Genome-Wide Association Study Identifies First Locus Associated with Susceptibility to Cerebral Venous Thrombosis.

OBJECTIVE: Cerebral venous thrombosis (CVT) is an uncommon form of stroke affecting mostly young individuals. Although genetic factors are thought to play a role in this cerebrovascular condition, its genetic etiology is not well understood. METHODS: A genome-wide association study was performed to identify genetic variants influencing susceptibility to CVT. A 2-stage genome-wide study was undertaken in 882 Europeans diagnosed with CVT and 1,205 ethnicity-matched control subjects divided into discovery and independent replication datasets. RESULTS: In the overall case-control cohort, we identified highly significant associations with 37 single nucleotide polymorphisms (SNPs) within the 9q34.2 region. The strongest association was with rs8176645 (combined p = 9.15 × 10-24 ; odds ratio [OR] = 2.01, 95% confidence interval [CI] = 1.76-2.31). The discovery set findings were validated across an independent European cohort. Genetic risk score for this 9q34.2 region increases CVT risk by a pooled estimate OR = 2.65 (95% CI = 2.21-3.20, p = 2.00 × 10-16 ). SNPs within this region were in strong linkage disequilibrium (LD) with coding regions of the ABO gene. The ABO blood group was determined using allele combination of SNPs rs8176746 and rs8176645. Blood groups A, B, or AB, were at 2.85 times (95% CI = 2.32-3.52, p = 2.00 × 10-16 ) increased risk of CVT compared with individuals with blood group O. INTERPRETATION: We present the first chromosomal region to robustly associate with a genetic susceptibility to CVT. This region more than doubles the likelihood of CVT, a risk greater than any previously identified thrombophilia genetic risk marker. That the identified variant is in strong LD with the coding region of the ABO gene with differences in blood group prevalence provides important new insights into the pathophysiology of CVT. ANN NEUROL 2021;90:777-788.

Publisher Correction: Multiancestry genome-wide association study of 520,000 subjects identifies 32 loci associated with stroke and stroke subtypes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Non-genetic and genetic risk factors for adult cerebral venous thrombosis.

INTRODUCTION: A wide variety of non-genetic and genetic factors have been shown to associate with increased risk for cerebral venous thrombosis (CVT). However, there is a paucity of risk factor data and conclusions about their impact are often conflicting. Herein, we quantified the associations of non-genetic and genetic risk factors for CVT in adults. MATERIALS AND METHODS: Electronic databases were searched up to January 2017. Meta-analyses were performed (RevMan v5.3) to determine pooled odds ratios (ORs and 95% CIs) for risk factors, interstudy heterogeneity and publication bias. RESULTS: Twenty non-genetic (n = 2314) and 33 genetic (n = 2117) studies up to January 2017 met the selection criteria. For non-genetic factors, CVT risk increased in the presence of glucocorticosteroid therapy by 18.3-fold (3.3-102.6), alcohol consumption 2.7-fold (1.8-3.9), infection 7.5-fold (2.6-21.6), surgery 9.6-fold (1.1-83.5), hypercholesterolaemia 2.4-fold (1.3-4.4), hyperhomocysteinaemia 3.1-fold (2.1-4.6), antiphospholipid antibodies 7.0-fold (2.1-23.6), autoimmune diseases 5.6-fold (2.3-13.6), anaemia 4.0-fold (2.1-7.9), malignancy 3.2-fold (1.4-7.1) and pregnancy/puerperium 11.4-fold (5.7-24.3). Smoking, hypertension and diabetes did not associate with CVT risk. For genetic factors, CVT risk increased in the presence of factor V Leiden (G1691A) by 2.5-fold (1.9-3.3), protein C deficiency 10.7-fold (3.1-37.7), protein S deficiency 5.7-fold (1.4-22.4), antithrombin deficiency 3.8-fold (1.0-13.8), prothrombin (G20210A) 5.5-fold (4.0-7.27) and TAFI gene variant (C1040T) 1.6-fold (1.0-2.4). Prothrombin G20210A and factor V Leiden polymorphisms tended to have higher ORs for CVT than for ischaemic stroke. CONCLUSIONS: We provide quantitative data supporting a strong basis for genetic and non-genetic risk factors in CVT. Its genetic liability seems higher when compared with sporadic ischaemic stroke.

