Proceedings of the 27th International Stroke Genetics Consortium Workshop

1. Proceedings of the 27th International Stroke Genetics Consortium Workshop

Stéphanie Debette, MD, PhD ¹ , Aniket Mishra, PhD ¹ , Rufus Akinyemi, MD, PhD ² , and Mayowa Owolabi, MD, PhD ²

¹Bordeaux Population Health Inserm Center U1219, University of Bordeaux, France; and ²The Center for Genomic and Precision Medicine, University of Ibadan, Nigeria.

Abstract

The 27th workshop of the International Stroke Genetics Consortium (ISGC) was held at the historical site of the University of Bordeaux on September 22-23, 2022 in Bordeaux, France, and was co-hosted by Stéphanie Debette, Aniket Mishra (University of Bordeaux, France), Rufus Akinyemi, and Mayowa Owolabi (University of Ibadan, Nigeria). The ISGC is an international collaboration of physicians and scientists who have agreed to pool resources and expertise in an effort to unravel the genetic basis of stroke and its comorbidities. Founded in 2007 by a small group of stroke genetics investigators, the ISGC has grown to over 200 members representing over 50 countries in North and South America, Europe, Australia, Africa, and Asia. ISGC workshops are held semi-annually and provide a forum for ISGC investigators to report on progress of ongoing scientific projects and discuss new ideas that help advance the ISGC's research mission. The ISGC nurtures junior investigators by offering several travel awards to young investigators interested in stroke genetics. Many abstracts in these Proceedings reflect the contributions of active junior investigators. Herein, we present the Proceedings and official published abstracts of the 27th ISGC Workshop.

Presentations at the workshop included invited lectures, ISGC working group reports, and short presentations about ongoing research projects and new collaborative proposals. Invited speakers, contributing in the areas of cross-ancestry approaches to stroke genetics, large biobank initiatives, rare genetic variants, imaging tools for cerebrovascular genomics and genetic contributions to drug discovery, included Ananyo Choudhury (University of the Witwatersrand), on cutting-edge genetic studies in African populations; Elisabeth Tournier-Lasserve (Paris Cité University), one of the world's lead experts of monogenic diseases affecting cerebral small vessels; Michel Thiébaut de Schotten (University of Bordeaux), an international expert in neuroimaging and connectomics; and Vincent Mooser (McGill University), holder of the Canada excellence research chair in genomic medicine and expert in drug discovery and precision drug development.

The ISGC actively invites other like-minded collaborative scientists to join the consortium.

The next Workshops will be held in Melbourne, Australia in March 2023. We encourage you to visit our website (strokegenetics.org) for further details.

On behalf of the ISGC Steering Committee and its many members, we hope that you enjoy reviewing these Proceedings.

Acknowledgement Steering Committee- Jin-Moo Lee, Chair and Israel Fernandez-Cadenas, Co-Chair; Members: Stephanie Debette (Immediate Past-Chair), Ann-Katrin Giese (Junior member); Rufus Akinyemi, Jemma Hopewell, Steven Kittner, Jane Maguire, Paul Nyquist, Natalia Rost. Working Group Leaders- Acute Endophenotypes WG: Israel Fernandez-Cadenas, Jin-Moo Lee; Cognitive WG: Matt Pase, Brad Worrall; Imaging WG: Natalia Rost; Intracranial Aneurysm WG: Ynte Ruigrok, Philippe Bijlenga; Intracerebral Hemorrhage WG: Guido Falcone, Jonathan Rosand, Dan Woo; Meta/MegaStroke WG: Stephanie Debette, Martin Dichgans, Jemma Hopewell; Mutliomics WG: Carlos Cruchaga, Myriam Fornage; Neuro-CHARGE WG: Myriam Fornage, Sudha Seshadri; SiGN WG: Steven Kittner, Brackie Mitchel; Translational Science WG: Chris Anderson, Tom Van Agtmael.

