Sex-Stratified Genome-Wide Association Study in the Spanish Population Identifies a Novel Locus for Lacunar Stroke.

BACKGROUND: Ischemic stroke (IS) represents a significant health burden globally, necessitating a better understanding of its genetic underpinnings to improve prevention and treatment strategies. Despite advances in IS genetics, studies focusing on the Spanish population and sex-stratified analyses are lacking. METHODS: A case-control genome-wide association study was conducted with 9081 individuals (3493 IS cases and 5588 healthy controls). IS subtypes using Trial of ORG 10172 in Acute Stroke Treatment criteria were explored in a sex-stratified approach. Replication efforts involved the MEGASTROKE, GIGASTROKE, and the UK Biobank international cohorts. Post-genome-wide association study analysis included: in silico proteomic analysis, gene-based analysis, quantitative trait loci annotation, transcriptome-wide association analysis, and bioinformatic analysis using chromatin accessibility data. RESULTS: Identified as associated with IS and its subtypes were 4 significant and independent loci. Replication confirmed 5p15.2 as a new locus associated with small-vessel occlusion stroke, with rs59970332-T as the lead variant (beta [SE], 0.13 [0.02]; P=4.34×10-8). Functional analyses revealed CTNND2 given proximity and its implication in pathways involved in vascular integrity and angiogenesis. Integration of Hi-C data identified additional potentially modulated genes, and in silico proteomic analysis suggested a distinctive blood proteome profile associated with the lead variant. Gene-set enrichment analyses highlighted pathways consistent with small-vessel disease pathogenesis. Gene-based associations with known stroke-related genes such as F2 and FGG were also observed, reinforcing the relevance of our findings. CONCLUSIONS: We found CTNND2 as a potential key molecule in small-vessel occlusion stroke risk, and predominantly in males. This study sheds light on the genetic architecture of IS in the Spanish population, providing novel insights into sex-specific associations and potential molecular mechanisms. Further research, including replication in larger cohorts, is essential for a comprehensive understanding of these findings and for their translation to clinical practice.

Ischaemic stroke patients present sex differences in gut microbiota.

BACKGROUND: Gut microbiota plays a role in the pathophysiology of ischaemic stroke (IS) through the bidirectional gut-brain axis. Nevertheless, little is known about sex-specific microbiota signatures in IS occurrence. METHODS: A total of 89 IS patients and 12 healthy controls were enrolled. We studied the taxonomic differences of the gut microbiota between men and women with IS by shotgun metagenomic sequencing. To evaluate the causal effect of several bacteria on IS risk, we performed a two-sample Mendelian randomisation (MR) with inverse-variance weighting (IVW) using genome-wide association analysis (GWAS) summary statistics from two cohorts of 5959 subjects with genetic and microbiota data and 1,296,908 subjects with genetic and IS data, respectively. RESULTS: α-Diversity analysis measured using Observed Species (p = 0.017), Chao1 (p = 0.009) and Abundance-based Coverage Estimator (p = 0.012) indexes revealed that IS men have a higher species richness compared with IS women. Moreover, we found sex-differences in IS patients in relation to the phylum Fusobacteria, class Fusobacteriia, order Fusobacteriales and family Fusobacteriaceae (all Bonferroni-corrected p < 0.001). MR confirmed that increased Fusobacteriaceae levels in the gut are causally associated with an increased risk of IS (IVW p = 0.02, ß = 0.32). CONCLUSIONS: Our study is the first to indicate that there are gut microbiome differences between men and women with IS, identifying high levels of Fusobacteriaceae in women as a specific risk factor for IS. Incorporating sex stratification analysis is important in the design, analysis and interpretation of studies on stroke and the gut microbiota.

Multi-ancestry GWAS reveals excitotoxicity associated with outcome after ischaemic stroke.