Multiancestry genome-wide association study of 520,000 subjects identifies 32 loci associated with stroke and stroke subtypes.

Stroke has multiple etiologies, but the underlying genes and pathways are largely unknown. We conducted a multiancestry genome-wide-association meta-analysis in 521,612 individuals (67,162 cases and 454,450 controls) and discovered 22 new stroke risk loci, bringing the total to 32. We further found shared genetic variation with related vascular traits, including blood pressure, cardiac traits, and venous thromboembolism, at individual loci (n = 18), and using genetic risk scores and linkage-disequilibrium-score regression. Several loci exhibited distinct association and pleiotropy patterns for etiological stroke subtypes. Eleven new susceptibility loci indicate mechanisms not previously implicated in stroke pathophysiology, with prioritization of risk variants and genes accomplished through bioinformatics analyses using extensive functional datasets. Stroke risk loci were significantly enriched in drug targets for antithrombotic therapy.

Meta-Analysis of Therapeutic Hypothermia for Traumatic Brain Injury in Adult and Pediatric Patients.

OBJECTIVE: Therapeutic hypothermia has been used to attenuate the effects of traumatic brain injuries. However, the required degree of hypothermia, length of its use, and its timing are uncertain. We undertook a comprehensive meta-analysis to quantify benefits of hypothermia therapy for traumatic brain injuries in adults and children by analyzing mortality rates, neurologic outcomes, and adverse effects. DATA SOURCES: Electronic databases PubMed, Google Scholar, Web of Science, Cochrane Central Register of Controlled Trials, and ClinicalTrials.gov and manual searches of studies were conducted for relevant publications up until February 2016. STUDY SELECTION: Forty-one studies in adults (n = 3,109; age range, 18-81 yr) and eight studies in children (n = 454; age range, 3 mo to 18 yr) met eligibility criteria. DATA EXTRACTION: Baseline patient characteristics, enrollment time, methodology of cooling, target temperature, duration of hypothermia, and rewarming protocols were extracted. DATA SYNTHESIS: Risk ratios with 95% CIs were calculated. Compared with adults who were kept normothermic, those who underwent therapeutic hypothermia were associated with 18% reduction in mortality (risk ratio, 0.82; 95% CI, 0.70-0.96; p = 0.01) and a 35% improvement in neurologic outcome (risk ratio, 1.35; 95% CI, 1.18-1.54; p < 0.00001). The optimal management strategy for adult patients included cooling patients to a minimum of 33°C for 72 hours, followed by spontaneous, natural rewarming. In contrast, adverse outcomes were observed in children who underwent hypothermic treatment with a 66% increase in mortality (risk ratio, 1.66; 95% CI, 1.06-2.59; p = 0.03) and a marginal deterioration of neurologic outcome (risk ratio, 0.90; 95% CI, 0.80-1.01; p = 0.06). CONCLUSIONS: Therapeutic hypothermia is likely a beneficial treatment following traumatic brain injuries in adults but cannot be recommended in children.

Genetic variation at 16q24.2 is associated with small vessel stroke.

OBJECTIVE: Genome-wide association studies (GWAS) have been successful at identifying associations with stroke and stroke subtypes, but have not yet identified any associations solely with small vessel stroke (SVS). SVS comprises one quarter of all ischemic stroke and is a major manifestation of cerebral small vessel disease, the primary cause of vascular cognitive impairment. Studies across neurological traits have shown that younger-onset cases have an increased genetic burden. We leveraged this increased genetic burden by performing an age-at-onset informed GWAS meta-analysis, including a large younger-onset SVS population, to identify novel associations with stroke. METHODS: We used a three-stage age-at-onset informed GWAS to identify novel genetic variants associated with stroke. On identifying a novel locus associated with SVS, we assessed its influence on other small vessel disease phenotypes, as well as on messenger RNA (mRNA) expression of nearby genes, and on DNA methylation of nearby CpG sites in whole blood and in the fetal brain. RESULTS: We identified an association with SVS in 4,203 cases and 50,728 controls on chromosome 16q24.2 (odds ratio [OR; 95% confidence interval {CI}] = 1.16 [1.10-1.22]; p = 3.2 × 10-9 ). The lead single-nucleotide polymorphism (rs12445022) was also associated with cerebral white matter hyperintensities (OR [95% CI] = 1.10 [1.05-1.16]; p = 5.3 × 10-5 ; N = 3,670), but not intracerebral hemorrhage (OR [95% CI] = 0.97 [0.84-1.12]; p = 0.71; 1,545 cases, 1,481 controls). rs12445022 is associated with mRNA expression of ZCCHC14 in arterial tissues (p = 9.4 × 10-7 ) and DNA methylation at probe cg16596957 in whole blood (p = 5.3 × 10-6 ). INTERPRETATION: 16q24.2 is associated with SVS. Associations of the locus with expression of ZCCHC14 and DNA methylation suggest the locus acts through changes to regulatory elements. Ann Neurol 2017;81:383-394.

