DNA methylation and stroke prognosis: an epigenome-wide association study.

BACKGROUND AND AIMS: Stroke is the leading cause of adult-onset disability. Although clinical factors influence stroke outcome, there is a significant variability among individuals that may be attributed to genetics and epigenetics, including DNA methylation (DNAm). We aimed to study the association between DNAm and stroke prognosis. METHODS AND RESULTS: To that aim, we conducted a two-phase study (discovery-replication and meta-analysis) in Caucasian patients with ischemic stroke from two independent centers (BasicMar [discovery, N = 316] and St. Pau [replication, N = 92]). Functional outcome was assessed using the modified Rankin Scale (mRS) at three months after stroke, being poor outcome defined as mRS > 2. DNAm was determined using the 450K and EPIC BeadChips in whole-blood samples collected within the first 24 h. We searched for differentially methylated positions (DMPs) in 370,344 CpGs, and candidates below p-value < 10-5 were subsequently tested in the replication cohort. We then meta-analyzed DMP results from both cohorts and used them to identify differentially methylated regions (DMRs). After doing the epigenome-wide association study, we found 29 DMPs at p-value < 10-5 and one of them was replicated: cg24391982, annotated to thrombospondin-2 (THBS2) gene (p-valuediscovery = 1.54·10-6; p-valuereplication = 9.17·10-4; p-valuemeta-analysis = 6.39·10-9). Besides, four DMRs were identified in patients with poor outcome annotated to zinc finger protein 57 homolog (ZFP57), Arachidonate 12-Lipoxygenase 12S Type (ALOX12), ABI Family Member 3 (ABI3) and Allantoicase (ALLC) genes (p-value < 1·10-9 in all cases). DISCUSSION: Patients with poor outcome showed a DMP at THBS2 and four DMRs annotated to ZFP57, ALOX12, ABI3 and ALLC genes. This suggests an association between stroke outcome and DNAm, which may help identify new stroke recovery mechanisms.

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.

Epigenome-wide association study identifies novel genes associated with ischemic stroke.

BACKGROUND: DNA methylation has previously been associated with ischemic stroke, but the specific genes and their functional roles in ischemic stroke remain to be determined. Here we aimed to identify differentially methylated genes that play a functional role in ischemic stroke in a Chinese population. RESULTS: Genome-wide DNA methylation assessed with the Illumina Methylation EPIC Array in a discovery sample including 80 Chinese adults (40 cases vs. 40 controls) found that patients with ischemic stroke were characterized by increased DNA methylation at six CpG loci (individually located at TRIM6, FLRT2, SOX1, SOX17, AGBL4, and FAM84A, respectively) and decreased DNA methylation at one additional locus (located at TLN2). Targeted bisulfite sequencing confirmed six of these differentially methylated probes in an independent Chinese population (853 cases vs. 918 controls), and one probe (located at TRIM6) was further verified in an external European cohort (207 cases vs. 83 controls). Experimental manipulation of DNA methylation in engineered human umbilical vein endothelial cells indicated that the identified differentially methylated probes located at TRIM6, TLN2, and FLRT2 genes may play a role in endothelial cell adhesion and atherosclerosis. CONCLUSIONS: Altered DNA methylation of the TRIM6, TLN2, and FLRT2 genes may play a functional role in ischemic stroke in Chinese populations.

Single-nucleus RNA-sequencing of autosomal dominant Alzheimer disease and risk variant carriers.

Genetic studies of Alzheimer disease (AD) have prioritized variants in genes related to the amyloid cascade, lipid metabolism, and neuroimmune modulation. However, the cell-specific effect of variants in these genes is not fully understood. Here, we perform single-nucleus RNA-sequencing (snRNA-seq) on nearly 300,000 nuclei from the parietal cortex of AD autosomal dominant (APP and PSEN1) and risk-modifying variant (APOE, TREM2 and MS4A) carriers. Within individual cell types, we capture genes commonly dysregulated across variant groups. However, specific transcriptional states are more prevalent within variant carriers. TREM2 oligodendrocytes show a dysregulated autophagy-lysosomal pathway, MS4A microglia have dysregulated complement cascade genes, and APOEε4 inhibitory neurons display signs of ferroptosis. All cell types have enriched states in autosomal dominant carriers. We leverage differential expression and single-nucleus ATAC-seq to map GWAS signals to effector cell types including the NCK2 signal to neurons in addition to the initially proposed microglia. Overall, our results provide insights into the transcriptional diversity resulting from AD genetic architecture and cellular heterogeneity. The data can be explored on the online browser ( http://web.hararilab.org/SNARE/ ).

