Whether smoking is causally associated with risk of ischemic stroke and intracerebral hemorrhage is unknown. We used the Mendelian randomization design to explore the associations of genetic predisposition to smoking with ischemic stroke and its subtypes as well as intracerebral hemorrhage. Up to 372 single-nucleotide polymorphisms were used as instrumental variables for smoking initiation. We used summary statistics data for 438 847 individuals in the analyses of ischemic stroke (34 217 cases and 404 630 non-cases) and 3026 individuals in analyses of intracerebral hemorrhage (1545 cases and 1481 non-cases). Genetic predisposition to smoking initiation was statistically significantly positively associated with any ischemic stroke, large artery stroke, and small vessel stroke but not cardioembolic stroke or intracerebral hemorrhage. The odds ratios per one standard deviation higher log-odds of ever smoking regularly (smoking initiation) were 1.24 (95% CI 1.16-1.32; p = 1.310-10) for any ischemic stroke, 1.64 (95% CI 1.40-1.91; p = 2.810-10) for large artery stroke, 1.47 (95% CI 1.26-1.71; p = 1.110-6) for small vessel stroke, 1.12 (95% CI 0.99-1.27; p = 0.08) for cardioembolic stroke, and 1.13 (95% CI 0.81-1.58; p = 0.47) for intracerebral hemorrhage. This study provides genetic support for a causal association of smoking with ischemic stroke, particularly large artery and small vessel stroke.his work was supported by the Swedish Research Council for Health, Working Life and Welfare (Forskningsrådet för hälsa, arbetsliv och välfärd) and the Swedish Research Council. S.B. is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (grant number 204623/Z/16/Z)

Burgess, Stephen

Larsson, Susanna C

Michaelsson, Karl

English

We used the Mendelian randomization design to explore the potential causal association of smoking with ischemic stroke and intracerebral hemorrhage using summary statistics data for 34,217 ischemic stroke cases and 404,630 noncases, and 1,545 cases of intracerebral hemorrhage and 1,481 noncases. Genetic predisposition to smoking initiation (ever smoking regularly), based on up to 372 single-nucleotide polymorphisms, was statistically significantly positively associated with any ischemic stroke, large artery stroke, and small vessel stroke but not cardioembolic stroke or intracerebral hemorrhage. This study provides genetic support for a causal association of smoking with ischemic stroke, particularly large artery and small vessel stroke. ANN NEUROL 2019;86:468-471

Apollo (Cambridge)

