Stroke genetics informs drug discovery and risk prediction across ancestries.

Previous genome-wide association studies (GWASs) of stroke - the second leading cause of death worldwide - were conducted predominantly in populations of European ancestry1,2. Here, in cross-ancestry GWAS meta-analyses of 110,182 patients who have had a stroke (five ancestries, 33% non-European) and 1,503,898 control individuals, we identify association signals for stroke and its subtypes at 89 (61 new) independent loci: 60 in primary inverse-variance-weighted analyses and 29 in secondary meta-regression and multitrait analyses. On the basis of internal cross-ancestry validation and an independent follow-up in 89,084 additional cases of stroke (30% non-European) and 1,013,843 control individuals, 87% of the primary stroke risk loci and 60% of the secondary stroke risk loci were replicated (P < 0.05). Effect sizes were highly correlated across ancestries. Cross-ancestry fine-mapping, in silico mutagenesis analysis3, and transcriptome-wide and proteome-wide association analyses revealed putative causal genes (such as SH3PXD2A and FURIN) and variants (such as at GRK5 and NOS3). Using a three-pronged approach4, we provide genetic evidence for putative drug effects, highlighting F11, KLKB1, PROC, GP1BA, LAMC2 and VCAM1 as possible targets, with drugs already under investigation for stroke for F11 and PROC. A polygenic score integrating cross-ancestry and ancestry-specific stroke GWASs with vascular-risk factor GWASs (integrative polygenic scores) strongly predicted ischaemic stroke in populations of European, East Asian and African ancestry5. Stroke genetic risk scores were predictive of ischaemic stroke independent of clinical risk factors in 52,600 clinical-trial participants with cardiometabolic disease. Our results provide insights to inform biology, reveal potential drug targets and derive genetic risk prediction tools across ancestries.

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.

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.

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.

Role of PATJ in stroke prognosis by modulating endothelial to mesenchymal transition through the Hippo/Notch/PI3K axis.

Through GWAS studies we identified PATJ associated with functional outcome after ischemic stroke (IS). The aim of this study was to determine PATJ role in brain endothelial cells (ECs) in the context of stroke outcome. PATJ expression analyses in patient's blood revealed that: (i) the risk allele of rs76221407 induces higher expression of PATJ, (ii) PATJ is downregulated 24 h after IS, and (iii) its expression is significantly lower in those patients with functional independence, measured at 3 months with the modified Rankin scale ((mRS) ≤2), compared to those patients with marked disability (mRS = 4-5). In mice brains, PATJ was also downregulated in the injured hemisphere at 48 h after ischemia. Oxygen-glucose deprivation and hypoxia-dependent of Hypoxia Inducible Factor-1α also caused PATJ depletion in ECs. To study the effects of PATJ downregulation, we generated PATJ-knockdown human microvascular ECs. Their transcriptomic profile evidenced a complex cell reprogramming involving Notch, TGF-ß, PI3K/Akt, and Hippo signaling that translates in morphological and functional changes compatible with endothelial to mesenchymal transition (EndMT). PATJ depletion caused loss of cell-cell adhesion, upregulation of metalloproteases, actin cytoskeleton remodeling, cytoplasmic accumulation of the signal transducer C-terminal transmembrane Mucin 1 (MUC1-C) and downregulation of Notch and Hippo signaling. The EndMT phenotype of PATJ-depleted cells was associated with the nuclear recruitment of MUC1-C, YAP/TAZ, ß-catenin, and ZEB1. Our results suggest that PATJ downregulation 24 h after IS promotes EndMT, an initial step prior to secondary activation of a pro-angiogenic program. This effect is associated with functional independence suggesting that activation of EndMT shortly after stroke onset is beneficial for stroke recovery.

Causal role of lipid metabolome on the risk of ischemic stroke, its etiological subtypes, and long-term outcome: A Mendelian randomization study.

