ARTICLE OPEN ACCESS Genome-wide association meta-analysis of functional outcome after ischemic stroke Martin Söderholm, MD, PhD,* Annie Pedersen, MD,* Erik Lorentzen, MSc, Tara M. Stanne, PhD, Correspondence Steve Bevan, PhD, Maja Olsson, MSc, PhD, John W. Cole, MD, Israel Fernandez-Cadenas, MSc, PhD, Dr. Pedersen Graeme J. Hankey, MD, Jordi Jimenez-Conde, MD, PhD, Katarina Jood, MD, PhD, Jin-Moo Lee, MD, PhD, annie.pedersen@gu.se Robin Lemmens, MD, PhD, Christopher Levi, MD, Braxton D. Mitchell, PhD, MPH, Bo Norrving, MD, PhD, Kristiina Rannikmäe, MD, PhD, Natalia S. Rost, MD, MPH, Jonathan Rosand, MD, MSc, Peter M. Rothwell, MD, PhD, Rodney Scott, DSc, PhD, Daniel Strbian, MD, PhD, Jonathan W. Sturm, MBChB, PhD, Cathie Sudlow, MD, PhD, Matthew Traylor, MSc, PhD, Vincent Thijs, MD, PhD, Turgut Tatlisumak, MD, PhD, Daniel Woo, MD, Bradford B. Worrall, MD, Jane M. Maguire, RN, PhD,‡ Arne Lindgren, MD, PhD,‡ and Christina Jern, MD, PhD‡, on behalf of the International Stroke Genetics Consortium, the NINDS-SiGN Consortium, and the Genetics of Ischaemic Stroke Functional Outcome (GISCOME) Network ® Neurology 2019;92:e1271-e1283. doi:10.1212/WNL.0000000000007138 Abstract RELATED ARTICLE Objective Editorial To discover common genetic variants associated with poststroke outcomes using a genome- A SNP-it of stroke outcome wide association (GWA) study. Page 549 Methods The study comprised 6,165 patients with ischemic stroke from 12 studies in Europe, the United States, and Australia included in the GISCOME (Genetics of Ischaemic Stroke Functional Outcome) network. The primary outcome was modified Rankin Scale score after 60 to 190 days, evaluated as 2 dichotomous variables (0–2 vs 3–6 and 0–1 vs 2–6) and subsequently as an ordinal variable. GWA analyses were performed in each study independently and results were meta-analyzed. Analyses were adjusted for age, sex, stroke severity (baseline NIH Stroke Scale score), and ancestry. The significance level was p < 5 × 10−8. Results We identified one genetic variant associated with functional outcome with genome-wide sig- nificance (modified Rankin Scale scores 0–2 vs 3–6, p = 5.3 × 10−9). This intronic variant (rs1842681) in the LOC105372028 gene is a previously reported trans-expression quantitative trait locus for PPP1R21, which encodes a regulatory subunit of protein phosphatase 1. This ubiquitous phosphatase is implicated in brain functions such as brain plasticity. Several variants detected in this study demonstrated suggestive association with outcome (p < 10−5), some of which are within or near genes with experimental evidence of influence on ischemic stroke volume and/or brain recovery (e.g., NTN4, TEK, and PTCH1). Conclusions In this large GWA study on functional outcome after ischemic stroke, we report one significant variant and several variants with suggestive association to outcome 3 months after stroke onset with plausible mechanistic links to poststroke recovery. Future replication studies and explo- ration of potential functional mechanisms for identified genetic variants are warranted. *These authors contributed equally to this work. ‡These authors jointly supervised this work. Go to Neurology.org/N for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article. The Article Processing Charge was funded by the Swedish government (under the “Avtal om Läkarutbildning och Medicinsk Forskning, ALF”). This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. e1271 Glossary EC = endothelial cell; eQTL = expression quantitative trait locus; GISCOME = Genetics of Ischaemic Stroke Functional Outcome; GTEx = Genotype-Tissue Expression; GWA = genome-wide association; HGVD = Human Genetic Variation Database; mRS = modified Rankin Scale; NIHSS = NIH Stroke Scale; OR = odds ratio; PP1 = protein phosphatase 1; SNP = single-nucleotide polymorphism. Functional outcomes after ischemic stroke have a wide range date, no genome-wide association (GWA) study has been of interindividual variability, from complete recovery to per- published on functional outcome after overall ischemic stroke. sistent severe disability.1 Although a large number of factors Such a hypothesis-free approach may identify variants in- influence recovery after ischemic stroke, such as age and initial dicating novel pathways for poststroke pathophysiologic stroke severity, a discernible portion of this interindividual processes or recovery, and thereby suggest new targets for variation remains unexplained by clinical factors.1 The un- interventions and drug development. explored mechanisms behind this variation represent poten- tial keys in the search for a personalized poststroke The Genetics of Ischaemic Stroke Functional Outcome management approach that would include accurate prog- (GISCOME) network aims to identify genetic variants that nostic prediction as well as patient-tailored treatment, sec- may influence functional outcome after ischemic stroke.8 ondary prevention, and rehabilitation. Here, we present the results from a GWA study of functional outcome as assessed by the modified Rankin Scale (mRS) at 3 Genetic factors may account for part of the variability in stroke months after ischemic stroke. outcomes. Studies in humans and animals support a genetic influence on recovery after brain injury.2–4 In addition, genetic variants identified through candidate gene studies have been reported to associate with functional outcome after stroke, Methods e.g., within the brain-derived neurotrophic factor and Study population cyclooxygenase-2 genes,5–7 although these candidate gene The GISCOME study population and analysis plan have been study results have been inconsistent and need replication.1 To previously described in detail.8 In brief, the GISCOME study e1272 Neurology | Volume 92, Number 12 | March 19, 2019 Neurology.org/N population consists of patients with ischemic stroke of mainly 5061/dryad.s38kf65). Multivariable models were used for European ancestry aged 18 years and older from 12 study analyses of each outcome variable, under an additive genetic locations in Europe, the United States, and Australia. After the model. We aimed to explore genetic variants associated with publication of the GISCOME study protocol,8 2 additional functional outcome that were independent of stroke severity. sets of data from (1) the Sahlgrenska Academy Study on In this primary model, results were adjusted for age, sex, an- Ischemic Stroke, and (2) the Malmö Diet and Cancer Study cestry (up to the first 5 principal components [data available have been added (data available from Dryad, supplemental from Dryad, table e-3]), and baseline stroke severity as Methods and tables e-1 and e-2, doi.org/10.5061/dryad. assessed by the NIH Stroke Scale (NIHSS) at 0 to 10 days s38kf65). The GISCOME study includes centers with mRS post stroke onset, with preference to as close to day 0–1 as and genotype data contributing to the International Stroke possible.8 In addition, models without adjustment for baseline Genetics Consortium and the National Institute of Neuro- stroke severity were performed for each outcome for com- logical Disorders and Stroke – Stroke Genetics Network (SiGN) parison; however, unless otherwise stated, all results in this study efforts studying genetics of stroke risk. We did not have report refer to the primary model as described above. any additional cohorts available at the time of this study for replication. All dichotomized analyses were performed with logistic re- gression using PLINK software version 1.90b4.6.11 The full Standard protocol approvals, registrations, mRS was analyzed with ordinal logistic regression under and patient consents a cumulative logit model using the MATLAB mnrfit algorithm All participants provided written informed consent to par- (MATLAB Statistics and Machine Learning Toolbox Release ticipate. For participants who were unable to communicate, 2016b; The MathWorks, Inc., Natick, MA). Under the pro- consent was obtained from their next of kin. Local ethics portional odds assumption, the effect of a predictor single- committees approved the individual studies. nucleotide polymorphism (SNP) is invariant of the choice of outcome categories. As a result, estimates from all study Outcome cohorts, including those that did not separate mRS 0–2, are The mRS as close as possible to 90 days (60–190 days permit- comparable in ordinal regression. Furthermore, one unit ted) was selected to assess functional outcome, as described in change in the dosage of a variant with regression coefficient β the analysis plan.8 The majority of the included studies (≈80%) suggests a change in the odds of an mRS score lower than or assessed mRS at 3 months ± 2 weeks.8 In most studies, this was equal to x vs higher than x by a factor OR = e−β , equally for done by face-to-face interviews. In 3 studies (Lund Stroke all scores x. We tested deviations from proportional odds for Register, Malmö Diet and Cancer Study, and parts of the Sahl- all reported variants from the ordinal models. No significant grenska Academy Study on Ischemic Stroke), data from the deviations were found. Swedish quality register for stroke (Riksstroke) were used to assess mRS by a validated translation algorithm.8,9 This approach Inverse variance-weighted fixed-effects meta-analysis was prevented a differentiation between the mRS scores 0, 1, and 2. performed using METAL software.12 Variants with minor allele frequency <0.01 were excluded. Variants that were Based on our a priori analysis plan, we analyzed mRS as 2 missing in >50% of cohorts were also excluded. After filtering, dichotomous variables, (1) mRS 0–2 vs 3–6 and (2) mRS 0–1 approximately 8.5 million SNPs were included in each of the vs 2–6, and also as the full ordinal scale variable. The mRS 0–2 final meta-analyses. Quantile-quantile plots are shown in vs 3–6 and ordinal scale analyses included a larger number of figures 1 and 2 and in data available from Dryad (figures e-1 to participants. Analyses of mRS 0–1 vs 2–6 were performed to e-4, doi.org/10.5061/dryad.s38kf65), and genomic inflation explore whether a second dichotomization could identify any factors (λ) are displayed in data available from Dryad (table strong associations that were not picked up by the other e-4). There was no sign of population stratification based on analyses. In addition, we made an explorative effort to in- these measures. Heterogeneity of SNP effects between studies vestigate potential associations with infarcts in subcortical and was tested in the meta-analysis. cortical locations separately. Since information about infarct location was not available, we divided the cases into small Markers with p values <5 × 10−8 were considered significant vessel disease (referred to as lacunar stroke) and other sub- for association with outcome, while markers with p values types (referred to as nonlacunar stroke), according to subtype <1 × 10−5 were considered suggestive. To facilitate compar- classifications in TOAST (Trial of ORG 10172 in Acute ison of the results from the dichotomized and ordinal analy- Stroke Treatment).10 A total of 992 patients had lacunar ses, we present all effect sizes as odds ratios (ORs) per copy of stroke, 3,991 patients had nonlacunar stroke, and for 1,182 the minor allele; an OR >1 indicates a higher mRS score individuals, this information was missing. (worse outcome) per copy of the minor allele and an OR <1 indicates a lower mRS score. GWA analysis and meta-analysis The methods for genotyping, imputation, and quality control Investigation of expression quantitative trait loci of genotype data are described in data available from Dryad We explored associations of the markers with p values <1 × (supplemental Methods and tables e-1 and e-2, doi.org/10. 