Machine Learning Approximations to Predict Epigenetic Age Acceleration in Stroke Patients.

Age acceleration (Age-A) is a useful tool that is able to predict a broad range of health outcomes. It is necessary to determine DNA methylation levels to estimate it, and it is known that Age-A is influenced by environmental, lifestyle, and vascular risk factors (VRF). The aim of this study is to estimate the contribution of these easily measurable factors to Age-A in patients with cerebrovascular disease (CVD), using different machine learning (ML) approximations, and try to find a more accessible model able to predict Age-A. We studied a CVD cohort of 952 patients with information about VRF, lifestyle habits, and target organ damage. We estimated Age-A using Hannum's epigenetic clock, and trained six different models to predict Age-A: a conventional linear regression model, four ML models (elastic net regression (EN), K-Nearest neighbors, random forest, and support vector machine models), and one deep learning approximation (multilayer perceptron (MLP) model). The best-performing models were EN and MLP; although, the predictive capability was modest (R2 0.358 and 0.378, respectively). In conclusion, our results support the influence of these factors on Age-A; although, they were not enough to explain most of its variability.

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.

Intracranial Aneurysm Classifier Using Phenotypic Factors: An International Pooled Analysis.

Intracranial aneurysms (IAs) are usually asymptomatic with a low risk of rupture, but consequences of aneurysmal subarachnoid hemorrhage (aSAH) are severe. Identifying IAs at risk of rupture has important clinical and socio-economic consequences. The goal of this study was to assess the effect of patient and IA characteristics on the likelihood of IA being diagnosed incidentally versus ruptured. Patients were recruited at 21 international centers. Seven phenotypic patient characteristics and three IA characteristics were recorded. The analyzed cohort included 7992 patients. Multivariate analysis demonstrated that: (1) IA location is the strongest factor associated with IA rupture status at diagnosis; (2) Risk factor awareness (hypertension, smoking) increases the likelihood of being diagnosed with unruptured IA; (3) Patients with ruptured IAs in high-risk locations tend to be older, and their IAs are smaller; (4) Smokers with ruptured IAs tend to be younger, and their IAs are larger; (5) Female patients with ruptured IAs tend to be older, and their IAs are smaller; (6) IA size and age at rupture correlate. The assessment of associations regarding patient and IA characteristics with IA rupture allows us to refine IA disease models and provide data to develop risk instruments for clinicians to support personalized decision-making.

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.

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.

Epigenetic Clock Explains White Matter Hyperintensity Burden Irrespective of Chronological Age.

In this manuscript we studied the relationship between WMH and biological age (B-age) in patients with acute stroke. We included in this study 247 patients with acute stroke recruited at Hospital del Mar having both epigenetic (DNA methylation) and magnetic resonance imaging data. WMH were measured using a semi-automated method. B-age was calculated using two widely used methods: the Hannum and Horvath formulas. We used multiple linear regression models to interrogate the role of B-age on WMH volume after adjusting for chronological age (C-age) and other covariables. Average C-age of the sample was 68.4 (±11.8) and we observed a relatively high median WMH volume (median = 8.8 cm3, Q1-Q3 = 4.05-18.8). After adjusting for potential confounders, we observed a significant effect of B-ageHannum on WMH volume (ßHannum = 0.023, p-value = 0.029) independently of C-age, which remained significant (ßC-age = 0.021, p-value = 0.036). Finally, we performed a mediation analysis, which allowed us to discover that 42.7% of the effect of C-age on WMH is mediated by B-ageHannum. On the other hand, B-ageHoarvath showed no significant associations with WMH after being adjusted for C-age. In conclusion, we show for the first time that biological age, measured through DNA methylation, contributes substantially to explain WMH volumetric burden irrespective of chronological age.

Genomic and phenotypic insights from an atlas of genetic effects on DNA methylation.