During the first hours after stroke onset, neurological deficits can be highly unstable: some patients rapidly improve, while others deteriorate. This early neurological instability has a major impact on long-term outcome. Here, we aimed to determine the genetic architecture of early neurological instability measured by the difference between the National Institutes of Health Stroke Scale (NIHSS) within 6 h of stroke onset and NIHSS at 24 h. A total of 5876 individuals from seven countries (Spain, Finland, Poland, USA, Costa Rica, Mexico and Korea) were studied using a multi-ancestry meta-analyses. We found that 8.7% of NIHSS at 24 h of variance was explained by common genetic variations, and also that early neurological instability has a different genetic architecture from that of stroke risk. Eight loci (1p21.1, 1q42.2, 2p25.1, 2q31.2, 2q33.3, 5q33.2, 7p21.2 and 13q31.1) were genome-wide significant and explained 1.8% of the variability suggesting that additional variants influence early change in neurological deficits. We used functional genomics and bioinformatic annotation to identify the genes driving the association from each locus. Expression quantitative trait loci mapping and summary data-based Mendelian randomization indicate that ADAM23 (log Bayes factor = 5.41) was driving the association for 2q33.3. Gene-based analyses suggested that GRIA1 (log Bayes factor = 5.19), which is predominantly expressed in the brain, is the gene driving the association for the 5q33.2 locus. These analyses also nominated GNPAT (log Bayes factor = 7.64) ABCB5 (log Bayes factor = 5.97) for the 1p21.1 and 7p21.1 loci. Human brain single-nuclei RNA-sequencing indicates that the gene expression of ADAM23 and GRIA1 is enriched in neurons. ADAM23, a presynaptic protein and GRIA1, a protein subunit of the AMPA receptor, are part of a synaptic protein complex that modulates neuronal excitability. These data provide the first genetic evidence in humans that excitotoxicity may contribute to early neurological instability after acute ischaemic stroke.

Genetic Architecture of Ischaemic Strokes after COVID-19 Shows Similarities with Large Vessel Strokes.

We aimed to analyse whether patients with ischaemic stroke (IS) occurring within eight days after the onset of COVID-19 (IS-COV) are associated with a specific aetiology of IS. We used SUPERGNOVA to identify genome regions that correlate between the IS-COV cohort (73 IS-COV cases vs. 701 population controls) and different aetiological subtypes. Polygenic risk scores (PRSs) for each subtype were generated and tested in the IS-COV cohort using PRSice-2 and PLINK to find genetic associations. Both analyses used the IS-COV cohort and GWAS from MEGASTROKE (67,162 stroke patients vs. 454,450 population controls), GIGASTROKE (110,182 vs. 1,503,898), and the NINDS Stroke Genetics Network (16,851 vs. 32,473). Three genomic regions were associated (p-value < 0.05) with large artery atherosclerosis (LAA) and cardioembolic stroke (CES). We found four loci targeting the genes PITX2 (rs10033464, IS-COV beta = 0.04, p-value = 2.3 × 10-2, se = 0.02), previously associated with CES, HS6ST1 (rs4662630, IS-COV beta = -0.04, p-value = 1.3 × 10-3, se = 0.01), TMEM132E (rs12941838 IS-COV beta = 0.05, p-value = 3.6 × 10-4, se = 0.01), and RFFL (rs797989 IS-COV beta = 0.03, p-value = 1.0 × 10-2, se = 0.01). A statistically significant PRS was observed for LAA. Our results suggest that IS-COV cases are genetically similar to LAA and CES subtypes. Larger cohorts are needed to assess if the genetic factors in IS-COV cases are shared with the general population or specific to viral infection.

Biological Age Acceleration Is Lower in Women With Ischemic Stroke Compared to Men.

BACKGROUND: Stroke onset in women occurs later in life compared with men. The underlying mechanisms of these differences have not been established. Epigenetic clocks, based on DNA methylation (DNAm) profiles, are the most accurate biological age estimate. Epigenetic age acceleration (EAA) measures indicate whether an individual is biologically younger or older than expected. Our aim was to analyze whether sexual dichotomy at age of stroke onset is conditioned by EAA. METHODS: We used 2 DNAm datasets from whole blood samples of case-control genetic studies of ischemic stroke (IS), a discovery cohort of 374 IS patients (N women=163, N men=211), from GRECOS (Genotyping Recurrence Risk of Stroke) and SEDMAN (Dabigatran Study in the Early Phase of Stroke, New Neuroimaging Markers and Biomarkers) studies and a replication cohort of 981 IS patients (N women=411, N men=570) from BASICMAR register. We compared chronological age, 2 DNAm-based biomarkers of aging and intrinsic and extrinsic epigenetic age acceleration EAA (IEAA and extrinsic EAA, respectively), in IS as well as in individual IS etiologic subtypes. Horvath and Hannum epigenetic clocks were used to assess the aging rate. A proteomic study using the SOMAScan multiplex assay was performed on 26 samples analyzing 1305 proteins. RESULTS: Women present lower Hannum-extrinsic EAA values, whereas men have higher Hannum-extrinsic EAA values (women=-0.64, men=1.24, P=1.34×10-2); the same tendency was observed in the second cohort (women=-0.57, men=0.79, P=0.02). These differences seemed to be specific to cardioembolic and undetermined stroke subtypes. Additionally, 42 blood protein levels were associated with Hannum-extrinsic EAA (P<0.05), belonging to the immune effector process (P=1.54×10-6) and platelet degranulation (P<8.74×10-6) pathways. CONCLUSIONS: This study shows that sex-specific underlying biological mechanisms associated with stroke onset could be due to differences in biological age acceleration between men and women.