10−5 and proxy SNPs (r2 > 0.8 in 1000 Genomes Phase 1 Neurology.org/N Neurology | Volume 92, Number 12 | March 19, 2019 e1273 Figure 1 Manhattan and quantile-quantile plots of analysis for associations with dichotomized mRS at 3 months Outcome was measured as mRS 0–2 vs 3–6 at 3 months after ischemic stroke onset. Dotted lines show genome-wide significance (black, p <5 × 10−8) and suggestive association level (green, p < 10−5). Results are adjusted for age, sex, principal components, and baseline NIH Stroke Scale score. mRS = modified Rankin Scale. European population) with expression quantitative trait loci Data availability (eQTLs) in publicly available datasets encompassing numer- The datasets generated and analyzed during the current study ous tissues: Genotype-Tissue Expression (GTEx) V6,13 are available on reasonable request. GRASP2,14,15 Human Genetic Variation Database (HGVD),16 and BIOS.17 For eQTLs, p < 10−4 was considered significant. Gene-based analysis Results Gene-based tests were performed for each meta-analysis Characteristics such as age, sex, and stroke severity, as well as the using VEGAS2 with linkage disequilibrium structure based numbers of included cases for each mRS score and in each on the European population.18 All SNPs within ±10 kbp from outcome analysis, are displayed in table 1. The analyses of mRS the untranslated regions 39 and 59 of each gene were in- 0–2 vs 3–6 and the ordinal analyses included 6,021 stroke cases, cluded, to account for potential regulatory variants.18 The whereas analyses of mRS 0–1 vs 2–6 included 4,363 cases. number of genes included was approximately 23,000, which corresponds to a Bonferroni-corrected significance threshold One common variant on chromosome 18q11.2 (rs1842681, of p < 2.2 × 10−6. minor allele frequency: 0.23) was associated at genome-wide e1274 Neurology | Volume 92, Number 12 | March 19, 2019 Neurology.org/N Figure 2 Manhattan and quantile-quantile plots of analysis for associations with ordinal mRS at 3 months Outcome was measured as ordinal mRS at 3 months after ischemic stroke onset. Dotted lines show genome-wide significance (black, p <5 × 10−8) and suggestive association level (green, p < 10−5). Results are adjusted for age, sex, principal components, and baseline NIH Stroke Scale score. mRS = modified Rankin Scale. significance with outcome defined as mRS 0–2 vs 3–6 (OR known as PPP1R21, pHGVD = 1.67 ×10−7). PPP1R21 is for minor allele [A]: 1.40, p = 5.3 × 10−9) (table 2, figures 1, expressed in the brain (GTEx, June 1, 2018). 3, and 4). The effect was similar with and without adjustment for stroke severity (table 2), and the association was ob- No other SNP was significantly associated with mRS 0–2 vs served in the same direction, but with a somewhat lower 3–6, or with ordinal scale mRS (figures 1 and 2). The results effect size, for ordinal mRS (OR: 1.17, p = 1.5 × 10−4) and for the lead SNPs of the top 10 independent loci for each mRS 0–1 vs 2–6 (OR: 1.12, p = 7.4 × 10−2). In line with this, outcome are displayed in data available from Dryad (tables e-5 the distribution of the minor allele count for rs1842681 over and e-6, doi.org/10.5061/dryad.s38kf65). The results from mRS categories shows a threshold between mRS 2 and 3 analyses without severity adjustment are displayed in data (data available from Dryad, figure e-5, doi.org/10.5061/ available from Dryad (tables e-7 and e-8 and figures e-1, e-2, dryad.s38kf65). The variant is located in an intron of the e-6, and e-7). There were no signs of heterogeneity for the gene LOC105372028 (long noncoding RNA synonymous effects of the reported SNPs. All I2 values were <0.35 and all p with RP11-449D8.5 [Genome Reference Consortium Hu- for heterogeneity >0.08, except for one SNP (rs58448576) in man Build 38/hg38]) and is in a putative binding site of the analysis of mRS 0–1 vs 2–6, which had an I2 of 0.48 and a p several regulatory proteins (HaploReg, March 21, 2018). This value of 0.03 (without adjustment for multiple testing). In the variant has no eQTL reported in GTEx (June 1, 2018). gene-based analyses, no gene reached the predefined thresh- However, in HGVD, it has a trans-eQTL for KLRAQ1 (also old for significance (data available from Dryad, table e-9). Neurology.org/N Neurology | Volume 92, Number 12 | March 19, 2019 e1275 The analysis of mRS 0–1 vs 2–6 yielded no significant asso- Table 1 Characteristics of the study population and ciations. These results are presented in data available from numbers of included patients for each mRS score Dryad (tables e-12 and e-13 and figures e-3, e-4, and e-8, doi. and for each outcome (mRS 0–2 vs 3–6, 0–1 vs org/10.5061/dryad.s38kf65). 2–6, and the full ordinal scale) Total no. 6,165 Suggestive associations with outcome Sex, M/F, n 3,497/2,668 Thirty-three SNPs in 12 different loci (with at least 1 Mbp distance) were suggestively (p < 10−5) associated with mRS Age, y, median (IQR) 70 (60–80) 0–2 vs 3–6 (excluding the SNPs in the significant locus on NIHSS (0–10 d), median (IQR) 4 (2–8) chromosome 18q11.2), and 75 SNPs in 17 different loci with mRS score at 3 mo, n ordinal mRS (figures 1 and 2). Of these 29 independent loci, the top SNPs of 16 loci have either significant eQTLs for and/ 0–2a 972 or are located within or near (<100 kbp) genes that are 0 706 expressed in the brain (GTEx, June 1, 2018). Five genes that were not reported to be expressed in brain tissue were 1 1,126 expressed in arteries or lymphocytes (GTEx, June 1, 2018). 