Characterizing genetic influences on DNA methylation (DNAm) provides an opportunity to understand mechanisms underpinning gene regulation and disease. In the present study, we describe results of DNAm quantitative trait locus (mQTL) analyses on 32,851 participants, identifying genetic variants associated with DNAm at 420,509 DNAm sites in blood. We present a database of >270,000 independent mQTLs, of which 8.5% comprise long-range (trans) associations. Identified mQTL associations explain 15-17% of the additive genetic variance of DNAm. We show that the genetic architecture of DNAm levels is highly polygenic. Using shared genetic control between distal DNAm sites, we constructed networks, identifying 405 discrete genomic communities enriched for genomic annotations and complex traits. Shared genetic variants are associated with both DNAm levels and complex diseases, but only in a minority of cases do these associations reflect causal relationships from DNAm to trait or vice versa, indicating a more complex genotype-phenotype map than previously anticipated.

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 × 10-8) located in the RP11-362K2.2:RP11-767I20.1 gene and a suggestive variant (rs13297983: beta 0.07, p-value 6.10 × 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-ß, tau protein, white matter microstructure, inflammation, and matrix metalloproteinases.

The copy number variation and stroke (CaNVAS) risk and outcome study.

BACKGROUND AND PURPOSE: The role of copy number variation (CNV) variation in stroke susceptibility and outcome has yet to be explored. The Copy Number Variation and Stroke (CaNVAS) Risk and Outcome study addresses this knowledge gap. METHODS: Over 24,500 well-phenotyped IS cases, including IS subtypes, and over 43,500 controls have been identified, all with readily available genotyping on GWAS and exome arrays, with case measures of stroke outcome. To evaluate CNV-associated stroke risk and stroke outcome it is planned to: 1) perform Risk Discovery using several analytic approaches to identify CNVs that are associated with the risk of IS and its subtypes, across the age-, sex- and ethnicity-spectrums; 2) perform Risk Replication and Extension to determine whether the identified stroke-associated CNVs replicate in other ethnically diverse datasets and use biomarker data (e.g. methylation, proteomic, RNA, miRNA, etc.) to evaluate how the identified CNVs exert their effects on stroke risk, and lastly; 3) perform outcome-based Replication and Extension analyses of recent findings demonstrating an inverse relationship between CNV burden and stroke outcome at 3 months (mRS), and then determine the key CNV drivers responsible for these associations using existing biomarker data. RESULTS: The results of an initial CNV evaluation of 50 samples from each participating dataset are presented demonstrating that the existing GWAS and exome chip data are excellent for the planned CNV analyses. Further, some samples will require additional considerations for analysis, however such samples can readily be identified, as demonstrated by a sample demonstrating clonal mosaicism. CONCLUSION: The CaNVAS study will cost-effectively leverage the numerous advantages of using existing case-control data sets, exploring the relationships between CNV and IS and its subtypes, and outcome at 3 months, in both men and women, in those of African and European-Caucasian descent, this, across the entire adult-age spectrum.

Defining Minor Intracerebral Hemorrhage.

BACKGROUND AND PURPOSE: The minor stroke concept has not been analyzed in intracerebral hemorrhage (ICH) patients. Our purpose was to determine the optimal cut point on the NIH Stroke Scale (NIHSS) for defining a minor ICH (mICH) in patients with primary ICH. METHODS: An ICH was considered minor if associated with a favorable 3-month outcome (modified Rankin Scale score ≤2). For supratentorial ICH, the discovery cohort consisted of 478 patients prospectively admitted at University Hospital del Mar. Association between NIHSS at admission and 3-month outcome was evaluated with area under the curve-receiver operating characteristics (AUC-ROC) and Youden's index to identify the optimal NIHSS cutoff point to define mICH. External validation was performed in a cohort of 242 supratentorial ICH patients from University Hospital Sant Pau. For infratentorial location, patients from both hospitals (n = 85) were analyzed together. RESULTS: The best -NIHSS cutoff point defining supratentorial-mICH was 6 (AUC-ROC = 0.815 [0.774-0.857] in the discovery cohort and AUC-ROC = 0.819 [0.756-0.882] in the external validation cohort). For infratentorial ICH, the best cutoff point was 4 (AUC-ROC = 0.771 [0.664-0.877]). Using these cutoff points, 40.5% of all primary ICH cases were mICH. Of these, 70.2% were living independently at 3-month follow-up (72% for supratentorial ICH and 56.1% for infratentorial ICH) and 6.5% had died (5.3% for supratentorial ICH, and 14.6% for infratentorial ICH). For patients identified as non-mICH, good 3-month outcome was observed in 11.3% of cases; mortality was 51%. CONCLUSIONS: The definition of mICH using the NIHSS cutoff point of 6 for supratentorial ICH and 4 for infratentorial ICH is useful to identify good outcome in ICH patients.

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.