Single nucleotide variations in ZBTB46 are associated with post-thrombolytic parenchymal haematoma.

Haemorrhagic transformation is a complication of recombinant tissue-plasminogen activator treatment. The most severe form, parenchymal haematoma, can result in neurological deterioration, disability, and death. Our objective was to identify single nucleotide variations associated with a risk of parenchymal haematoma following thrombolytic therapy in patients with acute ischaemic stroke. A fixed-effect genome-wide meta-analysis was performed combining two-stage genome-wide association studies (n = 1904). The discovery stage (three cohorts) comprised 1324 ischaemic stroke individuals, 5.4% of whom had a parenchymal haematoma. Genetic variants yielding a P-value < 0.05 1 × 10-5 were analysed in the validation stage (six cohorts), formed by 580 ischaemic stroke patients with 12.1% haemorrhagic events. All participants received recombinant tissue-plasminogen activator; cases were parenchymal haematoma type 1 or 2 as defined by the European Cooperative Acute Stroke Study (ECASS) criteria. Genome-wide significant findings (P < 5 × 10-8) were characterized by in silico functional annotation, gene expression, and DNA regulatory elements. We analysed 7 989 272 single nucleotide polymorphisms and identified a genome-wide association locus on chromosome 20 in the discovery cohort; functional annotation indicated that the ZBTB46 gene was driving the association for chromosome 20. The top single nucleotide polymorphism was rs76484331 in the ZBTB46 gene [P = 2.49 × 10-8; odds ratio (OR): 11.21; 95% confidence interval (CI): 4.82-26.55]. In the replication cohort (n = 580), the rs76484331 polymorphism was associated with parenchymal haematoma (P = 0.01), and the overall association after meta-analysis increased (P = 1.61 × 10-8; OR: 5.84; 95% CI: 3.16-10.76). ZBTB46 codes the zinc finger and BTB domain-containing protein 46 that acts as a transcription factor. In silico studies indicated that ZBTB46 is expressed in brain tissue by neurons and endothelial cells. Moreover, rs76484331 interacts with the promoter sites located at 20q13. In conclusion, we identified single nucleotide variants in the ZBTB46 gene associated with a higher risk of parenchymal haematoma following recombinant tissue-plasminogen activator treatment.

Genome-Wide Studies in Ischaemic Stroke: Are Genetics Only Useful for Finding Genes?

Ischaemic stroke is a complex disease with some degree of heritability. This means that heritability factors, such as genetics, could be risk factors for ischaemic stroke. The era of genome-wide studies has revealed some of these heritable risk factors, although the data generated by these studies may also be useful in other disciplines. Analysis of these data can be used to understand the biological mechanisms associated with stroke risk and stroke outcome, to determine the causality between stroke and other diseases without the need for expensive clinical trials, or to find potential drug targets with higher success rates than other strategies. In this review we will discuss several of the most relevant studies regarding the genetics of ischaemic stroke and the potential use of the data generated.

ICA1L Is Associated with Small Vessel Disease: A Proteome-Wide Association Study in Small Vessel Stroke and Intracerebral Haemorrhage.