2 1,026 3 953 Among the suggestive associations, 3 are linked to genes with experimental evidence of influence on outcome from animal 4 659 models of stroke and are discussed below.19–21 First, 5 236 rs2236406, an intron variant in the PTCH1 gene, was identified in the mRS 0–2 vs 3–6 analysis (table 2, figures 3 and 4). The 6 487 gene-based analysis also showed a suggestive association for Outcomes, n PTCH1 with mRS 0–2 vs 3–6 (p = 6.8 × 10−5). Rs2236406 is mRS 0–2 vs 3–6 3,741 vs 2,280b an eQTL for long noncoding RNA RP11-435O5.5 (pGTEx = 5.7 × 10−7), which overlaps the PTCH1 gene. Second, in the Ordinal mRS 6,021b ordinal analysis, a suggestive association with mRS was found mRS 0–1 vs 2–6 1,796 vs 2,567b for rs13299556 (table 2, figures 3 and 4). Associations at low p values were also found for this variant with dichotomous mRS, Abbreviations: IQR = interquartile range; mRS = modified Rankin Scale; NIHSS = NIH Stroke Scale. although not reaching the level for suggestive association (table 2). a Cases for which data from the Swedish quality register for stroke (Riks- rs13299556 is an intron variant in the PLAA gene. There was stroke) were used to assess mRS. This approach prevented differentiation among mRS 0, 1, and 2. no strong association for PLAA in the gene-based analysis (ordinal mRS; p = 0.072). However, this variant, and variants in b Numbers refer to the primary, fully adjusted model, including age, sex, principal components, and baseline NIHSS score. Models without adjustment for baseline NIHSS score included 3,830 vs 2,335 (mRS high linkage disequilibrium with it (figure 4), are reported as 0–2 vs 3–6), 6,165 (ordinal), and 1,832 vs 2,620 (mRS 0–1 vs 2–6) participants. eQTLs for the nearby genes TEK and LRRC19 (pGTEx = 1.6 × 10−6 for both). The p values from the gene-based analysis for Table 2 Comparison between analyses of mRS 0–2 vs 3–6 and ordinal scale mRS of selected genetic variants with either a significant association with outcome or a suggestive association and a link to a gene with experimental support for influence on poststroke outcome mRS 0–2 vs 3–6 Ordinal mRS Primary Not adjusted for NIHSS Primary Not adjusted for NIHSS Marker MA MAF OR p Value OR p Value OR p Value OR p Value −9,a −7 −4 rs1842681 A 0.23 1.40 5.27 × 10 1.30 1.46 × 10 1.17 1.53 × 10 1.19 2.98 × 10−5 rs2236406 C 0.36 1.27 3.43 × 10−6,a 1.21 1.98 × 10−5 1.13 9.11 × 10−4 1.11 3.96 × 10−3 rs13299556 C 0.27 1.25 6.25 × 10−5 1.22 2.93 × 10−5 1.20 5.69 × 10−6 1.21 7.59 × 10−7,a rs78734480 C 0.03 0.57 4.79 × 10−4 0.66 2.34 × 10−3 0.58 1.03 × 10−6,a 0.62 5.00 × 10−6 Abbreviations: MA = minor allele; MAF = minor allele frequency; mRS = modified Rankin Scale; NIHSS = NIH Stroke Scale; OR = odds ratio. ORs with effects above unity indicate a higher mRS (worse outcome) per copy of the minor allele. Primary model includes age, sex, principal components, and baseline NIHSS score. a Indicates the outcome and model with the lowest p value for each marker. e1276 Neurology | Volume 92, Number 12 | March 19, 2019 Neurology.org/N Figure 3 Forest plots for functional outcome at 3 months by study cohort The plots show ORs and 95% confidence intervals for minor allele of (A) rs1842681 (mRS 0–2 vs 3–6), (B) rs2236406 (mRS 0–2 vs 3–6), (C) rs13299556 (ordinal mRS), and (D) rs78734480 (ordinal mRS). ORs with effects above unity indicate a higher mRS score (worse outcome) per copy of the minor allele. Results are adjusted for age, sex, principal components, and baseline NIH Stroke Scale score. For cohort abbreviations, see data available from Dryad (table e-1, doi.org/ 10.5061/dryad.s38kf65). mRS = modified Rankin Scale; OR = odds ratio. those genes were 0.0019 and 0.084, respectively. The variant Discussion is also predicted to alter a putative regulatory motif sequence (HaploReg, March 21, 2018). Third, a suggestive association This GWA study on functional outcome after ischemic stroke, with ordinal mRS was found for an intron variant in NTN4 including more than 6,000 patients, identified one significant (rs78734480; table 2, figures 3 and 4). This variant was also locus and several suggestive variants related to genes with associated with dichotomous mRS at a low p value, although a potential mechanism for influencing stroke recovery and not below the cutoff for suggestive association (table 2). outcomes. Replication of these findings in independent datasets as they become available is the essential next step. Analysis of lacunar and nonlacunar stroke The effect sizes of these genetic variants were generally small, To evaluate whether any strong associations could be identified and further studies should include even larger samples to specifically in patients with lacunar stroke (small vessel disease identify additional variants associated with stroke outcome strokes, n = 992), or in nonlacunar stroke (other etiologic sub- and to enable subgroup analyses. types including cortical infarcts, n = 3,991), we analyzed those groups separately. No genome-wide significant association was The genome-wide significant SNP (rs1842681) for mRS 0–2 detected (all p values >6 × 10−7). The top 5 findings differed vs 3–6 is an intronic variant in the gene LOC105372028 (long between these subgroups and are shown in data available from noncoding RNA), the function of which remains to be estab- Dryad (tables e-10 and e-11, doi.org/10.5061/dryad.s38kf65). lished. However, expression analyses show that the gene Neurology.org/N Neurology | Volume 92, Number 12 | March 19, 2019 e1277 Figure 4 Regional association plots for outcome at 3 months after ischemic stroke onset (A) Significant locus (rs1842681) showing association with mRS 0–2 vs 3–6, (B) rs2236406 (intron variant in the PTCH1 gene) showing suggestive association with mRS 0–2 vs 3–6, (C) rs13299556 (intron variant in the PLAA gene with eQTL for TEK, pGTEx = 1.6 × 10−6) showing suggestive association with ordinal