Short- and long-term outcome of patients with aneurysmal subarachnoid hemorrhage.

OBJECTIVE: To describe short-term and 5-year rates of mortality and poor outcome in patients with spontaneous aneurysmal subarachnoid hemorrhage (aSAH) who received repair treatment. METHODS: In this prospective observational study, mortality and poor outcome (modified Rankin Scale score 3-6) were analyzed in 311 patients with aSAH at 3 months, 1 year, and 5 years follow-up. Sensitivity analysis was performed according to treatment modality. In-hospital and 5-year complications were analyzed. RESULTS: Of 476 consecutive patients with spontaneous subarachnoid hemorrhage, 347 patients (72.9%) had aSAH. Of these, 311 (89.6%) were treated (242 endovascular, 69 neurosurgical), with a mean follow-up of 43.4 months (range, 1 to 145). Three-month, 1-year, and 5-year mortality was 18.4%, 22.9%, and 29.0%, and poor outcome was observed in 42.3%, 36.0%, and 36.0%, respectively. Adjusted poor outcome was lower in endovascular than in neurosurgical treatment at 3 months (odds ratio [OR] 0.36 [95% confidence interval [CI] 0.18-0.74]), with an absolute difference of 15.8% (number needed to treat = 6.3), and at 1 year (OR = 0.40 [95% CI 0.20-0.81]), with an absolute difference of 15.9% (number needed to treat = 6.3). Complications did not differ between the 2 procedures. However, mechanical ventilation was less frequent with the endovascular technique (OR 0.67 [95% CI 0.54-0.84]). CONCLUSIONS: Patients with aSAH treated according to current guidelines had a short-term mortality of 18.4% and 5-year mortality of 29%. The majority (64.0%) of patients remained alive without disabilities at 5-year follow-up. Patients prioritized to endovascular treatment had better outcomes than those referred to neurosurgery because endovascular coiling was not feasible.

DNA methylation of MMPs and TIMPs in atherothrombosis process in carotid plaques and blood tissues.

BACKGROUND AND PURPOSE: Polymorphisms and serum levels of Matrix Metalloproteinases (MMP) and Tissue Inhibitor of Metalloproteinases (TIMP) have been studied with regard to atheromatous plaques and ischemic stroke, while no studies of DNA methylation (DNAm) patterns of MMP or TIMP have been performed to that end. Here, we evaluate DNAm levels of the MMP and TIMP gene families in human carotid plaques and blood samples of atherothrombotic stroke patients. METHODS: We profiled the DNAm status of stable and ulcerated atherosclerotic plaques obtained as pair sets from three patients who underwent carotid endarterectomy surgery. We selected 415 CpG sites, mapping into MMPs and TIMPs genes for further study. Secondly, the statistically associated CpG sites were analyzed in blood samples from two separate atherothrombotic stroke cohorts (total sample size = 307), ischemic stroke-cohort 1 (ISC-1): 37 atherothrombotic patients and 6 controls, ischemic stroke-cohort 2 (ISC-2): 80 atherothrombotic patients and 184 controls. DNAm levels from plaque tissue and blood samples were evaluated using a high-density microarray Infinium, HumanMethylation450 BeadChip and Infinium MethylationEPIC BeadChip. RESULTS: Three CpG sites were statistically significantly associated with unstable plaque portions; cg02969624, q-value = 0.035 (TIMP2), and cg04316754, q-value = 0.037 (MMP24) were hypermethylated, while cg24211657 q-value = 0.035 (TIMP2) was hypomethylated. Association of cg04316754 (MMP24) methylation levels with atherothrombotic risk was also observed in blood tissue: ISC-1 p-values = 0.03, ISC-2 p-value = 1.9 × 10-04. CONCLUSIONS: The results suggest different DNAm status of MMP24 between stable and unstable atherothrombotic carotid plaques, and between atherothrombotic stroke and controls in blood samples.

Identification of 20 novel loci associated with ischaemic stroke. Epigenome-wide association study.

