseases in humans. Until recently, GWAS in ischaemic stroke have been limited by small sample sizes and have yielded few loci associated with ischaemic stroke. We did a large-scale GWAS to identify additional susceptibility genes for stroke and its subtypes. Methods To identify genetic loci associated with ischaemic stroke, we did a two-stage GWAS. In the first stage, we included 16 851 cases with state-of-the-art phenotyping data and 32 473 stroke-free controls. Cases were aged 16 to 104 years, recruited between 1989 and 2012, and subtypes of ischaemic stroke were recorded by centrally trained and certified investigators who used the web-based protocol, Causative Classification of Stroke (CCS). We constructed case-control strata by identifying samples that were genotyped on nearly identical arrays and were of similar genetic ancestral background. We cleaned and imputed data by use of dense imputation reference panels generated from whole-genome sequence data. We did genome-wide testing to identify stroke-associated loci within each stratum for each available phenotype, and we combined summary-level results using inverse variance-weighted fixed-effects meta-analysis. In the second stage, we did in-silico lookups of 1372 single nucleotide polymorphisms identified from the first stage GWAS in 20 941 cases and 364 736 unique stroke-free controls. The ischaemic stroke subtypes of these cases had previously been established with the Trial of Org 10 172 in Acute Stroke Treatment (TOAST) classification system, in accordance with local standards. Results from the two stages were then jointly analysed in a final meta-analysis. Findings We identified a novel locus (G allele at rs12122341) at 1p13.2 near TSPAN2 that was associated with large artery atherosclerosis-related stroke (first stage odds ratio [OR] 1·21, 95% CI 1·13–1·30, p=4·50 × 10 −8 ; joint OR 1·19, 1·12–1·26, p=1·30 × 10 −9 ). Our results also supported robust associations with ischaemic stroke for four other loci that have been reported in previous studies, including PITX2 (first stage OR 1·39, 1·29–1·49, p=3·26 × 10 −19 ; joint OR 1·37, 1·30–1·45, p=2·79 × 10 −32 ) and ZFHX3 (first stage OR 1·19, 1·11–1·27, p=2·93 × 10 −7 ; joint OR 1·17, 1·11–1·23, p=2·29 × 10 −10 ) for cardioembolic stroke, and HDAC9 (first stage OR 1·29, 1·18–1·42, p=3·50 × 10 −8 ; joint OR 1·24, 1·15–1·33, p=4·52 × 10 −9 ) for large artery atherosclerosis stroke. The 12q24 locus near ALDH2 , which has previously been associated with all ischaemic stroke but not with any specific subtype, exceeded genome-wide significance in the meta-analysis of small artery stroke (first stage OR 1·20, 1·12–1·28, p=6·82 × 10 −8 ; joint OR 1·17, 1·11–1·23, p=2·92 × 10 −9 ). Other loci associated with stroke in previous studies, including NINJ2 , were not confirmed. Interpretation Our results suggest that all ischaemic stroke-related loci previously implicated by GWAS are subtype specific. We identified a novel gene associated with large artery atherosclerosis stroke susceptibility. Follow-up studies will be necessary to establish whether the locus near TSPAN2 can be a target for a novel therapeutic approach to stroke prevention. In view of the subtype-specificity of the associations detected, the rich phenotyping data available in the Stroke Genetics Network (SiGN) are likely to be crucial for further genetic discoveries related to ischaemic stroke. Funding US National Institute of Neurological Disorders and Stroke, National Institutes of Health.

The development of carotid atheroma results in stenosis, which has been shown to increase the risk of ischemic stroke. This chapter summarizes cerebrovascular atherosclerotic phenotypes and the genetic behind those phenotypes.

Background and Purpose— Ischemic stroke (IS) shares many common risk factors with coronary artery disease (CAD). We hypothesized that genetic variants associated with myocardial infarction (MI) or CAD may be similarly involved in the etiology of IS. To test this hypothesis, we evaluated whether single-nucleotide polymorphisms (SNPs) at 11 different loci recently associated with MI or CAD through genome-wide association studies were associated with IS. Methods— Meta-analyses of the associations between the 11 MI-associated SNPs and IS were performed using 6865 cases and 11 395 control subjects recruited from 9 studies. SNPs were either genotyped directly or imputed; in a few cases a surrogate SNP in high linkage disequilibrium was chosen. Logistic regression was performed within each study to obtain study-specific βs and standard errors. Meta-analysis was conducted using an inverse variance weighted approach assuming a random effect model. Results— Despite having power to detect odds ratio of 1.09–1.14 for overall IS and 1.20–1.32 for major stroke subtypes, none of the SNPs were significantly associated with overall IS and/or stroke subtypes after adjusting for multiple comparisons. Conclusions— Our results suggest that the major common loci associated with MI risk do not have effects of similar magnitude on overall IS but do not preclude moderate associations restricted to specific IS subtypes. Disparate mechanisms may be critical in the development of acute ischemic coronary and cerebrovascular events.