Towards the genetic basis of cerebral venous thrombosis-the BEAST Consortium: a study protocol.

INTRODUCTION: Cerebral venous thrombosis (CVT) is a rare cerebrovascular condition accounting for <1% of all stroke cases and mainly affects young adults. Its genetic aetiology is not clearly elucidated. METHODS AND ANALYSIS: To better understand the genetic basis of CVT, we have established an international biobank of CVT cases, Biorepository to Establish the Aetiology of Sinovenous Thrombosis (BEAST) which aims to recruit highly phenotyped cases initially of European descent and later from other populations. To date we have recruited 745 CVT cases from 12 research centres. As an initial step, the consortium plans to undertake a genome-wide association analysis of CVT using the Illumina Infinium HumanCoreExome BeadChip to assess the association and impact of common and low-frequency genetic variants on CVT risk by using a case-control study design. Replication will be performed to confirm putative findings. Furthermore, we aim to identify interactions of genetic variants with several environmental and comorbidity factors which will likely contribute to improve the understanding of the biological mechanisms underlying this complex disease. ETHICS AND DISSEMINATION: BEAST meets all ethical standards set by local institutional review boards for each of the participating sites. The research outcomes will be published in international peer-reviewed open-access journals with high impact and visibility. The results will be presented at national and international meetings to highlight the contributions into improving the understanding of the mechanisms underlying this uncommon but important disease. This international DNA repository will become an important resource for investigators in the field of haematological and vascular disorders.

Low-frequency and common genetic variation in ischemic stroke: The METASTROKE collaboration.

OBJECTIVE: To investigate the influence of common and low-frequency genetic variants on the risk of ischemic stroke (all IS) and etiologic stroke subtypes. METHODS: We meta-analyzed 12 individual genome-wide association studies comprising 10,307 cases and 19,326 controls imputed to the 1000 Genomes (1 KG) phase I reference panel. We selected variants showing the highest degree of association (p < 1E-5) in the discovery phase for replication in Caucasian (13,435 cases and 29,269 controls) and South Asian (2,385 cases and 5,193 controls) samples followed by a transethnic meta-analysis. We further investigated the p value distribution for different bins of allele frequencies for all IS and stroke subtypes. RESULTS: We showed genome-wide significance for 4 loci: ABO for all IS, HDAC9 for large vessel disease (LVD), and both PITX2 and ZFHX3 for cardioembolic stroke (CE). We further refined the association peaks for ABO and PITX2. Analyzing different allele frequency bins, we showed significant enrichment in low-frequency variants (allele frequency <5%) for both LVD and small vessel disease, and an enrichment of higher frequency variants (allele frequency 10% and 30%) for CE (all p < 1E-5). CONCLUSIONS: Our findings suggest that the missing heritability in IS subtypes can in part be attributed to low-frequency and rare variants. Larger sample sizes are needed to identify the variants associated with all IS and stroke subtypes.

Candidate-gene analysis of white matter hyperintensities on neuroimaging.