Small vessel strokes (SVS) and intracerebral haemorrhages (ICH) are acute outcomes of cerebral small vessel disease (SVD). Genetic studies combining both phenotypes have identified three loci associated with both traits. However, the genetic cis-regulation at the protein level associated with SVD has not been studied before. We performed a proteome-wide association study (PWAS) using FUSION to integrate a genome-wide association study (GWAS) and brain proteomic data to discover the common mechanisms regulating both SVS and ICH. Dorsolateral prefrontal cortex (dPFC) brain proteomes from the ROS/MAP study (N = 376 subjects and 1443 proteins) and the summary statistics for the SVS GWAS from the MEGASTROKE study (N = 237,511) and multi-trait analysis of GWAS (MTAG)-ICH−SVS from Chung et al. (N = 240,269) were selected. We performed PWAS and then a co-localization analysis with COLOC. The significant and nominal results were validated using a replication dPFC proteome (N = 152). The replicated results (q-value < 0.05) were further investigated for the causality relationship using summary data-based Mendelian randomization (SMR). One protein (ICA1L) was significantly associated with SVS (z-score = −4.42 and p-value = 9.6 × 10−6) and non-lobar ICH (z-score = −4.8 and p-value = 1.58 × 10−6) in the discovery PWAS, with a high co-localization posterior probability of 4. In the validation PWAS, ICA1L remained significantly associated with both traits. The SMR results for ICA1L indicated a causal association of protein expression levels in the brain with SVS (p-value = 3.66 × 10−5) and non-lobar ICH (p-value = 1.81 × 10−5). Our results show that the association of ICA1L with SVS and non-lobar ICH is conditioned by the cis-regulation of its protein levels in the brain.

Causal Effect of MMP-1 (Matrix Metalloproteinase-1), MMP-8, and MMP-12 Levels on Ischemic Stroke: A Mendelian Randomization Study.

Background and Purpose: MMP (matrix metalloproteinase) levels have been widely associated with ischemic stroke risk and poststroke outcome. However, their role as a risk factor or as a subeffect because of ischemia is uncertain. Methods: We performed a literature search of genome-wide studies that evaluate serum/plasma levels of MMPs. We used a 2-sample Mendelian randomization approach to evaluate the causality of MMP levels on ischemic stroke risk or poststroke outcome, using 2 cohorts: MEGASTROKE (n=440 328) and GODs (n=1791). Results: Genome-wide association studies of MMP-1, MMP-8, and MMP-12 plasma/serum levels were evaluated. A significant association, which was also robust in the sensitivity analysis, was found with all ischemic strokes: MMP-12 (odds ratio=0.90 [95% CI, 0.86­0.94]; q value=7.43×10−5), and with subtypes of stroke, large-artery atherosclerosis: MMP-1 (odds ratio=0.95 [95% CI, 0.92­0.98]; q value=0.01) and MMP-12 (odds ratio=0.71 [95% CI, 0.65­0.77]; q value=5.11×10−14); small-vessel occlusion: MMP-8 (odds ratio=1.24 [95% CI, 1.06­1.45]; q value=0.03). No associations were found in relation to stroke outcome. Conclusions: Our study suggests a causal link between lower serum levels of MMP-12 and the risk of ischemic stroke, lower serum levels of MMP-1 and MMP-12 and the risk of large-artery stroke and higher serum levels of MMP-8 and the risk of lacunar stroke.

Early Neurological Change After Ischemic Stroke Is Associated With 90-Day Outcome.

BACKGROUND AND PURPOSE: Large-scale observational studies of acute ischemic stroke (AIS) promise to reveal mechanisms underlying cerebral ischemia. However, meaningful quantitative phenotypes attainable in large patient populations are needed. We characterize a dynamic metric of AIS instability, defined by change in National Institutes of Health Stroke Scale score (NIHSS) from baseline to 24 hours baseline to 24 hours (NIHSSbaseline - NIHSS24hours = ΔNIHSS6-24h), to examine its relevance to AIS mechanisms and long-term outcomes. METHODS: Patients with NIHSS prospectively recorded within 6 hours after onset and then 24 hours later were enrolled in the GENISIS study (Genetics of Early Neurological Instability After Ischemic Stroke). Stepwise linear regression determined variables that independently influenced ΔNIHSS6-24h. In a subcohort of tPA (alteplase)-treated patients with large vessel occlusion, the influence of early sustained recanalization and hemorrhagic transformation on ΔNIHSS6-24h was examined. Finally, the association of ΔNIHSS6-24h with 90-day favorable outcomes (modified Rankin Scale score 0-2) was assessed. Independent analysis was performed using data from the 2 NINDS-tPA stroke trials (National Institute of Neurological Disorders and Stroke rt-PA). RESULTS: For 2555 patients with AIS, median baseline NIHSS was 9 (interquartile range, 4-16), and median ΔNIHSS6-24h was 2 (interquartile range, 0-5). In a multivariable model, baseline NIHSS, tPA-treatment, age, glucose, site, and systolic blood pressure independently predicted ΔNIHSS6-24h (R2=0.15). In the large vessel occlusion subcohort, early sustained recanalization and hemorrhagic transformation increased the explained variance (R2=0.27), but much of the variance remained unexplained. ΔNIHSS6-24h had a significant and independent association with 90-day favorable outcome. For the subjects in the 2 NINDS-tPA trials, ΔNIHSS3-24h was similarly associated with 90-day outcomes. CONCLUSIONS: The dynamic phenotype, ΔNIHSS6-24h, captures both explained and unexplained mechanisms involved in AIS and is significantly and independently associated with long-term outcomes. Thus, ΔNIHSS6-24h promises to be an easily obtainable and meaningful quantitative phenotype for large-scale genomic studies of AIS.