BRIEF COMMUNICATIONSmoking and Stroke:A MendelianRandomization StudySusanna C. Larsson, PhD ,1,2Stephen Burgess, PhD,3,4 andKarl Michaëlsson, MD, PhD2We used the Mendelian randomization design to explorethe potential causal association of smoking with ischemicstroke and intracerebral hemorrhage using summary statis-tics data for 34,217 ischemic stroke cases and 404,630noncases, and 1,545 cases of intracerebral hemorrhage and1,481 noncases. Genetic predisposition to smoking initia-tion (ever smoking regularly), based on up to 372 single-nucleotide polymorphisms, was statistically significantlypositively associated with any ischemic stroke, large arterystroke, and small vessel stroke but not cardioembolic strokeor intracerebral hemorrhage. This study provides geneticsupport for a causal association of smoking with ischemicstroke, particularly large artery and small vessel stroke.ANN NEUROL 2019;00:1–4Evidence from observational studies indicates that smokingincreases the risk of stroke.1 Even smoking as little as 1 cig-arette per day is associated with an approximately 25% to 30%increased risk of stroke.1 However, the role of smoking forischemic stroke subtypes and intracerebral hemorrhage isunclear. Moreover, because data on smoking in relation tostroke originate from observational studies, which are vulnera-ble to confounding, the causal nature of the associationbetween smoking and stroke risk remains elusive.We thereforeimplemented the Mendelian randomization (MR) design toassess the association of genetic predisposition to smoking withischemic stroke and its main subtypes as well as intracerebralhemorrhage. MR is a method that can be used to strengthencausal inference in observational studies by utilizing geneticvariants associated with the exposure as instrumental variablesfor the exposure.2Subjects and MethodsOutcome DataThe current study is based on publicly available summary statisticsdata for genetic associations with ischemic stroke and its subtypes fromthe MEGASTROKE consortium, including data for 438,847European-descent individuals (34,217 ischemic stroke cases and404,630 noncases).3 The cases were subtyped according to the Trialof Org 10172 in Acute Stroke Treatment criteria4 into large-artery(n = 4,373), small vessel (n = 5,386), and cardioembolic (n = 7,193)stroke. Summary statistics data for intracerebral hemorrhage wereavailable from a genome-wide association meta-analysis of 3,026European-descent individuals (1,545 cases and 1,481 noncases).5 Thepresent MR study was approved by the Swedish Ethical ReviewAuthority.Selection of Instrumental VariablesA genome-wide association meta-analysis of 1,232,091 individualsidentified 378 conditionally independent single-nucleotide poly-morphisms (SNPs) at 259 loci (defined as a 1Mb region surround-ing the top p value) associated with ever smoking regularly(hereafter referred to as smoking initiation) at genome-wide signifi-cance.6 Conditionally independent SNPs had been identified usingthe novel, partial correlation-based score statistic method.7 The378 SNPs explained 2.3% of the phenotypic variation in smokinginitiation. Thirteen and 113 SNPs were unavailable in the ischemicstroke and intracerebral hemorrhage datasets, respectively. A proxySNP in linkage disequilibrium (R2 > 0.7) with the specified SNP wasidentified for 7 and 61 of the missing SNPs for ischemic stroke andintracerebral hemorrhage datasets, respectively. Thus, the smokinginitiation analyses included 372 SNPs in the analysis of ischemicstroke and 326 SNPs in the analysis of intracerebral hemorrhage.As an additional analysis, we used 126 SNPs associated with ameasure of lifetime smoking exposure that takes into accountsmoking status and, among ever smokers, duration, heaviness, andcessation.8 These SNPs were broadly distinct from the SNPspredicting smoking initiation (only 9 SNPs overlapped) and stronglyassociated with the positive control of lung cancer.8 All 126 SNPswere available in the ischemic stroke dataset, whereas 109 SNPs(including 41 proxy variants) were available for analysis of intracere-bral hemorrhage. In a further secondary analysis, we used 53 condi-tionally independent genome-wide significant SNPs associated withcigarettes smoked per day in a genome-wide association meta-analysis of 337,334 ever smokers,6 which were available in the ische-mic stroke dataset.There was partial sample overlap between the 3 smoking pheno-types. The genome-wide association studies of smoking initiation, life-time smoking exposure, and cigarettes per day included, respectively,From the 1Unit of Cardiovascular and Nutritional Epidemiology, Instituteof Environmental Medicine, Karolinska Institute, Stockholm, Sweden;2Department of Surgical Sciences, Uppsala University, Uppsala, Sweden;3Medical Research Council Biostatistics Unit, University of Cambridge,Cambridge, United Kingdom; and 4Department of Public Health andPrimary Care, University of Cambridge, Cambridge, United Kingdom.Address correspondence to Dr Larsson, Unit of Cardiovascular and Nutri-tional Epidemiology, Institute of Environmental Medicine, KarolinskaInstitute, 171 77 Stockholm, Sweden. E-mail: susanna.larsson@ki.seReceived Apr 17, 2019, and in revised form Jun 11, 2019. Accepted forpublication Jun 18, 2019.View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.25534.