The Vitamin Intervention for Stroke Prevention (VISP) trial was a multi-center, controlled, double blinded clinical trial, designed to determine whether the daily intake of high dose folic acid, vitamins B 6 and B 12 reduced recurrent cerebral infarction. As part of GARNET (the Genomics and Randomized Trials Network), we have performed a Genome-Wide Association Study (GWAS), using the Illumina Omni 1M SNP platform in 2,100 ischemic stroke patients from VISP. Extensive quality control (QC) measures were performed, resulting in a total of 737,081 SNPs for analysis. We have performed GWA analyses for baseline quantitative measures of creatinine, total cholesterol (TC), high density lipoprotein (HDL), triglycerides (TG), C-reactive protein (CRP), Factor 1+2 thrombin-antithrombin (TAT), tissue plasminogen activator (tPA), thrombomodulin (TM) and von Willebrand factor (vWF), using linear regression approaches implemented in PLINK. Principal component analysis (PCA), implemented in KING, was utilized to address confounders due to population substructure. Inverse normal transformation was performed for each of the quantitative traits, prior to analysis. Genome wide association analyses were performed using age, sex and the top 10 principal components as covariates. There is no inflation in our GWAS scan results (GC lambda ≤ 1.012 in all scans). We observed three associated loci that exceed genome wide significance (≤6.8×10 -8 ): in or near the CETP gene for HDL (6.5×10 -10 ), the CRP gene for CRP (7.4×10 -10 ), and at the ABO locus for vWF (8.7×10 -31 ). The most strongly associated SNPs for other traits were closest to the following genes: GABRA3 for creatinine ( rs994424 , 5.7×10 -6 ), SPATA13 for TC (rs9553189, 3.2×10 -7 ), between ARMCX2 and NXF5 genes on the X chromosome for TG (rs2213369, 1.7×10 -6 ); the ZIC4 gene for TAT (rs17565826, 4.1×10-6); LOC283089 for tPA (rs3011337, 3.6×10 -6 ); and JAZF1 for TM (rs10258132, 1.1×10 -6 ). Putative novel associations with these traits will require confirmation in larger independent samples. Our GWAS scan has successfully replicated associations for SNPs in the CETP, CRP and ABO genes for HDL, CRP and vWF respectively in an ischemic stroke population.

Background Previously we performed a linkage scan of 638 African American affected sibling pairs (ASP) with type 2 diabetes (T2D) enriched for end-stage renal disease (ESRD). Ordered subset linkage analysis (OSA) revealed a linkage peak on chromosome 7p in the subset of families with earlier age of T2D diagnosis. Methods We fine mapped this region by genotyping 11 additional polymorphic markers in the same ASP and investigated a total of 68 single nucleotide polymorphisms (SNPs) in functional candidate genes (GCK1, IL6, IGFBP1 and IGFBP3) for association with age of T2D diagnosis, age of ESRD diagnosis, duration of T2D to onset of ESRD, body mass index (BMI) in African American cases and T2D-ESRD in an African American case-control cohort. OSA of fine mapping markers supported linkage at 28 cM on 7p (near D7S3051) in early-onset T2D families (max. LOD = 3.61, P = 0.002). SNPs in candidate genes and 70 ancestry-informative markers (AIMs) were evaluated in 577 African American T2D-ESRD cases and 596 African American controls. Results The most significant association was observed between ESRD age of diagnosis and SNP rs730497, located in intron 1 of the GCK1 gene (recessive T2D age-adjusted P = 0.0006). Nominal associations were observed with GCK1 SNPs and T2D age of diagnosis (BMI-adjusted P = 0.014 to 0.032). Also, one IGFBP1 and four IGFBP3 SNPs showed nominal genotypic association with T2D-ESRD (P = 0.002-0.049). After correcting for multiple tests, only rs730497 remanined significant. Conclusion Variant rs730947 in the GCK1 gene appears to play a role in early ESRD onset in African Americans.