PATJ Low Frequency Variants Are Associated With Worse Ischemic Stroke Functional Outcome.

RATIONALE: Ischemic stroke is among the leading causes of adult disability. Part of the variability in functional outcome after stroke has been attributed to genetic factors but no locus has been consistently associated with stroke outcome. OBJECTIVE: Our aim was to identify genetic loci influencing the recovery process using accurate phenotyping to produce the largest GWAS (genome-wide association study) in ischemic stroke recovery to date. METHODS AND RESULTS: A 12-cohort, 2-phase (discovery-replication and joint) meta-analysis of GWAS included anterior-territory and previously independent ischemic stroke cases. Functional outcome was recorded using 3-month modified Rankin Scale. Analyses were adjusted for confounders such as discharge National Institutes of Health Stroke Scale. A gene-based burden test was performed. The discovery phase (n=1225) was followed by open (n=2482) and stringent joint-analyses (n=1791). Those cohorts with modified Rankin Scale recorded at time points other than 3-month or incomplete data on previous functional status were excluded in the stringent analyses. Novel variants in PATJ (Pals1-associated tight junction) gene were associated with worse functional outcome at 3-month after stroke. The top variant was rs76221407 (G allele, ß=0.40, P=1.70×10-9). CONCLUSIONS: Our results identify a set of common variants in PATJ gene associated with 3-month functional outcome at genome-wide significance level. Future studies should examine the role of PATJ in stroke recovery and consider stringent phenotyping to enrich the information captured to unveil additional stroke outcome loci.

Contribution of "Omic" Studies to the Understanding of Cadasil. A Systematic Review.

CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) is a small vessel disease caused by mutations in NOTCH3 that lead to an odd number of cysteines in the epidermal growth factor (EGF)-like repeat domain, causing protein misfolding and aggregation. The main symptoms are migraines, psychiatric disorders, recurrent strokes, and dementia. Omic technologies allow the massive study of different molecules for understanding diseases in a non-biased manner or even for discovering targets and their possible treatments. We analyzed the progress in understanding CADASIL that has been made possible by omics sciences. For this purpose, we included studies that focused on CADASIL and used omics techniques, searching bibliographic resources, such as PubMed. We excluded studies with other phenotypes, such as migraine or leukodystrophies. A total of 18 articles were reviewed. Due to the high prevalence of NOTCH3 mutations considered pathogenic to date in genomic repositories, one can ask whether all of them produce CADASIL, different degrees of the disease, or whether they are just a risk factor for small vessel disease. Besides, proteomics and transcriptomics studies found that the molecules that are significantly altered in CADASIL are mainly related to cell adhesion, the cytoskeleton or extracellular matrix components, misfolding control, autophagia, angiogenesis, or the transforming growth factor ß (TGFß) signaling pathway. The omics studies performed on CADASIL have been useful for understanding the biological mechanisms and could be key factors for finding potential drug targets.

Genome-Wide Association Study of White Blood Cell Counts in Patients With Ischemic Stroke.

Background and Purpose- Immune cells play a key role in the first 24h poststroke (acute phase), being associated with stroke outcome. We aimed to find genetic risk factors associated with leukocyte counts during the acute phase of stroke. Methods- Ischemic stroke patients with leukocyte counts data during the first 24h were included. Genome-wide association study and gene expression studies were performed. Results- Our genome-wide association study, which included 2064 (Discovery) and 407 (Replication) patients, revealed a new locus (14q24.3) associated with leukocyte counts. After Joint analysis (n=2471) 5 more polymorphisms reached genome-wide significance (P<5×10-8). The 14q24.3 locus was associated with acute stroke outcome (rs112809786, P=0.036) and with ACOT1 and PTGR2 gene expression. Previous polymorphisms associated with leukocyte counts in general-population did not show any significance in our study. Conclusions- We have found the first locus associated with leukocyte counts in ischemic stroke, also associated with acute outcome. Genetic analysis of acute endophenotypes could be useful to find the genetic factors associated with stroke outcome. Our findings suggested a different modulation of immune cells in stroke compared with healthy conditions.