© 2019 The Authors. Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association. 1This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distributionand reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.383,631 (of the total 1,232,091 individuals), 462,690, and337,334 individuals from UK Biobank.Statistical AnalysisAnalyses were performed using the mrrobust9 and Mendelian-Randomization10 packages. The principal analysis was conductedusing the inverse-variance weighted method, under a random-effects model, and supplemented with the weighted medianapproach.11 The MR-Pleiotropy RESidual Sum and Outlier andMR-Egger regression methods were used to identify outliers andpleiotropy.11,12 Smoking initiation is genetically correlated withalcohol consumption (rg = 0.34) and education (rg = −0.40).6 Ina multivariable MR analysis, we included SNPs associated withthose exposures6,13 along with SNPs for smoking initiation. Tocorrect for multiple testing, we applied a Bonferroni-correctedsignificance threshold of α = 0.01 (where α = 0.05/5 outcomes).ResultsGenetic predisposition to smoking initiation was statisticallysignificantly positively associated with any ischemic stroke,large artery stroke, and small vessel stroke but not car-dioembolic stroke or intracerebral hemorrhage in analysesusing the inverse-variance weighted method (Fig 1). Resultswere similar in a sensitivity analysis based on the weightedmedian method; the odds ratios per unit increase in log oddsof ever smoking regularly were 1.22 (95% confidence inter-val [CI] = 1.12–1.34, p = 7.6 × 10−6) for any ischemicstroke, 1.59 (95% CI = 1.26–1.99, p = 6.7 × 10−5) for largeartery stroke, 1.35 (95% CI = 1.09–1.66, p = 0.005) forsmall vessel stroke, 1.11 (95% CI = 0.94–1.32, p = 0.21) forcardioembolic stroke, and 0.83 (95% CI = 0.50–1.37,p = 0.47) for intracerebral hemorrhage. One outlier SNPwas identified in the analyses of any ischemic stroke butnone in the analyses of the other outcomes. Exclusion of theoutlier did not essentially change the results for smoking andischemic stroke (odds ratio = 1.23, 95% CI = 1.15–1.31,p = 6.5 × 10−10). There was no indication of directionalpleiotropy (all p > 0.24). Restricting the analysis to 1 SNP ineach locus yielded similar results (odds ratio of ischemicstroke = 1.23, 95% CI = 1.14–1.33, p = 7.0 × 10−8). Theassociation between genetic predisposition to smoking initia-tion and ischemic stroke was weaker but remained statisti-cally significant in multivariable MR analysis that includedSNPs for alcohol consumption and education (oddsratio = 1.14, 95% CI = 1.07–1.23, p = 1.8 × 10−4).Findings were similar in analyses of lifetime smokingexposure (Fig 2). Genetically predicted cigarettes smokedper day was positively associated with ischemic stroke, butthe association was not statistically significant in the inverse-variance weighted analysis. The odds ratio was 1.14(95% CI = 0.98–1.33, p = 0.09) per 1 standard deviation(corresponding to about 8 cigarettes per day among currentsmokers in the UK Biobank) increase in cigarettes per day.DiscussionThis study provides genetic support for a causal associationbetween smoking and increased risk of ischemic stroke, par-ticularly large artery and small vessel stroke. We found nocausal association between smoking and cardioembolic strokeor intracerebral hemorrhage.Cigarette smoke can adversely affect the cardiovascularsystem through its content of oxidant gases (eg, oxides of nitro-gen and free radicals) and other toxic substances, which mayincrease stroke risk through endothelial dysfunction, lipidFIGURE 1: Associations of genetic predisposition to smoking initiation with ischemic stroke and its subtypes and intracerebralhemorrhage. Odds ratios are expressed per unit increase in log odds of ever smoking regularly (smoking initiation). Estimates wereobtained using the inverse variance-weighted method under a random-effects model. CI = confidence interval; OR = odds ratio.2 Volume 00, No. 0ANNALS of Neurologyoxidation, inflammation, platelet activation, thrombogenesis,and enhanced coagulability.14 Additionally, nicotine mayreduce cerebral blood flow.14Notable strengths of this MR study are that the analyseswere based on large sample sizes for ischemic stroke and arobust genetic instrument for smoking initiation, comprising372 SNPs. Thus, we had high statistical power to detect weakassociations with ischemic stroke. Nonetheless, the analyses ofintracerebral hemorrhage were based on a small sample sizeand we cannot rule out that we may have overlooked a weakassociation between smoking and intracerebral hemorrhage.Population stratification can affect the findings of MR studiesbut we reduced this bias by using summary statistics data forindividuals of European ancestry only.A possible criticism of the use of smoking initiation