Contact Email: stephanie.debette@u-bordeaux.fr

2. The influence of vascular risk factors and genetic factors on penetrance of variants causing monogenic stroke

B.P.H. Cho, MSc ¹ , E.L. Harshfield, PhD ¹ , M. Al-Thani, BSc ¹ , D.J. Tozer, PhD ¹ , S. Bell, PhD ¹ , and H.S. Markus, F Med Sci ¹

Objective To determine the frequency of pathogenic variants in the 3 most common monogenic cerebral small vessel disease and their associations with both prevalent and incident stroke and dementia. Background NOTCH3, HTRA1 and COL4A1/2 variants identical to those causing monogenic forms of stroke appear more common than expected in the general population. However, their association with stroke and dementia risk, and factors that may modify their penetrance in the community remain unclear. Design/Methods HTRA1 and COL4A1/2 variants, reported in familial cerebral small vessel disease, were examined for pathogenicity using the American College of Medical Genetics and Genomics guidelines. The variants, along with the established pathogenic cysteine-altering variants in NOTCH3, were identified in UK Biobank whole-exome sequencing data. Frequency of stroke, dementia and other clinical manifestations of familial stroke, and MRI features were compared between variant carriers and non-carriers. Statistical tests for interaction were performed for Framingham cardiovascular risk score, ischemic stroke polygenic risk score and variant status. Results Of 454,787 participants, 973 carried NOTCH3 variants, 546 carried HTRA1 variants, and 336 carried COL4A1/2 variants. After adjustment for covariates, variant carriers were at least 66% more likely to have a history of stroke. Additionally, NOTCH3 carriers had increased risk of vascular dementia (OR = 5.41, 95% CI 3.10–8.72), HTRA1 carriers an increased risk of all-cause dementia (OR = 2.15, 95% CI 1.27–3.39), and COL4A1/2 carriers an increased risk of intracerebral hemorrhage (OR = 3.55, 95% CI 1.33–7.50). NOTCH3 and HTRA1 carriers had MRI features consistent with monogenic SVD. Cardiovascular risk factors and variant location affected the penetrance of these associations. Conclusions Pathogenic variants in NOTCH3, HTRA1 and COL4A1/2 are risk factors for apparently “sporadic” stroke and dementia in the community. Intensive cardiovascular risk factor modification is likely to reduce stroke and dementia risk in individuals with these variants.

Contact Email: phc37@medschl.cam.ac.uk

3. Epigenetic and integrative cross-omics analyses of cerebral white matter hyperintensities on MRI

Y. Yang ¹ , M.J. Knol ² , R. Wang ³ , A. Mishra ⁴ , D. Liu ⁵ , M.M.B. Breteler ⁵ , M.A. Ikram ² , H.J. Grabe ⁶ , J. Wardlaw ⁷ , W.T. Longstreth Jr. ⁸ , L.J. Launer ⁹ , S. Seshadri ¹⁰ , S. Debette ⁴ , and M. Fornage ¹ ; for NeuroCHARGE