mRS, and (D) rs78734480 (intron variant in the NTN4 gene) showing suggestive association with ordinal mRS. Results are adjusted for age, sex, principal components, and baseline NIH Stroke Scale score. LOC105372028 (indicated in red, panel A) has been overlaid from the Genome Reference Consortium Human Build (GRCh) 38/hg38, as it was missing from GRCh37/hg19. The rs1842681 variant is intronic of the LOC105372028 gene (chromosome 18: 24725781–24766645). Position for rs1842681 in GRCh38/hg38, 18:24761199. eQTL = expression quantitative trait locus; GTEx = Genotype-Tissue Expression; mRS = modified Rankin Scale; SNP = single-nucleotide polymorphism. expression of LOC105372028 is highest in brain tissue (Na- needed to corroborate this finding and clarify the potential role tional Center for Biotechnology Information, ncbi.nlm.nih. of this locus for functional outcome after stroke. gov/gene/?term=LOC105372028) and rs1842681 is located in a putative regulatory element binding site (HaploReg, March Twenty-nine independent loci were associated with ischemic 21, 2018). Furthermore, rs1842681 is a trans-eQTL for stroke outcome at the predefined suggestive association level. KLRAQ1, also known as PPP1R21, which encodes a regulatory Three of these loci are linked to genes with experimental subunit of protein phosphatase 1 (PP1). PP1 is a ubiquitous evidence of influence on outcome from animal models of phosphatase implicated in many brain functions including stroke.19–21 First, the intronic variant in PLAA (rs13299556) learning and memory formation.22,23 PP1 is also a key regulator was both one of the top findings in the ordinal analysis and of Ca2+/calmodulin (CaM)-dependent protein kinase II associated with the dichotomized outcome at a low p value. It (CaMKII) signaling, which is crucial for Ca2+-mediated neu- is located in a putative transcription factor binding site and is ronal plasticity in the brain.24 Thus, although speculative, reported to be an eQTL for the expression of both LRRC19 rs1842681 may modulate expression of PPP1R21, which in and TEK. LRRC19 encodes a protein with a potential role in turn could affect brain plasticity and thereby outcome post regulating neurite outgrowth,25 and might thus influence stroke, a hypothesis that requires validation through functional stroke recovery. TEK encodes a tyrosine kinase pre- experiments. The association for rs1842681 with outcome was dominantly expressed in endothelial cells (ECs). Focal observed after adjusting for baseline NIHSS score, suggesting upregulation of TEK has been demonstrated in capillaries at a mechanism that is independent of initial stroke severity. the border of infarcted myocardium, and expression is in- However, additional data from further studies are clearly duced by hypoxia and proinflammatory cytokines in human e1278 Neurology | Volume 92, Number 12 | March 19, 2019 Neurology.org/N ECs in vitro.26 TEK is activated by angiopoietin-1 and this Several additional variants with suggestive association to promotes migration, sprouting, and survival of ECs.27,28 Of outcome in this study are linked to genes that may potentially note, results from a rodent ischemic stroke model suggest that be mechanistically involved in processes affecting stroke upregulation of angiopoietin-1 and TEK improves stroke outcomes. For instance, RUNX1 encodes a runt-related outcome,19 and one clinical study reported an association proangiogenic transcription factor with high expression in between high plasma levels of angiopoietin-1 and poor out- arteries (GTEx, June 1, 2018) that is upregulated after come as assessed by mRS at 3 months after ischemic stroke.29 stroke.39 In mice, expression of RUNX1 is induced in putative Angiopoietin-2 is the antagonist of angiopoietin-1.30 neural stem or progenitor cells after brain injury and sug- Angiopoietin-2 gain-of-function mice have enhanced blood- gested to promote neuronal differentiation.40 TNR encodes brain barrier permeability and increased brain infarct sizes a protein that is involved in neuronal plasticity and is highly upon permanent middle cerebral occlusion compared to wild- and exclusively expressed in the brain.41,42 Examples of genes type mice, and both phenotypes were rescued by activation of with a possible stroke subtype-specific significance among TEK signaling.31 The same study reported increased circu- suggestive findings are MTHFS, which is involved in folate lating serum concentrations of angiopoietin-2 in patients with and homocysteine metabolism, pathways with potential in- acute ischemic stroke compared to controls.31 Finally, in an fluence on cerebral small vessel disease,43 and SCML4, in ischemic stroke model, mice with type 2 diabetes mellitus which the variant rs74514008 was suggestively associated with showed increased angiopoietin-2 but decreased angiopoietin- ordinal mRS in the present study. SCML4 has shown asso- 1/TEK protein expression compared to wild-type mice, sug- ciation with coronary artery disease in a recent GWA study, gesting that the TEK signaling may be involved in diabetes- and subsequent functional characterization suggested a role in induced vascular damage post stroke.32 atherosclerosis.44 Second, an intronic variant in the PTCH1 gene showed sug- To further explore the SNPs associated with outcomes in gestive association with mRS 0–2 vs 3–6. PTCH1 is involved different subgroups, such as certain stroke subtypes, patients in sonic hedgehog signaling, a pathway that for instance with diabetes, or those receiving recanalization therapies reduces oxidative stress on neurons and regulates ischemia- would be of great interest. Mechanisms of neuron injury and induced neuronal progenitor proliferation.33,34 In rats with recovery probably have similarities regardless of the