BACKGROUND: White matter hyperintensities (WMH) are a common radiographic finding and may be a useful endophenotype for small vessel diseases. Given high heritability of WMH, we hypothesised that certain genotypes may predispose individuals to these lesions and consequently, to an increased risk of stroke, dementia and death. We performed a meta-analysis of studies investigating candidate genes and WMH to elucidate the genetic susceptibility to WMH and tested associated variants in a new independent WMH cohort. We assessed a causal relationship of WMH to methylene tetrahydrofolate reductase (MTHFR). METHODS: Database searches through March 2014 were undertaken and studies investigating candidate genes in WMH were assessed. Associated variants were tested in a new independent ischaemic cohort of 1202 WMH patients. Mendelian randomization was undertaken to assess a causal relationship between WMH and MTHFR. RESULTS: We identified 43 case-control studies interrogating eight polymorphisms in seven genes covering 6,314 WMH cases and 15,461 controls. Fixed-effects meta-analysis found that the C-allele containing genotypes of the aldosterone synthase CYP11B2 T(-344)C gene polymorphism were associated with a decreased risk of WMH (OR=0.61; 95% CI, 0.44 to 0.84; p=0.003). Using mendelian randomisation the association among MTHFR C677T, homocysteine levels and WMH, approached, but did not reach, significance (expected OR=1.75; 95% CI, 0.90-3.41; observed OR=1.68; 95% CI, 0.97-2.94). Neither CYP11B2 T(-344)C nor MTHFR C677T were significantly associated when tested in a new independent cohort of 1202 patients with WMH. CONCLUSIONS: There is a genetic basis to WMH but anonymous genome wide and exome studies are more likely to provide novel loci of interest.

Genetic overlap between diagnostic subtypes of ischemic stroke.

BACKGROUND AND PURPOSE: Despite moderate heritability, the phenotypic heterogeneity of ischemic stroke has hampered gene discovery, motivating analyses of diagnostic subtypes with reduced sample sizes. We assessed evidence for a shared genetic basis among the 3 major subtypes: large artery atherosclerosis (LAA), cardioembolism, and small vessel disease (SVD), to inform potential cross-subtype analyses. METHODS: Analyses used genome-wide summary data for 12 389 ischemic stroke cases (including 2167 LAA, 2405 cardioembolism, and 1854 SVD) and 62 004 controls from the Metastroke consortium. For 4561 cases and 7094 controls, individual-level genotype data were also available. Genetic correlations between subtypes were estimated using linear mixed models and polygenic profile scores. Meta-analysis of a combined LAA-SVD phenotype (4021 cases and 51 976 controls) was performed to identify shared risk alleles. RESULTS: High genetic correlation was identified between LAA and SVD using linear mixed models (rg=0.96, SE=0.47, P=9×10(-4)) and profile scores (rg=0.72; 95% confidence interval, 0.52-0.93). Between LAA and cardioembolism and SVD and cardioembolism, correlation was moderate using linear mixed models but not significantly different from zero for profile scoring. Joint meta-analysis of LAA and SVD identified strong association (P=1×10(-7)) for single nucleotide polymorphisms near the opioid receptor µ1 (OPRM1) gene. CONCLUSIONS: Our results suggest that LAA and SVD, which have been hitherto treated as genetically distinct, may share a substantial genetic component. Combined analyses of LAA and SVD may increase power to identify small-effect alleles influencing shared pathophysiological processes.

Effect of genetic variants associated with plasma homocysteine levels on stroke risk.

BACKGROUND AND PURPOSE: Elevated total plasma homocysteine (tHcy) levels are known to be associated with increased risk of ischemic stroke (IS). Given that both tHcy and IS are heritable traits, we investigated a potential genetic relationship between homocysteine levels and stroke risk by assessing 18 polymorphisms previously associated with tHcy levels for their association with IS and its subtypes. METHODS: Previous meta-analysis results from an international stroke collaborative network, METASTROKE, were used to assess association of the 18 tHcy-associated single-nucleotide polymorphisms (SNPs) in 12 389 IS cases and 62 004 controls. We also investigated the associations in regions located within 50 kb from the 18 tHcy-related SNPs and the association of a genetic risk score, including the 18 SNPs. RESULTS: One SNP located in the RASIP1 gene and a cluster of 3 SNPs located at and near SLC17A3 were significantly associated with IS (P<0.0003) after correcting for multiple testing. For stroke subtypes, the sentinel SNP located upstream of MUT was significantly associated with small-vessel disease (P=0.0022), whereas 1 SNP located in MTHFR was significantly associated with large-vessel disease (P=0.00019). A genetic risk score, including the 18 SNPs, did not show significant association with IS or its subtypes. CONCLUSIONS: This study found several potential associations with IS and its subtypes: an association of an MUT variant with small-vessel disease, an MTHFR variant with large-vessel disease, and associations of RASIP1 and SLC17A3 variants with overall IS.