BACKGROUND AND AIMS: The lipid profile is consistently associated with coronary artery disease (CAD) and ischemic stroke (IS). However, the lipoprotein subfractions have not been deeply explored in stroke subtypes, especially in IS outcome. METHODS: We performed two-sample Mendelian randomization (MR) analysis using 92 lipid traits measured by nuclear magnetic resonance in 115,000 subjects from the UK Biobank. Data for genetic associations with IS, its subtypes, and long-term outcome (LTO) were obtained from three cohorts of European ancestry: GIGASTROKE (73,652 cases, 1,234,808 controls), GODS (n = 1791) and GISCOME (n = 6165). Results obtained using CARDIoGRAMPlusC4D were used to identify differences with CAD. RESULTS: Genetically determined low concentration of medium high-density lipoprotein (HDL) particles (odds ratio (OR) = 0.92, 95% CI 0.88-0.96; p = 3.6 × 10-4) and its cholesterol content (OR = 0.92, 95% CI 0.88-0.96; p = 1.9 × 10-4) showed causal associations with an increased risk of stroke. Genetic predisposition to high apolipoprotein (apo)B to apoA-I ratio was causally associated with an increased risk of IS (OR = 1.12, 95% CI 1.06-1.18, p = 1.1 × 10-4), and a highly suggestive association was found between non-esterified cholesterol in low-density lipoprotein (LDL) and increased risk of atherothrombotic stroke (LAS) (OR = 1.35, 95% CI 1.10-1.66; p = 4.0 × 10-3). Low cholesterol in small and medium LDL was suggestively associated with poor LTO. CONCLUSIONS: Our results support that low medium HDL concentration was causally associated with an increased stroke risk, while high levels of non-esterified cholesterol in LDL were suggestively associated with an increased risk of LAS and with a better LTO.

Altered methylation pattern in EXOC4 is associated with stroke outcome: an epigenome-wide association study.

BACKGROUND AND PURPOSE: The neurological course after stroke is highly variable and is determined by demographic, clinical and genetic factors. However, other heritable factors such as epigenetic DNA methylation could play a role in neurological changes after stroke. METHODS: We performed a three-stage epigenome-wide association study to evaluate DNA methylation associated with the difference between the National Institutes of Health Stroke Scale (NIHSS) at baseline and at discharge (ΔNIHSS) in ischaemic stroke patients. DNA methylation data in the Discovery (n = 643) and Replication (n = 62) Cohorts were interrogated with the 450 K and EPIC BeadChip. Nominal CpG sites from the Discovery (p value < 10-06) were also evaluated in a meta-analysis of the Discovery and Replication cohorts, using a random-fixed effect model. Metabolic pathway enrichment was calculated with methylGSA. We integrated the methylation data with 1305 plasma protein expression levels measured by SOMAscan in 46 subjects and measured RNA expression with RT-PCR in a subgroup of 13 subjects. Specific cell-type methylation was assessed using EpiDISH. RESULTS: The meta-analysis revealed an epigenome-wide significant association in EXOC4 (p value = 8.4 × 10-08) and in MERTK (p value = 1.56 × 10-07). Only the methylation in EXOC4 was also associated in the Discovery and in the Replication Cohorts (p value = 1.14 × 10-06 and p value = 1.3 × 10-02, respectively). EXOC4 methylation negatively correlated with the long-term outcome (coefficient = - 4.91) and showed a tendency towards a decrease in EXOC4 expression (rho = - 0.469, p value = 0.091). Pathway enrichment from the meta-analysis revealed significant associations related to the endocytosis and deubiquitination processes. Seventy-nine plasma proteins were differentially expressed in association with EXOC4 methylation. Pathway analysis of these proteins showed an enrichment in natural killer (NK) cell activation. The cell-type methylation analysis in blood also revealed a differential methylation in NK cells. CONCLUSIONS: DNA methylation of EXOC4 is associated with a worse neurological course after stroke. The results indicate a potential modulation of pathways involving endocytosis and NK cells regulation.

DNA Methylation and Ischemic Stroke Risk: An Epigenome-Wide Association Study.

BACKGROUND: Ischemic stroke (IS) risk heritability is partly explained by genetics. Other heritable factors, such as epigenetics, could explain an unknown proportion of the IS risk. The objective of this study is to evaluate DNA methylation association with IS using epigenome-wide association studies (EWAS). METHODS: We performed a two-stage EWAS comprising 1,156 subjects. Differentially methylated positions (DMPs) and differentially methylated regions (DMRs) were assessed using the Infinium 450K and EPIC BeadChip in the discovery cohort (252 IS and 43 controls). Significant DMPs were replicated in an independent cohort (618 IS and 243 controls). Stroke subtype associations were also evaluated. Differentially methylated cell-type (DMCT) was analyzed in the replicated CpG sites using EpiDISH. We additionally performed pathway enrichment analysis and causality analysis with Mendelian randomization for the replicated CpG sites. RESULTS: A total of 957 CpG sites were epigenome-wide-significant (p ≤ 10-7) in the discovery cohort, being CpG sites in the top signals (logFC = 0.058, p = 2.35 × 10-22; logFC = 0.035, p = 3.22 × 10-22, respectively). ZFHX3 and MAP3K1 were among the most significant DMRs. In addition, 697 CpG sites were replicated considering Bonferroni-corrected p-values (p < 5.22 × 10-5). All the replicated DMPs were associated with risk of cardioembolic, atherothrombotic, and undetermined stroke. The DMCT analysis demonstrated that the significant associations were driven by natural killer cells. The pathway enrichment analysis showed overrepresentation of genes belonging to certain pathways including oxidative stress. ZFHX3 and MAP3K1 methylation was causally associated with specific stroke-subtype risk. CONCLUSION: Specific DNA methylation pattern is causally associated with IS risk. These results could be useful for specifically predicting stroke occurrence and could potentially be evaluated as therapeutic targets.