etiology, experimentally induced middle cerebral artery occlusion, in- but mechanism of recovery after cortical and subcortical hibition of the sonic hedgehog signaling caused increased stroke may be different. Therefore, we analyzed mRS 0–2 vs infarct size, and PTCH1 was downregulated when sonic 3–6 in patients with lacunar and nonlacunar stroke separately. hedgehog signaling was inhibited.20,35 The association be- The top 5 independent loci differed between the 2 groups, but tween PTCH1 and stroke outcome observed in the present no genome-wide significant association was detected. Given study may thus potentially be explained by an increased in- the small sample sizes, especially for lacunar stroke, these farct size. However, the effect of this variant was somewhat analyses are clearly exploratory. Thus, in further studies with stronger when adjusting for baseline NIHSS score, which may a higher number of patients, an important aim will be to suggest a potential influence also on recovery processes. further explore the genetic associations in specific subgroups. Third, a variant in NTN4 showed suggestive association with The strengths of the present study include a relatively large ordinal mRS and was also associated with dichotomous mRS sample size, well defined endpoints, extensive genetic data, and at a low p value. NTN4 encodes a member of the netrin that analyses were adjusted for NIHSS score to assess the family of proteins expressed in brain tissue (ncbi.nlm.nih. influence on outcome independent of baseline stroke severity. gov/gene/59277) and with functions in processes with bi- We performed dichotomous outcome analyses to identify ge- ologically plausible roles in stroke recovery such as angio- netic variants associated with being dependent or independent genesis, neurite growth, and migration.36,37 In a mouse in activities of daily living, which reflects a clinically important model, NTN4 was upregulated in blood vessels and astro- difference in functional outcome. We also performed ordinal cytes in the ischemic core after stroke, and intra- analysis, which aims to identify variants that have a similar effect cerebroventricular administration of NTN4 led to across different degrees of functional outcome. To detect such improvements both in angiogenesis and in poststroke out- variants, the ordinal model provides greater power.8 Another come, possibly through increased collateral blood flow strength of the ordinal approach is the ability to compensate for leading to improved survival of neurons partially affected by any differences in mRS assessment between studies. ischemia.21 In another study, NTN4 contributed to thala- mocortical axon branching in rodents and the expression was There are also several limitations that should be considered. altered by neuronal activity in the cortex, implying that Although this study included more than 6,000 patients with NTN4 might act as a positive regulator for thalamocortical ischemic stroke, only one significant locus was identified, and axon branching through activity-dependent expression.38 thus, the findings do not explain the assumed genetic variation Although highly speculative, this gene could thus have a role for ischemic stroke outcomes. This implies that the effect sizes in the plastic process of axonal outgrowth and restoration of of individual SNPs on outcome of ischemic stroke are small synaptic architecture post stroke. and that our sample size might be insufficient even to detect Neurology.org/N Neurology | Volume 92, Number 12 | March 19, 2019 e1279 common genetic variants. Another important limitation is the Clinical Neuroscience Institute of Neuroscience and Physi- explorative nature of the results, which clearly require future ology (K.J., T.T.), Sahlgrenska Academy at the University of replication in independent cohorts as they become available. Gothenburg; Department of Clinical Genetics and Genomics The use of the mRS as the primary outcome metrics is a clear (A.P., C.J.) and Department of Neurology (K.J.), Sahlgrenska limitation in that it is a crude measurement of outcome, es- University Hospital, Gothenburg; Bioinformatics Core Fa- pecially when dichotomized. However, the mRS represents cility (E.L.), University of Gothenburg, Sweden; School of a well-established, formalized, and easily available measure of Life Sciences (S.B.), University of Lincoln, UK; Department poststroke functional outcome, and the dichotomizations of Neurology (J.C.), University of Maryland School of Med- represent differences of great importance clinically and for the icine and Baltimore VAMC; Stroke Pharmacogenomics and patient. The mRS scores were assessed at different time points Genetics (I.F.-C.), Institut d’investigació Biomedica de Sant ranging from 60 to 190 days, potentially diluting any detect- Pau, Hospital de Sant Pau; Neurovascular Research Labora- able associations, as functional outcome status may vary over tory and Neurovascular Unit (I.F.-C.), Neurology and Med- time. However, the majority of included studies assessed mRS icine Departments, Universitat Autònoma de Barcelona, Vall at 3 months ± 2 weeks after stroke onset. We did not have d’Hebrón Hospital, Barcelona, Spain; Medical School (G.J.H.), information on premorbid mRS and this would have provided The University of Western Australia, Perth; Department of a valuable means to calculate the change in functional ability. Neurology (J.J.-C.), Institut Hospital del Mar d’Investigació Moreover, stroke severity was scored at different time points. Mèdica (IMIM), Barcelona; Universitat Autònoma de Barce- However, in a majority of cases, NIHSS was scored early and lona (J.J.-C.), Barcelona, Spain; Department of Neurology in only 160 individuals later than day 3 after admission. As (J.