OBJECTIVE—Recently, variants in the TCF7L2 gene have been reported to be associated with type 2 diabetes across multiple Europid populations, but only one small sample of African-American type 2 diabetic patients has been examined. Our objective was to investigate the importance of TCF7L2 in a larger African-American case-control population. RESEARCH DESIGN AND METHODS—We investigated single nucleotide polymorphisms (SNPs) in six known type 2 diabetes genes in 577 African-American case subjects with type 2 diabetes enriched for nephropathy and 596 African-American control subjects. Additionally, we genotyped 70 ancestry-informative markers (AIMs) to apply adjustments for differences in ancestral proportions. RESULTS—The most significant associations were observed with TCF7L2 intron 3 SNPs rs7903146 (additive P = 4.10 × 10−6, odds ratio [OR] 1.51; admixture-adjusted Pa = 3.77 × 10−6) and rs7901695 (P = 0.001, OR 1.30; Pa = 0.003). The 2-SNP haplotype containing these SNPs was also associated with type 2 diabetes (P = 3 × 10−5). Modest associations were also seen with TCF7L2 intron 4 SNPs rs7895340, rs11196205, and rs12255372 (0.01 < P < 0.05; 0.03 < Pa < 0.08), as well as with ATP-sensitive inwardly rectifying potassium channel subunit Kir6.2 (KCNJ11) and hepatocyte nuclear factor 4-α (HNF4A) SNPs (0.01 < P < 0.05; 0.01 < Pa < 0.41). No significant associations were detected with genotyped calpain 10 (CAPN10), peroxisome proliferator–activated receptor γ (PPARG), and transcription factor 1 (TCF1) SNPs. CONCLUSIONS—This study indicates that variants in the TCF7L2 gene significantly contribute to diabetes susceptibility in African-American populations.

In a genome-wide scan for type 2 diabetes (T2DM) in African American (AA) families, ordered subsets analysis (OSA) provided evidence for linkage to chromosome 20p in a subset with later age at diagnosis (max LOD 2.57, P =0.008). The proprotein convertase subtilisin/kexin-type 2 ( PCSK2 ) gene is within the LOD-1 interval of this linkage peak. Twenty-nine single nucleotide polymorphisms (SNPs) were genotyped across this gene in 380 unrelated AA individuals with T2DM and end-stage renal disease (T2DM–ESRD), 278 AA controls, 96 European Americans (EA) and 120 Yoruba Nigerian (YRI) controls. In addition, 22 ancestry-informative markers (AIMs) were genotyped in all AA subjects, 120 YRI, and 282 EA controls. ADMIXMAP was used to model the distributions of admixture and generate score tests of allelic and haplotypic association. Association with T2DM was observed among 4 SNPs: rs2021785 (admixture-adjusted P a =0.00014), rs1609659 ( P a =0.028), rs4814597 ( P a =0.039) and rs2269023 ( P a =0.043). None of the PCSK2 SNPs were associated with age at T2DM diagnosis. A variant in the PCKS2 gene, rs2021785, appears to play a role in susceptibility to T2DM in this AA population.

Background Variants of uncoupling protein genes UCP1 and UCP2 have been associated with a range of traits. We wished to evaluate contributions of known UCP1 and UCP2 variants to metabolic traits in the Insulin Resistance and Atherosclerosis (IRAS) Family Study. Methods We genotyped five promoter or coding single nucleotide polymorphisms (SNPs) in 239 African American (AA) participants and 583 Hispanic participants from San Antonio (SA) and San Luis Valley. Generalized estimating equations using a sandwich estimator of the variance and exchangeable correlation to account for familial correlation were computed for the test of genotypic association, and dominant, additive and recessive models. Tests were adjusted for age, gender and BMI (glucose homeostasis and lipid traits), or age and gender (obesity traits), and empirical P-values estimated using a gene dropping approach. Results UCP1 A-3826G was associated with AIRg in AA (P = 0.006) and approached significance in Hispanic families (P = 0.054); and with HDL-C levels in SA families (P = 0.0004). Although UCP1 expression is reported to be restricted to adipose tissue, RT-PCR indicated that UCP1 is expressed in human pancreas and MIN-6 cells, and immunohistochemistry demonstrated co-localization of UCP1 protein with insulin in human islets. UCP2 A55V was associated with waist circumference (P = 0.045) in AA, and BMI in SA (P = 0.018); and UCP2 G-866A with waist-to-hip ratio in AA (P = 0.016). Conclusion This study suggests a functional variant of UCP1 contributes to the variance of AIRg in an AA population; the plausibility of this unexpected association is supported by the novel finding that UCP1 is expressed in islets.

