Elsevier

The Lancet Neurology

Volume 15, Issue 2, February 2016, Pages 174-184
The Lancet Neurology

Articles
Loci associated with ischaemic stroke and its subtypes (SiGN): a genome-wide association study

https://doi.org/10.1016/S1474-4422(15)00338-5Get rights and content

Summary

Background

The discovery of disease-associated loci through genome-wide association studies (GWAS) is the leading genetic approach to the identification of novel biological pathways underlying diseases in humans. Until recently, GWAS in ischaemic stroke have been limited by small sample sizes and have yielded few loci associated with ischaemic stroke. We did a large-scale GWAS to identify additional susceptibility genes for stroke and its subtypes.

Methods

To identify genetic loci associated with ischaemic stroke, we did a two-stage GWAS. In the first stage, we included 16 851 cases with state-of-the-art phenotyping data and 32 473 stroke-free controls. Cases were aged 16 to 104 years, recruited between 1989 and 2012, and subtypes of ischaemic stroke were recorded by centrally trained and certified investigators who used the web-based protocol, Causative Classification of Stroke (CCS). We constructed case-control strata by identifying samples that were genotyped on nearly identical arrays and were of similar genetic ancestral background. We cleaned and imputed data by use of dense imputation reference panels generated from whole-genome sequence data. We did genome-wide testing to identify stroke-associated loci within each stratum for each available phenotype, and we combined summary-level results using inverse variance-weighted fixed-effects meta-analysis. In the second stage, we did in-silico lookups of 1372 single nucleotide polymorphisms identified from the first stage GWAS in 20 941 cases and 364 736 unique stroke-free controls. The ischaemic stroke subtypes of these cases had previously been established with the Trial of Org 10 172 in Acute Stroke Treatment (TOAST) classification system, in accordance with local standards. Results from the two stages were then jointly analysed in a final meta-analysis.

Findings

We identified a novel locus (G allele at rs12122341) at 1p13.2 near TSPAN2 that was associated with large artery atherosclerosis-related stroke (first stage odds ratio [OR] 1·21, 95% CI 1·13–1·30, p=4·50 × 10−8; joint OR 1·19, 1·12–1·26, p=1·30 × 10−9). Our results also supported robust associations with ischaemic stroke for four other loci that have been reported in previous studies, including PITX2 (first stage OR 1·39, 1·29–1·49, p=3·26 × 10−19; joint OR 1·37, 1·30–1·45, p=2·79 × 10−32) and ZFHX3 (first stage OR 1·19, 1·11–1·27, p=2·93 × 10−7; joint OR 1·17, 1·11–1·23, p=2·29 × 10−10) for cardioembolic stroke, and HDAC9 (first stage OR 1·29, 1·18–1·42, p=3·50 × 10−8; joint OR 1·24, 1·15–1·33, p=4·52 × 10−9) for large artery atherosclerosis stroke. The 12q24 locus near ALDH2, which has previously been associated with all ischaemic stroke but not with any specific subtype, exceeded genome-wide significance in the meta-analysis of small artery stroke (first stage OR 1·20, 1·12–1·28, p=6·82 × 10−8; joint OR 1·17, 1·11–1·23, p=2·92 × 10−9). Other loci associated with stroke in previous studies, including NINJ2, were not confirmed.

Interpretation

Our results suggest that all ischaemic stroke-related loci previously implicated by GWAS are subtype specific. We identified a novel gene associated with large artery atherosclerosis stroke susceptibility. Follow-up studies will be necessary to establish whether the locus near TSPAN2 can be a target for a novel therapeutic approach to stroke prevention. In view of the subtype-specificity of the associations detected, the rich phenotyping data available in the Stroke Genetics Network (SiGN) are likely to be crucial for further genetic discoveries related to ischaemic stroke.

Funding

US National Institute of Neurological Disorders and Stroke, National Institutes of Health.

Introduction

Worldwide, stroke is the second leading cause of death1 and a major contributor to dementia and age-related cognitive decline. About 15 million people have a stroke each year.1 Most survivors are left with a permanent disability, which makes stroke the world's leading cause of adult incapacity.2 Strokes result from the sudden occlusion or rupture of a blood vessel supplying the brain, and so are categorised accordingly as ischaemic (vessel occlusion) or haemorrhagic (vessel rupture) on the basis of neuroimaging results. Up to 85% of all strokes are ischaemic.