GRECOS Project (Genotyping Recurrence Risk of Stroke): The Use of Genetics to Predict the Vascular Recurrence After Stroke.

BACKGROUND AND PURPOSE: Vascular recurrence occurs in 11% of patients during the first year after ischemic stroke (IS) or transient ischemic attack. Clinical scores do not predict the whole vascular recurrence risk; therefore, we aimed to find genetic variants associated with recurrence that might improve the clinical predictive models in IS. METHODS: We analyzed 256 polymorphisms from 115 candidate genes in 3 patient cohorts comprising 4482 IS or transient ischemic attack patients. The discovery cohort was prospectively recruited and included 1494 patients, 6.2% of them developed a new IS during the first year of follow-up. Replication analysis was performed in 2988 patients using SNPlex or HumanOmni1-Quad technology. We generated a predictive model using Cox regression (GRECOS score [Genotyping Reurrence Risk of Stroke]) and generated risk groups using a classification tree method. RESULTS: The analyses revealed that rs1800801 in the MGP gene (hazard ratio, 1.33; P=9×10-03), a gene related to artery calcification, was associated with new IS during the first year of follow-up. This polymorphism was replicated in a Spanish cohort (n=1.305); however, it was not significantly associated in a North American cohort (n=1.683). The GRECOS score predicted new IS (P=3.2×10-09) and could classify patients, from low risk of stroke recurrence (1.9%) to high risk (12.6%). Moreover, the addition of genetic risk factors to the GRECOS score improves the prediction compared with previous Stroke Prognosis Instrument-II score (P=0.03). CONCLUSIONS: The use of genetics could be useful to estimate vascular recurrence risk after IS. Genetic variability in the MGP gene was associated with vascular recurrence in the Spanish population.

Systematic Review of Cysteine-Sparing NOTCH3 Missense Mutations in Patients with Clinical Suspicion of CADASIL.

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is caused by mutations in the NOTCH3 gene, affecting the number of cysteines in the extracellular domain of the receptor, causing protein misfolding and receptor aggregation. The pathogenic role of cysteine-sparing NOTCH3 missense mutations in patients with typical clinical CADASIL syndrome is unknown. The aim of this article is to describe these mutations to clarify if any could be potentially pathogenic. Articles on cysteine-sparing NOTCH3 missense mutations in patients with clinical suspicion of CADASIL were reviewed. Mutations were considered potentially pathogenic if patients had: (a) typical clinical CADASIL syndrome; (b) diffuse white matter hyperintensities; (c) the 33 NOTCH3 exons analyzed; (d) mutations that were not polymorphisms; and (e) Granular osmiophilic material (GOM) deposits in the skin biopsy. Twenty-five different mutations were listed. Four fulfill the above criteria: p.R61W; p.R75P; p.D80G; and p.R213K. Patients carrying these mutations had typical clinical CADASIL syndrome and diffuse white matter hyperintensities, mostly without anterior temporal pole involvement. Cysteine-sparing NOTCH3 missense mutations are associated with typical clinical CADASIL syndrome and typical magnetic resonance imaging (MRI) findings, although with less involvement of the anterior temporal lobe. Hence, these mutations should be further studied to confirm their pathological role in CADASIL.

TRAF3 Epigenetic Regulation Is Associated With Vascular Recurrence in Patients With Ischemic Stroke.

BACKGROUND AND PURPOSE: Clopidogrel is one of the most used antiplatelet drugs in patients with cardiovascular disease. However, 16% to 50% of patients have a high on-clopidogrel platelet reactivity and an increased risk of ischemic events. The pathogenesis of high on-treatment platelet reactivity in patients with stroke is only partially explained by genetic variations. This study aims to find differentially methylated sites across the genome associated with vascular recurrence in ischemic stroke patients treated with clopidogrel. METHODS: From a cohort of 1900 patients with ischemic stroke, we selected 42 patients treated with clopidogrel, including 21 with a recurrent vascular event and 21 without vascular recurrence during the first year of follow-up. Over 480 000 DNA methylation sites were analyzed across the genome. Differentially methylated CpG sites were identified by nonparametric testing using R. Replication analysis was performed in a new cohort of 191 subjects and results were correlated with platelet reactivity in a subset of 90 subjects using light transmission aggregometry. RESULTS: A total of 73 differentially methylated CpG sites (P<1×10(-05)) were identified; 3 of them were selected for further replication: cg03548645 (P=1.42×10(-05), TRAF3), cg09533145 (P=7.81×10(-06), ADAMTS2), and cg15107336 (P=1.89×10(-05), XRCC1). The cg03548645 CpG remained significant in the replication study (P=0.034), a deep analysis of this region revealed another methylation site associated with vascular recurrence, P=0.037. Lower cg03548645 (TRAF3) DNA methylation levels were correlated with an increased platelet aggregation (ρ=-0.29, P=0.0075). CONCLUSIONS: This study suggests for the first time that epigenetics may significantly contribute to the variability of clopidogrel response and recurrence of ischemic events in patients with stroke.