The estrogen receptor-α gene (ESR1) was selected as a positional candidate under a type 2 diabetes linkage peak at 6q24-27. A total of 42 ESR1 single nucleotide polymorphisms (SNPs) were genotyped in 380 African-American type 2 diabetic case subjects with end-stage renal disease (ESRD) and 276 African-American control subjects. A total of 22 ancestry informative markers were also genotyped, and the program Admixmap was used to adjust allelic and haplotypic association tests for individual estimates of admixture. The most significant association with type 2 diabetes–ESRD was with rs1033182 in intron 2 (P = 0.013, admixture-adjusted Pa = 0.021). Genotyping 17 SNPs across a region of ESR1 intron 1–intron 2 in an expanded population of 851 case and 635 control subjects supported association with rs1033182 (P = 0.004, Pa = 0.027) and with an independent six-SNP haplotype of high linkage disequilibrium spanning 6.4 kb (P < 0.0001, Pa < 0.0001). The same 17 ESR1 SNPs were genotyped in 300 European-American type 2 diabetes–ESRD case subjects and 310 European-American control subjects. Two intron 2 SNPs, rs2431260 (P = 0.015) and rs1709183 (P = 0.019), and a four-SNP haplotype containing these SNPs (P = 0.033) were associated with type 2 diabetes and/or ESRD. Results suggest that intron 1 and intron 2 of the ESR1 gene may contain functionally important regions related to type 2 diabetes or ESRD risk.

Circulating homocysteine levels (tHcy), a product of the folate one carbon metabolism pathway (FOCM) through the demethylation of methionine, are heritable and are associated with an increased risk of common diseases such as stroke, cardiovascular disease (CVD), cancer and dementia. The FOCM is the sole source of de novo methyl group synthesis, impacting many biological and epigenetic pathways. However, the genetic determinants of elevated tHcy (hyperhomocysteinemia), dysregulation of methionine metabolism and the underlying biological processes remain unclear. We conducted independent genome-wide association studies and a meta-analysis of methionine metabolism, characterized by post-methionine load test tHcy, in 2,710 participants from the Framingham Heart Study (FHS) and 2,100 participants from the Vitamin Intervention for Stroke Prevention (VISP) clinical trial, and then examined the association of the identified loci with incident stroke in FHS. Five genes in the FOCM pathway (GNMT [p = 1.60×10−63], CBS [p = 3.15×10−26], CPS1 [p = 9.10×10−13], ALDH1L1 [p = 7.3×10−13] and PSPH [p = 1.17×10−16]) were strongly associated with the difference between pre- and post-methionine load test tHcy levels (ΔPOST). Of these, one variant in the ALDH1L1 locus, rs2364368, was associated with incident ischemic stroke. Promoter analyses reveal genetic and epigenetic differences that may explain a direct effect on GNMT transcription and a downstream affect on methionine metabolism. Additionally, a genetic-score consisting of the five significant loci explains 13% of the variance of ΔPOST in FHS and 6% of the variance in VISP. Association between variants in FOCM genes with ΔPOST suggest novel mechanisms that lead to differences in methionine metabolism, and possibly the epigenome, impacting disease risk. These data emphasize the importance of a concerted effort to understand regulators of one carbon metabolism as potential therapeutic targets., Author Summary Elevated homocysteine (tHcy) is strongly associated with risk for common disorders such as stroke, cardiovascular disease and Alzheimer disease. Lowering tHcy levels has proven to have variable success in reducing clinical risk, so the question remains, “Are we correctly targeting these disorders by lowering tHcy?” Understanding folate one-carbon metabolism pathway (FOCM) genetic variation will aid us in developing new targets for therapy. The FOCM is essential in regulation of the epigenome, which controls genes in ways beyond nucleotide sequence. We present data generated from stroke-only and general populations where we identify strong association of genetic risk factors for variation in one-carbon metabolism function, characterized by the post-methionine load test. We show that GNMT harbors genetic and epigenetic differences that influence gene function, which may have downstream effects on the epigenome of the cell, affecting disease risk. We developed a genetic risk score that predicts post-methionine load homocysteine levels that may be useful in clinic. Finally, we identified a novel association between ischemic stroke and ALDH1L1, which emphasizes the clinical importance of this work. Our results highlight the importance of a concerted effort to target the FOCM (beyond tHcy) and parallel pathways in future pharmacogenetic work using the genetic variation we describe here.