Although hypertension, atrial fibrillation, diabetes mellitus, and cigarette smoking are known risk factors for stroke,3 a substantial proportion of the risk remains unexplained and might be attributable to inherited genetic variation. Discovery of genetic variants that predispose to stroke is a crucial first step toward the development of improved diagnostic tests for stroke and novel therapies that might reduce the disease burden. Genome-wide association studies (GWAS) have thus far identified only a few confirmed loci,4, 5, 6, 7 which together account for a small proportion of the heritable risk.8

Research in context

Evidence before this study

We searched PubMed with the search terms “stroke” and “genome wide association study” for reports published before Oct 19, 2015. We only included peer-reviewed reports in English. Compared with the rapid pace of genetic discovery for other common disorders, only four loci (PITX2, HDAC9, ZFHX3, and 12q24.2) have been convincingly implicated by genome-wide association studies (GWAS) in ischaemic stroke. GWAS of stroke have been limited by small sample sizes and concerns about phenotypic heterogeneity.

Added value of this study

To our knowledge, the National Institute of Neurological Disorders and Stroke (NINDS)-Stroke Genetics Network (SiGN) project is the largest and most comprehensive study of ischaemic stroke so far. Discovery analyses were done in 16 851 cases and 32 473 controls and findings were followed up in an additional 20 941 cases and 364 736 controls. Furthermore, the project implemented the Causative Classification of Stroke (CCS) system to subtype cases and generated a rich phenotypic database. Trial of Org 10 172 in Acute Stroke Treatment (TOAST)-based subtypes were also available, allowing for the first ever analysis of the genetic overlap between TOAST and CCS subtypes.

Implications of all the available evidence

Our data show that increasing sample size and applying a standardised subtyping method can reveal additional information about the underlying genetic architecture of stroke. Because we had access to phenotype information generated by two different subtyping methods, we also showed that there is moderate to strong genetic correlation between the CCS and TOAST subtyping methods, suggesting that future studies might benefit from liberal inclusion of cases, regardless of subtyping approach. Also, our results show that all discovered loci, including the 12q24.12 locus, which was previously implicated in all ischaemic stroke, are specific to a single subtype, suggesting that these subtypes will have at least partly distinct genetic signatures. Because of the subtype-specificity of genetic associations in stroke, substantially larger samples of stroke subtypes will probably be needed to expand the number of identified stroke loci to that of other common diseases.

Ischaemic stroke occurs when the blood flow to a region of the brain is interrupted because of blockage of a blood vessel. Because vessel occlusion can occur through different mechanisms, ischaemic stroke can be subtyped on the basis of the presumed mechanism: large artery atherosclerosis, cardioembolism, or small artery occlusion. With one exception, all associations for ischaemic stroke detected in GWAS have been subtype-specific, suggesting the need for studies that are powered to detect subtype-specific associations. The National Institute of Neurological Disorders and Stroke (NINDS) Stroke Genetics Network (NINDS-SiGN)9 is the largest and most comprehensive GWAS of stroke and its subtypes to date. We sought to detect new associations of polymorphisms with risk of ischaemic stroke and its subtypes and to provide evidence for previously reported associations.

Section snippets

Study design

We did a two-stage joint association analysis of ischaemic stroke and its subtypes. The first stage consisted of a GWAS, and the second stage was an in-silico association analysis of the top single nucleotide polymorphisms (SNPs) identified in the first stage in a set of independent samples of cases and controls. We then analysed both stages together to identify loci that exceeded the threshold for genome-wide significance (1 × 10−8). Compared with separate discovery and replication analyses,

Results

After data quality control (appendix p 4 and pp 114–26), we included 16 851 stroke cases and 32 473 controls in the first stage of our analyses. The first stage GWAS revealed 1372 SNPs in 268 loci associated with ischaemic stroke or a specific subtype in any of the CCS or TOAST traits at p<1 × 10−6. We included an additional independent set of 20 941 cases and 364 736 controls in the second stage, which enabled the joint analysis of 37 893 cases and 397 209 controls across five primary

Discussion

Our results show a novel association between a genetic locus and large artery atherosclerosis. The lead SNP, rs12122341, is located in an intergenic region 23·6 kb upstream of TSPAN2, the gene encoding tetraspanin-2 (figure 2) This SNP is in linkage disequilibrium with intronic and untranslated region variants in TSPAN2 (r2>0·3 in 1000 Genomes Project samples of European ancestry), but is located in a DNA sequence immediately adjacent to TSPAN2 that can be bound by several transcription factor

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