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 (Delta NIHSS) 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 x 10(-08)) and in MERTK (p value = 1.56 x 10(-07)). Only the methylation in EXOC4 was also associated in the Discovery and in the Replication Cohorts (p value = 1.14 x 10(-06) and p value = 1.3 x 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

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 x 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 x 10(-8)) were characterized by in silica 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 x 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 x 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 silica 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.Peer reviewe

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

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 ) in the discovery cohort, being CpG sites in the top signals (logFC = 0.058, p = 2.35 × 10 ; logFC = 0.035, p = 3.22 × 10 , 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 ). 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.The EPIGENESIS project (Carlos III Institute—PI17/02089, Marató TV3 and Fundació MútuaTerrassa), the MAESTRO project—PI18/01338 (Carlos III Institute), the iBioStroke project (Eranet-Neuron, European research grants), the EPINEXO project—PI20/00678 (Carlos III Institute), the SEDMAN Study (Boehringer Ingelheim), the APHAS project (Pfizer/Bristol-Myers Squibb), and the European Regional Development Fund (ERDF)/Fondo Europeo de Desarrollo Regional (FEDER) and 2017SGR-1427 (AGAUR). I.F.-C is the recipient of a research contract from the Miguel Servet Program (CP12/03298) from the Carlos III Institute. J.C.-M. is supported by an AGAUR contract (Agència de Gestió d'Ajuts Universitaris i de Recerca; FI_DGR 2019, grant number 2019_FI_B 00853) co-financed by Fons Social Europeu (FSE). M.L. is supported by a PFIS contract (Contratos Predoctorales de Formación en Investigación en Salud): FI19/00309. C.G.-F is supported by a Sara Borrell contract (CD20/00043) from the Carlos III Institute and Fondo Europeo de Desarrollo Regional (ISCIII-FEDER). E.M. is supported by a Río Hortega contract (CM18/00198) from the Carlos III Institute

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

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. 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. 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 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

RP11-362K2.2 : RP11-767I20.1 Genetic Variation Is Associated with Post-Reperfusion Therapy Parenchymal Hematoma. A GWAS Meta-Analysis

Stroke is one of the most common causes of death and disability. Reperfusion therapies are the only treatment available during the acute phase of stroke. Due to recent clinical trials, these therapies may increase their frequency of use by extending the time-window administration, which may lead to an increase in complications such as hemorrhagic transformation, with parenchymal hematoma (PH) being the more severe subtype, associated with higher mortality and disability rates. Our aim was to find genetic risk factors associated with PH, as that could provide molecular targets/pathways for their prevention/treatment and study its genetic correlations to find traits sharing genetic background. We performed a GWAS and meta-analysis, following standard quality controls and association analysis (fastGWAS), adjusting age, NIHSS, and principal components. FUMA was used to annotate, prioritize, visualize, and interpret the meta-analysis results. The total number of patients in the meta-analysis was 2034 (216 cases and 1818 controls). We found rs79770152 having a genome-wide significant association (beta 0.09, p-value 3.90 x 10(-8)) located in the RP11-362K2.2:RP11-767I20.1 gene and a suggestive variant (rs13297983: beta 0.07, p-value 6.10 x 10(-8)) located in PCSK5 associated with PH occurrence. The genetic correlation showed a shared genetic background of PH with Alzheimer's disease and white matter hyperintensities. In addition, genes containing the ten most significant associations have been related to aggregated amyloid-beta, tau protein, white matter microstructure, inflammation, and matrix metalloproteinases.Peer reviewe

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 NIH stroke scale score (NIHSS) from baseline to 24 hours (baseline – 24h = ΔNIHSS(6–24h)), to examine its relevance to AIS mechanisms and long-term outcomes. METHODS: Patients with NIHSS prospectively recorded within 6h after onset and then 24h later were enrolled in the GENISIS (Genetics of Early Neurological InStability after Ischemic Stroke) study. Stepwise linear regression determined variables that independently influenced ΔNIHSS(6–24h). In a subcohort of tPA-treated patients with large vessel occlusion (LVO), the influence of early sustained recanalization and hemorrhagic transformation (HT) on ΔNIHSS(6–24h) was examined. Finally, the association of ΔNIHSS(6–24h) with 90-day favorable outcomes (modified Rankin scale score 0–2) was assessed. Independent analysis was performed using data from the two NINDS tPA stroke trials. RESULTS: For 2555 AIS patients, median baseline NIHSS was 9 (IQR 4–16) and median ΔNIHSS(6–24h) was 2 (IQR 0–5). In a multivariable model, baseline NIHSS, tPA treatment, age, glucose, site and systolic blood pressure independently predicted ΔNIHSS(6–24h) (R(2)=0.15). In the LVO subcohort, early sustained recanalization and HT increased the explained variance (R(2)=0.27), but much of the variance remained unexplained. ΔNIHSS(6–24h) had significant and independent association with 90-day favorable outcome. For the subjects in the two NINDS tPA trials, ΔNIHSS(3–24h) was similarly associated with 90-day outcomes. CONCLUSIONS: The dynamic phenotype, ΔNIHSS(6–24h), captures both explained and unexplained mechanisms involved in AIS, and is significantly and independently associated with long-term outcomes. Thus, ΔNIHSS(6–24h) promises to be an easily obtainable and meaningful quantitative phenotype for large-scale genomic studies of AIS