as aphenotype is that it does not reflect exposure to cigarettesmoke. Therefore, in a complementary analysis we investi-gated the association between a lifetime smoking index, whichtakes into account smoking intensity, and found broadly simi-lar results. In an additional secondary analysis, we observed asuggestive, nonsignificant, dose-response association betweencigarettes smoked per day and ischemic stroke. However, thatanalysis suffered from low precision due to low phenotypicvariation explained by the SNPs (about 1.1%–1.2%). More-over, we were unable to stratify the analysis by smoking statusand could not examine nonlinear associations.In conclusion, this MR study found support forcausal associations between smoking and increased risk ofischemic stroke as a whole, large artery stroke, and smallvessel stroke, but not cardioembolic stroke and intracerebralhemorrhage.AcknowledgmentThis work was supported by the Swedish Research Coun-cil for Health, Working Life and Welfare (Forskningsrådetför hälsa, arbetsliv och välfärd) and the Swedish ResearchCouncil. S.B. is supported by a Sir Henry Dale Fellowshipjointly funded by the Wellcome Trust and the Royal Soci-ety (grant number 204623/Z/16/Z). The funding sourceshad no role in the design and conduct of the study, in thecollection, analysis, and interpretation of the data, and inthe preparation, review, or approval of the manuscript.Funding of the MEGASTROKE project is specified atmegastroke.org/acknowledgements.html.We thank the MEGASTROKE and InternationalStroke Genetics consortia for providing summary statisticsdata for ischemic stroke and intracerebral hemorrhage.Author ContributionsS.C.L., S.B., and K.M. contributed to the conception anddesign of the study. S.C.L. contributed to the analysis ofdata, preparing the figures, and drafting the text.The investigator list of MEGASTROKE is availableat http://megastroke.org/authors.html.Potential Conflicts of InterestNothing to report.References1. Hackshaw A, Morris JK, Boniface S, et al. Low cigarette con-sumption and risk of coronary heart disease and stroke: meta-FIGURE 2: Associations of genetically predicted lifetime smoking index with ischemic stroke and its subtypes and intracerebralhemorrhage. Odds ratios are expressed per 1 standard deviation increase of the lifetime smoking index. Estimates were obtainedusing the inverse variance-weighted method under a random-effects model. CI = confidence interval; OR = odds ratio.3Larsson et al: Smoking and Strokeanalysis of 141 cohort studies in 55 study reports. BMJ 2018;360:j5855.2. Davey Smith G, Ebrahim S. ’Mendelian randomization’: can geneticepidemiology contribute to understanding environmental determi-nants of disease? Int J Epidemiol 2003;32:1–22.3. Malik R, Chauhan G, Traylor M, et al. Multiancestry genome-wideassociation study of 520,000 subjects identifies 32 loci associatedwith stroke and stroke subtypes. Nat Genet 2018;50:524–537.4. Adams HP Jr, Bendixen BH, Kappelle LJ, et al. Classification of sub-type of acute ischemic stroke. Definitions for use in a multicenterclinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment.Stroke 1993;24:35–41.5. Woo D, Falcone GJ, Devan WJ, et al. Meta-analysis of genome-wideassociation studies identifies 1q22 as a susceptibility locus for intra-cerebral hemorrhage. Am J Hum Genet 2014;94:511–521.6. Liu M, Jiang Y, Wedow R, et al. Association studies of up to 1.2 mil-lion individuals yield new insights into the genetic etiology oftobacco and alcohol use. Nat Genet 2019;51:237–244.7. Jiang Y, Chen S, McGuire D, et al. Proper conditional analysis in thepresence of missing data: application to large scale meta-analysis oftobacco use phenotypes. PLoS Genet 2018;14:e1007452.8. Wootton RE, Richmond RC, Stuijfzand BG, et al. Causal effects oflifetime smoking on risk for depression and schizophrenia: evidencefrom a Mendelian randomisation study. bioRxiv 2018;381301; doi:https://doi.org/10.1101/381301.9. Spiller W, Davies NM, Palmer TM. Software application profile:mrrobust—a tool for performing two-sample summary Mendelianrandomization analyses. bioRxiv 2017;142125; doi: https://doi.org/10.1101/142125.10. Yavorska OO, Burgess S. MendelianRandomization: an R packagefor performing Mendelian randomization analyses using summarizeddata. Int J Epidemiol 2017;46:1734–1739.11. Burgess S, Bowden J, Fall T, et al. Sensitivity analyses for robustcausal inference from Mendelian randomization analyses with multi-ple genetic variants. Epidemiology 2017;28:30–42.12. Verbanck M, Chen CY, Neale B, Do R. Detection of widespread hori-zontal pleiotropy in causal relationships inferred from Mendelian ran-domization between complex traits and diseases. Nat Genet 2018;50:693–698.13. Lee JJ, Wedow R, Okbay A, et al. Gene discovery and polygenicprediction from a genome-wide association study of educationalattainment in 1.1 million individuals. Nat Genet 2018;50:1112–1121.14. Rigotti NA, Clair C. Managing tobacco use: the neglected cardiovas-cular disease risk factor. Eur Heart J 2013;34:3259–3267.4 Volume 00, No. 0ANNALS of Neurology