DNA methylation is dynamic, varies throughout the life course, and its levels are influenced by lifestyle and environmental factors, as well as by genetic variation. The leading genetic variants at stroke risk loci identified to date explain roughly 1-2% of stroke heritability. Most of these single nucleotide polymorphisms are situated within a regulatory sequence marked by DNase I hypersensitivity sites, which would indicate involvement of an epigenetic mechanism. To detect epigenetic variants associated with stroke occurrence and stroke subtypes. A two-stage case-control epigenome-wide association study was designed. The discovery sample with 401 samples included 218 ischaemic stroke (IS) patients, assessed at Hospital del Mar (Barcelona, Spain) and 183 controls from the REGICOR cohort. In two independent samples (N = 226 and N = 166), we replicated 22 CpG sites differentially methylated in IS in 21 loci, including 2 CpGs in locus ZFHX3, which includes known genetic variants associated with stroke. The pathways associated with these loci are inflammation and angiogenesis. The meta-analysis identified 384 differentially methylated CpGs, including loci of known stroke and vascular risk genetic variants, enriched by loci involved in lipid metabolism, adipogenesis, circadian clock, and glycolysis pathways. We identified a set of 22 CpGs in 21 loci associated with IS. Our analysis suggests that DNA methylation changes may contribute to orchestrating gene expression that contributes to IS.

Validation of a clinical-genetics score to predict hemorrhagic transformations after rtPA.

OBJECTIVE: To validate the Genot-PA score, a clinical-genetic logistic regression score that stratifies the thrombolytic therapy safety, in a new cohort of patients with stroke. METHODS: We enrolled 1,482 recombinant tissue plasminogen activator (rtPA)-treated patients with stroke in Spain and Finland from 2003 to 2016. Cohorts were analyzed on the basis of ethnicity and therapy: Spanish patients treated with IV rtPA within 4.5 hours of onset (cohort A and B) or rtPA in combination with mechanical thrombectomy within 6 hours of onset (cohort C) and Finnish participants treated with IV rtPA within 4.5 hours of onset (cohort D). The Genot-PA score was calculated, and hemorrhagic transformation (HT) and parenchymal hematoma (PH) risks were determined for each score stratum. RESULTS: Genot-PA score was tested in 1,324 (cohort A, n = 726; B, n = 334; C, n = 54; and D, n = 210) patients who had enough information to complete the score. Of these, 213 (16.1%) participants developed HT and 85 (6.4%) developed PH. In cohorts A, B, and D, HT occurrence was predicted by the score (p = 2.02 × 10-6, p = 0.023, p = 0.033); PH prediction was associated in cohorts A through C (p = 0.012, p = 0.034, p = 5.32 × 10-4). Increased frequency of PH events from the lowest to the highest risk group was found (cohort A 4%-15.7%, cohort B 1.5%-18.2%, cohort C 0%-100%). The best odds ratio for PH prediction in the highest-risk group was obtained in cohort A (odds ratio 5.16, 95% confidence interval 1.46-18.08, p = 0.009). CONCLUSION: The Genot-PA score predicts HT in patients with stroke treated with IV rtPA. Moreover, in an exploratory study, the score was associated with PH risk in mechanical thrombectomy-treated patients.

Long-Term Stroke Recurrence after Transient Ischemic Attack: Implications of Etiology.

BACKGROUND AND PURPOSE: To analyze long-term stroke recurrence (SR) characteristics after transient ischemic attack (TIA) according to initial etiological classification. METHODS: A prospective cohort of 706 TIA patients was followed up in a single tertiary stroke center. Endpoint was SR. Etiologic subgroup was determined according to the evidence-based causative classification system. Location of TIA and SR was recorded as right, left, or posterior territory. Disability stroke recurrence (DSR) was defined as modified Rankin Scale (mRS) score >1 or a onepoint increase in those with previous mRS >1 at 3-month follow-up. RESULTS: During a follow-up of 3,493 patient-years (mean follow-up of 58.9±35.9 months), total SR was 125 (17.7%), corresponding to 3.6 recurrences per 100 patient-years. The etiology subgroups with a higher risk of SR were the unclassified (more than one cause) and large-artery atherosclerosis (LAA) categories. Of the SR cases, 88 (70.4%) had the same etiology as the index TIA; again, LAA etiology was the most frequent (83.9%). Notably, cardioaortic embolism was the most frequent cause (62.5%) of SR in the subgroup of 24 patients with undetermined TIA. Overall, SR occurred in the same territory in 74 of 125 patients (59.2%), with significant differences between etiological TIA subgroups (P=0.015). Eighty-two of 125 (65.6%) with SR had DSR, without differences between etiologies (P=0.453). CONCLUSIONS: SR occurred mainly with the same etiology and location as initial TIA, although undetermined TIA was associated with a high proportion of cardioaortic embolism SR. More than half of the recurrences caused some disability, regardless of etiology.