Genome-wide analysis of blood pressure variability and ischemic stroke.

BACKGROUND AND PURPOSE: Visit-to-visit variability in blood pressure (vBP) is associated with ischemic stroke. We sought to determine whether such variability has genetic causes and whether genetic variants associated with BP variability are also associated with ischemic stroke. METHODS: A Genome Wide Association Study (GWAS) for loci influencing BP variability was undertaken in 3802 individuals from the Anglo-Scandinavian Cardiac Outcome Trial (ASCOT) study, in which long-term visit-to-visit and within-visit BP measures were available. Because BP variability is strongly associated with ischemic stroke, we genotyped the sentinel single nucleotide polymorphism in an independent ischemic stroke population comprising 8624 cases and 12 722 controls and in 3900 additional (Scandinavian) participants from the ASCOT study to replicate our findings. RESULTS: The ASCOT discovery GWAS identified a cluster of 17 correlated single nucleotide polymorphisms within the NLGN1 gene (3q26.31) associated with BP variability. The strongest association was with rs976683 (P=1.4×10(-8)). Conditional analysis of rs976683 provided no evidence of additional independent associations at the locus. Analysis of rs976683 in patients with ischemic stroke found no association for overall stroke (odds ratio, 1.02; 95% CI, 0.97-1.07; P=0.52) or its subtypes: cardioembolic (odds ratio, 1.07; 95% CI, 0.97-1.16; P=0.17), large vessel disease (odds ratio, 0.98; 95% CI, 0.89-1.07; P=0.60), and small vessel disease (odds ratio, 1.07; 95% CI, 0.97-1.17; P=0.19). No evidence for association was found between rs976683 and BP variability in the additional (Scandinavian) ASCOT participants (P=0.18). CONCLUSIONS: We identified a cluster of single nucleotide polymorphisms at the NLGN1 locus showing significant association with BP variability. Follow-up analyses did not support an association with risk of ischemic stroke and its subtypes.

Common genetic loci influencing plasma homocysteine concentrations and their effect on risk of coronary artery disease.

BACKGROUND: The strong observational association between total homocysteine (tHcy) concentrations and risk of coronary artery disease (CAD) and the null associations in the homocysteine-lowering trials have prompted the need to identify genetic variants associated with homocysteine concentrations and risk of CAD. OBJECTIVE: We tested whether common genetic polymorphisms associated with variation in tHcy are also associated with CAD. DESIGN: We conducted a meta-analysis of genome-wide association studies (GWAS) on tHcy concentrations in 44,147 individuals of European descent. Polymorphisms associated with tHcy (P < 10(⁻8) were tested for association with CAD in 31,400 cases and 92,927 controls. RESULTS: Common variants at 13 loci, explaining 5.9% of the variation in tHcy, were associated with tHcy concentrations, including 6 novel loci in or near MMACHC (2.1 × 10⁻9), SLC17A3 (1.0 × 10⁻8), GTPB10 (1.7 × 10⁻8), CUBN (7.5 × 10⁻¹°), HNF1A (1.2 × 10⁻¹²)), and FUT2 (6.6 × 10⁻9), and variants previously reported at or near the MTHFR, MTR, CPS1, MUT, NOX4, DPEP1, and CBS genes. Individuals within the highest 10% of the genotype risk score (GRS) had 3-µmol/L higher mean tHcy concentrations than did those within the lowest 10% of the GRS (P = 1 × 10⁻³6). The GRS was not associated with risk of CAD (OR: 1.01; 95% CI: 0.98, 1.04; P = 0.49). CONCLUSIONS: We identified several novel loci that influence plasma tHcy concentrations. Overall, common genetic variants that influence plasma tHcy concentrations are not associated with risk of CAD in white populations, which further refutes the causal relevance of moderately elevated tHcy concentrations and tHcy-related pathways for CAD.