A Polygenic Risk Score Based on a Cardioembolic Stroke Multitrait Analysis Improves a Clinical Prediction Model for This Stroke Subtype.

Background: Occult atrial fibrillation (AF) is one of the major causes of embolic stroke of undetermined source (ESUS). Knowing the underlying etiology of an ESUS will reduce stroke recurrence and/or unnecessary use of anticoagulants. Understanding cardioembolic strokes (CES), whose main cause is AF, will provide tools to select patients who would benefit from anticoagulants among those with ESUS or AF. We aimed to discover novel loci associated with CES and create a polygenetic risk score (PRS) for a more efficient CES risk stratification. Methods: Multitrait analysis of GWAS (MTAG) was performed with MEGASTROKE-CES cohort (n = 362,661) and AF cohort (n = 1,030,836). We considered significant variants and replicated those variants with MTAG p-value < 5 × 10-8 influencing both traits (GWAS-pairwise) with a p-value < 0.05 in the original GWAS and in an independent cohort (n = 9,105). The PRS was created with PRSice-2 and evaluated in the independent cohort. Results: We found and replicated eleven loci associated with CES. Eight were novel loci. Seven of them had been previously associated with AF, namely, CAV1, ESR2, GORAB, IGF1R, NEURL1, WIPF1, and ZEB2. KIAA1755 locus had never been associated with CES/AF, leading its index variant to a missense change (R1045W). The PRS generated has been significantly associated with CES improving discrimination and patient reclassification of a model with age, sex, and hypertension. Conclusion: The loci found significantly associated with CES in the MTAG, together with the creation of a PRS that improves the predictive clinical models of CES, might help guide future clinical trials of anticoagulant therapy in patients with ESUS or AF.

Contribution of Common Genetic Variants to Risk of Early Onset Ischemic Stroke.

BACKGROUND AND OBJECTIVES: Current genome-wide association studies of ischemic stroke have focused primarily on late onset disease. As a complement to these studies, we sought to identifythe contribution of common genetic variants to risk of early onset ischemic stroke. METHODS: We performed a meta-analysis of genome-wide association studies of early onset stroke (EOS), ages 18-59, using individual level data or summary statistics in 16,730 cases and 599,237 non-stroke controls obtained across 48 different studies. We further compared effect sizes at associated loci between EOS and late onset stroke (LOS) and compared polygenic risk scores for venous thromboembolism between EOS and LOS. RESULTS: We observed genome-wide significant associations of EOS with two variants in ABO, a known stroke locus. These variants tag blood subgroups O1 and A1, and the effect sizes of both variants were significantly larger in EOS compared to LOS. The odds ratio (OR) for rs529565, tagging O1, 0.88 (95% CI: 0.85-0.91) in EOS vs 0.96 (95% CI: 0.92-1.00) in LOS, and the OR for rs635634, tagging A1, was 1.16 (1.11-1.21) for EOS vs 1.05 (0.99-1.11) in LOS; p-values for interaction = 0.001 and 0.005, respectively. Using polygenic risk scores, we observed that greater genetic risk for venous thromboembolism, another prothrombotic condition, was more strongly associated with EOS compared to LOS (p=0.008). DISCUSSION: The ABO locus, genetically predicted blood group A, and higher genetic propensity for venous thrombosis are more strongly associated with EOS than with LOS, supporting a stronger role of prothrombotic factors in EOS.

Pharmacogenetics studies in stroke patients treated with rtPA: a review of the most interesting findings.

Recombinant tissue-plasminogen activator (rtPA) is the only drug used during the acute phase of stroke. Despite its important benefits, a percentage of patients suffer symptomatic hemorrhagic transformations or a lack of early recanalization rates. These undesirable effects are associated with acute neurological and long-term functional deterioration. For the past 20 years, pharmacogenetic studies have tried to find the genetic risk factors associated with rtPA response. Most of these studies have used a gene-candidate strategy; however, recent genome-wide association studies have emerged indicating that genetic predisposition could modulate rtPA response. This review summarizes the most interesting findings in this field, including which genes and genetic variations are associated with hemorrhagic transformations and recanalization rates after thrombolytic therapy.