PPM1A Methylation Is Associated With Vascular Recurrence in Aspirin-Treated Patients.

BACKGROUND AND PURPOSE: Despite great efforts by pharmacogenetic studies, the causes of aspirin failure to prevent the recurrence of ischemic events remain unclear. Our aim was to study whether epigenetics could be associated with the risk of vascular recurrence in aspirin-treated stroke patients. METHODS: We performed an epigenetic joint analysis study in 327 patients treated with aspirin. In the discovery stage, we performed a nested case-control study in 38 matched ischemic stroke patients in whom 450 000 methylation sites were analyzed. Nineteen patients presented vascular recurrence after stroke, and 19 matched patients did not present vascular recurrence during the first year of follow-up. In a second stage, 289 new patients were analyzed by EpiTYPER. RESULTS: The following 3 differentially methylated candidate CpG sites, were identified in the discovery stage and analyzed in the second stage: cg26039762 (P=9.69×10(-06), RAF1), cg04985020 (P=3.47×10(-03), PPM1A), and cg08419850 (P=3.47×10(-03), KCNQ1). Joint analysis identified an epigenome-wide association for cg04985020 (PPM1A; P=1.78×10(-07)), with vascular recurrence in patients treated with aspirin. CONCLUSIONS: The pattern of differential methylation in PPM1A is associated with vascular recurrence in aspirin-treated stroke patients.

An Inflammatory Polymorphisms Risk Scoring System for the Differentiation of Ischemic Stroke Subtypes.

UNLABELLED: Inflammation has been associated with atherothrombotic stroke and recently with cardioembolic stroke. Different genetic risk factors have been specifically associated with the subtypes of ischemic stroke (cardioembolic, atherothrombotic, and lacunar). However, there are no studies that have generated genetic risk scores for the different subtypes of ischemic stroke using polymorphisms associated with inflammation. METHODS: We have analyzed 68 polymorphisms of 30 inflammatory mediator genes in 2,685 subjects: 1,987 stroke cases and 698 controls. We generated a genetic scoring system with the most significant polymorphisms weighted by the odds ratio of every polymorphism and taken into consideration the stroke subtype. RESULTS: Three polymorphisms, rs1205 (CRP gene), rs1800779, and rs2257073 (NOS3 gene), were associated with cardioembolic stroke (p value <0.05). The score generated was only associated with the cardioembolic stroke subtype (p value: 0.001) and was replicated in an independent cohort (p value: 0.017). The subjects with the highest score presented a cardioembolic stroke in 92.2% of the cases (p value: 0.002). CONCLUSION: The genetics of inflammatory markers is more closely associated with cardioembolic strokes than with atherothrombotic or lacunar strokes. The genetic risk scoring system could be useful in the prediction and differentiation of ischemic stroke; however, it might be specific to particular ischemic stroke subtypes.

Identification of Genetic Loci Associated With Intracerebral Hemorrhage Using a Multitrait Analysis Approach.