-M.L.), Washington University School of Medicine, St. previously described,8 we lack data on some important factors Louis, MO; Department of Neurosciences (R.L.), Experi- that influence outcomes such as acute therapies and re- mental Neurology, KU Leuven, University of Leuven; Center habilitation, which is why these factors could not be accounted for Brain & Disease Research (R.L.), Laboratory of Neurobi- for in the analyses. However, data from the cohorts with ology, VIB, Leuven; Department of Neurology (R.L.), Uni- available data on treatment with tissue plasminogen activator versity Hospitals Leuven, Belgium; School of Medicine and (about half of the cohorts) show that this treatment was given Public Health (C.L., R.S., J.S.), Hunter Medical Research In- to 14% of the patients. We can also not exclude the possibility stitute (C.L., R.S.), and Priority Research Centre for Stroke and of selection bias since participation in the study required in- Traumatic Brain Injury (C.L.), University of Newcastle, Aus- dividual informed consent, and data entry also requested tralia; Department of Medicine (B.D.M.), University of availability of functional status follow-up data. In line with this, Maryland, Baltimore; Geriatric Research, Education, and our study sample mainly reflects milder strokes (median Clinical Center (B.D.M.), Veterans Affairs Medical Center, NIHSS score of 4, table 1), which may hamper the detection of Baltimore, MD; Centre for Clinical Brain Sciences (K.R., C.S.), factors influencing a greater dynamic range of recovery. Lastly, University of Edinburgh, UK; J. Philip Kistler Stroke Research because our study population was of European ancestry, the Center, Department of Neurology (N.R., J.R.), Massachusetts results are not necessarily generalizable to other stroke pop- General Hospital, Harvard Medical School, Boston; Program in ulations. Further studies should ideally include a series of Medical and Population Genetics (J.R.), Broad Institute of prespecified collection time points, specific clinical variables of MIT and Harvard, Cambridge; Center for Genomic Medicine importance for stroke outcome, and a battery of outcome (J.R.), Massachusetts General Hospital, Boston; Stroke Pre- metrics that assess several domains of recovery including motor vention Research Unit (P.M.R.), Nuffield Department of impairment, aphasia, neglect, and cognitive impairment. Clinical Neurosciences, University of Oxford, UK; Department of Neurology (D.S., T.T.), Helsinki University Hospital, Fin- In this first large international GWA study on functional outcome land; Department of Clinical Neurosciences (M.T.), University after overall ischemic stroke, we report one significant variant and of Cambridge, UK; Stroke Division (V.T.), Florey Institute for several variants with suggestive association to outcome at 3 Neuroscience and Mental Health, University of Melbourne, months after stroke onset with plausible links to poststroke re- Heidelberg; Department of Neurology (V.T.), Austin Health, covery. Future studies on common variants should include larger Heidelberg, Australia; Department of Neurology and Re- sample sizes, enabling subgroup analyses, as well as replication of habilitation (D.W.), University of Cincinnati College of Med- the present results and elaboration of potential mechanisms. icine, OH; Departments of Neurology and Health Evaluation Sciences (B.B.W.), University of Virginia, Charlottesville; University of Technology Sydney (J.M.M.), Faculty of Health, Australia; and Hunter Medical Research Centre (J.M.M.), PRC Author Affiliation Stroke and Brain Injury, Newcastle, Australia. From the Department of Clinical Sciences Malmö (M.S.) and Department of Clinical Sciences Lund, Neurology (B.N., A.L.), Lund University; Department of Neurology and Re- Acknowledgment habilitation Medicine (M.S., B.N., A.L.), Neurology, Skåne The authors thank the Swedish quality register for stroke, University Hospital, Lund and Malmö; Institute of Bio- Riksstroke, collaboration for providing information about medicine (A.P., T.M.S., M.O., C.J.) and Department of outcome measures for some of the included studies. e1280 Neurology | Volume 92, Number 12 | March 19, 2019 Neurology.org/N Study funding The Stroke Genetics Network (SiGN) study was funded by Appendix Authors a cooperative agreement grant from the US National In- Author Location Contribution stitute of Neurological Disorders and Stroke (NINDS), Martin Lund University, Lund; Analysis and NIH (U01 NS069208 and R01 NS100178). SAHLSIS was Söderholm, Skane University Hospital, interpretation of the supported by the Swedish Heart and Lung Foundation MD, PhD Lund and Malmö, Sweden data, drafting the manuscript for (HLF-20160316), the Swedish Research Council (K2014- intellectual content 64X-14605-12-5), the Swedish Stroke Association, the Swedish state (under the “Avtal om Läkarutbildning och Annie Pedersen, University of Gothenburg, Sweden Analysis and interpretation of the Medicinsk Forskning, ALF”) (ALFGBG-720081). Austra- MD data, drafting the lian Stroke Genetics Collaboration study was supported by manuscript for intellectual content the National Health and Medical Research Council, Aus- tralia. Stroke Pharmacogenomics and Genetics group was Erik University of Gothenburg, Design and Lorentzen, Sweden conceptualization of the supported by Invictus plus network, Generation project, and MSc study, analysis and Miguel Servet programme from Instituto de Salud Carlos III, interpretation of the data, drafting part of the GODs project and Epigenesis project from Marató de TV3 methods section of the Foundation and Agaur from Generalitat de Catalunya Gov- manuscript ernment. Arne Lindgren was supported by the Swedish Heart Tara M. University of Gothenburg, Analysis of the data, and Lung