Common variants at 6p21.1 are associated with large artery atherosclerotic stroke.

Genome-wide association studies (GWAS) have not consistently detected replicable genetic risk factors for ischemic stroke, potentially due to etiological heterogeneity of this trait. We performed GWAS of ischemic stroke and a major ischemic stroke subtype (large artery atherosclerosis, LAA) using 1,162 ischemic stroke cases (including 421 LAA cases) and 1,244 population controls from Australia. Evidence for a genetic influence on ischemic stroke risk was detected, but this influence was higher and more significant for the LAA subtype. We identified a new LAA susceptibility locus on chromosome 6p21.1 (rs556621: odds ratio (OR)=1.62, P=3.9×10(-8)) and replicated this association in 1,715 LAA cases and 52,695 population controls from 10 independent population cohorts (meta-analysis replication OR=1.15, P=3.9×10(-4); discovery and replication combined OR=1.21, P=4.7×10(-8)). This study identifies a genetic risk locus for LAA and shows how analyzing etiological subtypes may better identify genetic risk alleles for ischemic stroke.

Bio-repository of DNA in stroke: a study protocol of three ancestral populations.

Stroke is a leading cause of death and disability in the world. Identifying the genes underlying stroke risk may help us to improve our understanding of the mechanisms that cause stroke and also identify novel therapeutic targets. To have sufficient power to disentangle the genetic component of stroke, large-scale highly phenotyped DNA repositories are necessary. The BRAINS (Bio-repository of DNA in stroke) study aims to recruit subjects with all subtypes of stroke as well as controls from UK, India, Sri Lanka and Qatar. BRAINS-UK will include 1500 stroke patients of European ancestry as well as British South Asians. BRAINS-South Asia aims to recruit 3000 stroke subjects and 3000 controls from across India and Sri Lanka. BRAINS-Middle East aims to enrol 1500 stroke patients from Qatar. The controls for BRAINS-Middle East will be recruited from a population-based Qatari Biobank. With the addition of new recruitment centres in India and Qatar, we present an updated version of the BRAINS study protocol. This is the first international DNA biobank for stroke patients and controls from the Middle East. By investigating the influence of genetic factors on stroke risk in European, South Asian and Middle Eastern populations, BRAINS has the potential to improve our understanding of genetic differences between these groups and may lead to new population-specific therapeutic targets.

The basis of differential responses to folic acid supplementation.

BACKGROUND/AIMS: Elevated levels of total homocysteine (tHcy) are associated with an increased risk of many common diseases. Supplementation with folic acid has been shown to significantly reduce tHcy levels. We used the classical twin model to partition the variability in changes in plasma tHcy levels through folic acid supplementation into genetic, environmental, and confounding epidemiological factors. METHODS: We carried out an intervention study of folic acid using 101 healthy, female, identical and non-identical twins aged 50-80 years. Each twin was administered folic acid (0.8 mg/day) for 6 weeks. Total plasma folate, cobalamin and tHcy were measured at both baseline and after dosing. We calculated the heritability and tested for associations between the MTHFR C677T functional variant and response to folic acid supplementation. RESULTS: Supplementation with folic acid led to a significant reduction in tHcy levels. The mean tHcy changed from 12.14 to 10.42 µmol/l after supplementation (p < 10(-5)). Moreover, the change in tHcy levels was highly heritable (64%), not associated with the C677T functional variant at MTHFR and not confounded by age, BMI or diet. CONCLUSIONS: Our results highlight the need to identify genetic factors associated with biomarkers of response to folate supplementation.

Genetic loci influencing kidney function and chronic kidney disease.

Using genome-wide association, we identify common variants at 2p12-p13, 6q26, 17q23 and 19q13 associated with serum creatinine, a marker of kidney function (P = 10(-10) to 10(-15)). Of these, rs10206899 (near NAT8, 2p12-p13) and rs4805834 (near SLC7A9, 19q13) were also associated with chronic kidney disease (P = 5.0 x 10(-5) and P = 3.6 x 10(-4), respectively). Our findings provide insight into metabolic, solute and drug-transport pathways underlying susceptibility to chronic kidney disease.