Smoking and stroke: A mendelian randomization study

Susanna C. Larsson

Stephen Burgess

Karl Michaëlsson

Crossref

We used the Mendelian randomization design to explore the potential causal association of smoking with ischemic stroke and intracerebral hemorrhage using summary statistics data for 34,217 ischemic stroke cases and 404,630 noncases, and 1,545 cases of intracerebral hemorrhage and 1,481 noncases. Genetic predisposition to smoking initiation (ever smoking regularly), based on up to 372 single-nucleotide polymorphisms, was statistically significantly positively associated with any ischemic stroke, large artery stroke, and small vessel stroke but not cardioembolic stroke or intracerebral hemorrhage. This study provides genetic support for a causal association of smoking with ischemic stroke, particularly large artery and small vessel stroke. ANN NEUROL 2019;86:468-47

Larsson, Susanna C.

Michaëlsson, Karl

Digitala Vetenskapliga Arkivet - Academic Archive On-line

Smoking and stroke : A mendelian randomization study

Smoking and stroke: A mendelian randomization study.

We used the Mendelian randomization design to explore the potential causal association of smoking with ischemic stroke and intracerebral hemorrhage using summary statistics data for 34,217 ischemic stroke cases and 404,630 noncases, and 1,545 cases of intracerebral hemorrhage and 1,481 noncases. Genetic predisposition to smoking initiation (ever smoking regularly), based on up to 372 single-nucleotide polymorphisms, was statistically significantly positively associated with any ischemic stroke, large artery stroke, and small vessel stroke but not cardioembolic stroke or intracerebral hemorrhage. This study provides genetic support for a causal association of smoking with ischemic stroke, particularly large artery and small vessel stroke. ANN NEUROL 2019;86:468-471</p

Larsson, Susanna C.,

Burgess, Stephen,

Michaëlsson, Karl,

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Smoking and stroke [Elektronisk resurs] : A mendelian randomization study

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Smoking and stroke: A mendelian randomization study

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Digitala Vetenskapliga Arkivet - Academic Archive On-line

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