Foundation, Region Skåne, Skåne University Hos- Stanne, PhD Sweden revising the manuscript for intellectual content pital, the Freemasons Lodge of Instruction EOS in Lund, Lund University, the Foundation of Färs & Frosta—one of Spar- Steve Bevan, University of Lincoln, UK Analysis of the data, PhD revising the manuscript banken Skåne’s ownership Foundations, and the Swedish for intellectual content Stroke Association. Martin Söderholm was supported by grants from the Swedish Stroke Association, the Foundation Maja Olsson, University of Gothenburg, Analysis of the data, MSc, PhD Sweden revising the manuscript of Färs & Frosta—one of Sparbanken Skåne’s ownership for intellectual content Foundations, and the Swedish government (under the “Avtal John W. Cole, University of Maryland Major role in the om Läkarutbildning och Medicinsk Forskning, ALF”). Annie MD School of Medicine and acquisition of data Pedersen was supported by grants from the Swedish gov- Baltimore VAMC ernment (under the “Avtal om Läkarutbildning och Medi- Israel Hospital de Sant Pau, Spain; Design or cinsk Forskning, ALF”) and the Gothenburg Foundation for Fernandez- Vall d’Hebrón Hospital, conceptualization of the Cadenas, Barcelona study, major role in the Neurological Research. Natalia Rost was in part supported MSc, PhD acquisition of data, by NIH-NINDS (R01NS086905 and R01NS082285). revising the manuscript for intellectual content Daniel Strbian was supported by the Finnish Subsidiary Governmental Fund (VTR). The authors thank NINDS for Graeme J. The University of Western Major role in the Hankey, MD Australia, Perth acquisition of data, funding the genotyping of patients included in the SiGN revising the manuscript study (U01 NS069208 and R01 NS100178) and Sólveig for intellectual content Grétarsdóttir for genotyping a subsample of the SAHLSIS Jordi Institut Hospital del Mar Design or cohort. Jimenez- d’Investigació Mèdica conceptualization of the Conde, MD, (IMIM), Barcelona; study, major role in the PhD Universitat Autònoma de acquisition of data Disclosure Barcelona, Spain M. Söderholm, A. Pedersen, E. Lorentzen, T. Stanne, S. Bevan, M. Olsson, J. Cole, I. Fernandez-Cadenas, G. Hankey, J. Katarina University of Gothenburg, Major role in the Jood, MD, Sweden acquisition of data, Jimenez-Conde, K. Jood, J. Lee, R. Lemmens, C. Levi, B. PhD revising the manuscript Mitchell, B. Norrving, K. Rannikmäe, N. Rost, J. Rosand, P. for intellectual content Rothwell, R. Scott, D. Strbian, J. Sturm, C. Sudlow, M. Traylor, Jin-Moo Lee, Washington University Design or V. Thijs, T. Tatlisumak, D. Woo, B. Worrall, and J. Maguire MD, PhD School of Medicine, St. conceptualization of the Louis, MO study, major role in the report no disclosures relevant to the manuscript. A. Lindgren acquisition of data, reports personal fees for advisory board, speech, seminar par- revising the manuscript for intellectual content ticipation from Bayer, AstraZeneca, Boehringer Ingelheim, BMS Pfizer, ReNeuron. C. Jern reports no disclosures relevant Robin University of Leuven; VIB, Design or Lemmens, Leuven; University conceptualization of the to the manuscript. Go to Neurology.org/N for full disclosures. MD, PhD Hospitals Leuven, Belgium study, major role in the acquisition of data, Publication history revising the manuscript for intellectual content Received by Neurology July 6, 2018. Accepted in final form November 9, 2018. Continued Neurology.org/N Neurology | Volume 92, Number 12 | March 19, 2019 e1281 Appendix (continued) Appendix (continued) Author Location Contribution Author Location Contribution Christopher University of Technology Major role in the Bradford B. University of Virginia, Design or Levi, MD Sydney; University of acquisition of data Worrall, MD Charlottesville conceptualization of the Newcastle, Australia study, major role in the acquisition of data Braxton D. University of Maryland, Major role in the Mitchell, Baltimore; Veterans Affairs acquisition of data, Jane M. University of Technology Design and PhD, MPH Medical Center, Baltimore, revising the manuscript Maguire, RN, Sydney; Hunter Medical conceptualization of the MD for intellectual content PhD Research Centre, study, major role in the Newcastle, Australia acquisition of data, Bo Norrving, Lund University, Lund; Major role in the interpretation of the data, MD, PhD Skane University Hospital, acquisition of data, revising the manuscript for Lund and Malmö, Sweden revising the manuscript intellectual content for intellectual content Arne Lund University, Lund; Design and Kristiina University of Edinburgh, UK Major role in the Lindgren, Skane University Hospital, conceptualization of the Rannikmäe, acquisition of data MD, PhD Lund and Malmö, Sweden study, major role in the MD, PhD acquisition of data, analysis and Natalia S. Harvard Medical School, Design or interpretation of the Rost, MD, Boston MA conceptualization of the data, revising the MPH study, major role in the manuscript for acquisition of data, intellectual content revising the manuscript for intellectual content Christina University of Gothenburg, Design and Jern, MD, PhD Sweden conceptualization of the Jonathan Harvard Medical School, Major role in the study, major role in the Rosand, MD, Boston; Broad Institute of acquisition of data, acquisition of data, MSc MIT and Harvard, revising the manuscript analysis and Cambridge; Massachusetts for intellectual content interpretation of the General Hospital, Boston data, revising the manuscript for Peter M. University of Oxford, UK Major role in the intellectual content Rothwell, acquisition of data MD, PhD References Rodney University of Technology Major role in the 1. Lindgren A, Maguire J. Stroke recovery genetics. Stroke 2016;47:2427–2434. 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