BACKGROUND AND OBJECTIVES: Genome-wide association studies (GWASs) have only 2 loci associated with spontaneous intracerebral hemorrhage (ICH): APOE for lobar and 1q22 for nonlobar ICH. We aimed to discover new loci through an analysis that combines correlated traits (multi-trait analysis of GWAS [MTAG]) and explore a gene-based analysis, transcriptome-wide association study (TWAS), and proteome-wide association study (PWAS) to understand the biological mechanisms of spontaneous ICH providing potential therapeutic targets. METHODS: We use the published MTAG of ICH (patients with spontaneous intraparenchymal bleeding) and small-vessel ischemic stroke. For all ICH, lobar ICH, and nonlobar ICH, a pairwise MTAG combined ICH with traits related to cardiovascular risk factors, cerebrovascular diseases, or Alzheimer disease (AD). For the analysis, we assembled those traits with a genetic correlation ≥0.3. A new MTAG combining multiple traits was performed with those traits whose pairwise MTAG yielded new GWAS-significant single nucleotide polymorphisms (SNPs), with a posterior-probability of model 3 (GWAS-pairwise) ≥0.6. We perform TWAS and PWAS that correlate the genetic component of expression or protein levels with the genetic component of a trait. We use the ICH cohort from UK Biobank as replication. RESULTS: For all ICH (1,543 ICH, 1,711 controls), the mean age was 72 ± 2 in cases and 70 ± 2 in controls, and half of them were women. Replication cohort: 700 ICH and 399,717 controls. Novel loci were found only for all ICH (the trait containing lobar and nonlobar ICH), combining data of ICH and small vessel stroke, white matter hyperintensities volume, fractional anisotropy, mean diffusivity, and AD. We replicated 6 SNPs belonging to 2q33.2 (ICA1L, ß = 0.20, SE = 0.03, p value = 8.91 × 10-12), 10q24.33 (OBFC1, ß = -0.12, SE = 0.02, p value = 1.67 × 10-8), 13q34 (COL4A2, ß = 0.02, SE = 0.02, p value = 2.34 × 10-11), and 19q13.32 (APOC1, ß = -0.19, SE = 0.03, p value = 1.38 × 10-12; APOE, ß = 0.21, SE = 0.03, p value = 2.70 × 10-11; PVRL2:CTB-129P6.4, ß = 0.15, SE = 0.03, p value = 1.38 × 10-8); 2 genes (SH3PXD2A, Z-score = 4.83, p value = 6.67 × 10-7; and APOC1, Z-score: = 5.11, p value = 1.60 × 10-7); and ICA1L transcript (Z-score = 6.8, p value = 9.1 × 10-12) and protein levels (Z-score = -5.8, p value = 6.7 × 10-9). DISCUSSION: Our results reinforce the role of APOE in ICH risk, replicate previous ICH-associated loci (2q33 and 13q34), and point to new ICH associations with OBFC1, PVRL2:CTB-129P6.4, APOC1, and SH3PXD2A. Our study used data from European subjects, our main limitation. These molecules could be potential targets for future studies for modulating ICH risk.

A multitrait genetic study of hemostatic factors and hemorrhagic transformation after stroke treatment.

BACKGROUND: Thrombolytic recombinant tissue plasminogen activator (r-tPA) treatment is the only pharmacologic intervention available in the ischemic stroke acute phase. This treatment is associated with an increased risk of intracerebral hemorrhages, known as hemorrhagic transformations (HTs), which worsen the patient's prognosis. OBJECTIVES: To investigate the association between genetically determined natural hemostatic factors' levels and increased risk of HT after r-tPA treatment. METHODS: Using data from genome-wide association studies on the risk of HT after r-tPA treatment and data on 7 hemostatic factors (factor [F]VII, FVIII, von Willebrand factor [VWF], FXI, fibrinogen, plasminogen activator inhibitor-1, and tissue plasminogen activator), we performed local and global genetic correlation estimation multitrait analyses and colocalization and 2-sample Mendelian randomization analyses between hemostatic factors and HT. RESULTS: Local correlations identified a genomic region on chromosome 16 with shared covariance: fibrinogen-HT, P = 2.45 × 10-11. Multitrait analysis between fibrinogen-HT revealed 3 loci that simultaneously regulate circulating levels of fibrinogen and risk of HT: rs56026866 (PLXND1), P = 8.80 × 10-10; rs1421067 (CHD9), P = 1.81 × 10-14; and rs34780449, near ROBO1 gene, P = 1.64 × 10-8. Multitrait analysis between VWF-HT showed a novel common association regulating VWF and risk of HT after r-tPA at rs10942300 (ZNF366), P = 1.81 × 10-14. Mendelian randomization analysis did not find significant causal associations, although a nominal association was observed for FXI-HT (inverse-variance weighted estimate [SE], 0.07 [-0.29 to 0.00]; odds ratio, 0.87; 95% CI, 0.75-1.00; raw P = .05). CONCLUSION: We identified 4 shared loci between hemostatic factors and HT after r-tPA treatment, suggesting common regulatory mechanisms between fibrinogen and VWF levels and HT. Further research to determine a possible mediating effect of fibrinogen on HT risk is needed.