Objective To identify variation in DNA methylation (DNAm) that influences cerebral white matter hyperintensities (WMH) burden. Background Cerebral WMH on MRI are markers of cerebral small vessel disease (cSVD), a major risk factor for stroke, dementia, and death. Despite the successful identification of multiple genetic variants associated with this highly heritable condition, its genetic architecture remains incompletely understood. More specifically, the role of DNAm has received little attention. Design/Methods We investigated the association of blood DNAm at approximately 450,000 CpG sites with WMH burden in 9,732 middle to late age adults from 14 cohorts. Integrative cross-omics analyses followed to elucidate the role of identified DNAm in cSVD. Results We identified 12 single-CpG and 46 region-based DNAm associations with WMH burden. Our top discovery single CpG, cg24202936 (p = 7.6 × 10⁻⁸), was associated with F2 expression in blood (p = 6.4 × 10⁻⁵), and colocalized with FOLH1 expression in brain (posterior probability (PP) = 0.75). Some single-CpG loci (cg17417856 and cg06809326) with suggestive evidence (p < 1 × 10⁻⁵) were also identified as differentially methylated regions (DMRs) in PRMT1 and in CCDC144NL-AS1. Mendelian randomization analyses showed that cg06809326 is causally associated with WMH burden (OR [95% CI]: 1.39 [1.03–1.87], p = 0.03) and that expression of CCDC144NL-AS1 mediates this association. DMR analysis, joint epigenetic association analysis, and multi-omics colocalization analysis consistently identified a role of DNAm near SH3PXD2A, a locus previously identified in genome-wide association studies of WMH. Gene set enrichment analyses revealed functions of the WMH-associated DNAm loci in the blood-brain barrier (BBB) and in the immune response. Integrative cross-omics analysis identified 19 key regulatory genes in 2 WMH-associated networks related to extracellular matrix (ECM) organization, and lipid and lipoprotein metabolism. A drug repositioning analysis indicated antihyperlipidemic agents, more specifically peroxisome proliferator-activated receptor alpha, as possible target drugs for WMH. Conclusions Our epigenome-wide association study and integrative cross-omics analyses implicate novel genes influencing WMH burden, which converged on pathways related to the immune response and to a compromised BBB possibly due to disrupted cell-cell and cell-ECM interactions. The results also suggest that antihyperlipidemic therapy may contribute to lowering risk for WMH possibly through protection against BBB disruption.

Contact Email: myriam.fornage@uth.tmc.edu

4. Prospective case recruitment and improved imputation in China Kadoorie Biobank enable GWAS identification of novel loci associated with intracerebral haemorrhage

R.G. Walters, PhD ^1,2 , K. Lin, PhD ¹ , I.Y. Millwood, DPhil ^1,2 , I. Turnbull, MRCP ¹ , and Z. Chen, DPhil ^1,2

Objective To identify genetic loci associated with risk of intracerebral haemorrhage (ICH), we have conducted genome-wide association studies (GWAS) in the China Kadoorie Biobank (CKB). Background GWAS provide an invaluable means of identifying biological pathways potentially involved in disease risk. This approach has been highly successful when applied to investigation of ischaemic stroke (IS) and its subtypes. By contrast, very few loci associated with ICH have been identified, partly due to limited numbers of available cases; not only is ICH less common than IS, but it has high associated mortality and morbidity, impeding retrospective recruitment of ICH cases. Design/Methods CKB is a population-based prospective cohort of >512,000 adults recruited in 2004–2008 from 10 geographically diverse regions across China. By 1 January 2019, electronic linkage to death and disease registries and to the national health insurance system recorded over 12,000 incident cases of ICH, of which approximately 45% were fatal. Genotyping has been completed for >100,000 CKB participants, with imputation of 51.6M variants (info >0.3) using reference panels from TopMed and the Westlake Biobank for Chinese. Using SAIGE software, we conducted GWAS for 6,499 cases of ICH and 61,950 stroke-free controls. Results GWAS of ICH yielded 2 novel associations near to PCSK9 (chr1 p32.3; MAF = 0.3%; OR = 3.7 [95% CI 2.3–5.9]; p = 2.0E-08) and KBTBD6 (chr3 q14.11; MAF = 0.5%; OR = 2.8 [1.9–3.9]; p = 2.5E-08). Both lead variants are extremely rare or absent in European populations. There was no replication of previously-reported ICH associations, although a signal at COL4A1/COL4A2 distinct from that previously-reported was identified at suggestive significance (p = 8.2E-07). Conclusions In conclusion, incident disease follow-up in the prospective CKB cohort has captured large numbers of ICH cases, enabling GWAS at sufficient power to identify associated loci at genome-wide significance. Novel findings compared with previous results from individuals of European ancestry emphasize the importance of conducting GWAS in ancestrally-diverse populations.

Contact Email: robin.walters@ndph.ox.ac.uk

5. Exploiting family history increases association power for common and rare variant association analyses in stroke

R. Malik ¹ , M.K. Georgakis ^1,2,3,4,5 , C.D. Anderson ^2,3,4,6 , and M. Dichgans ^1,7,8

Objective To explore whether including family history of stroke increases power not only for common variant discovery, but also for rare variant discovery. Background While the number of common variants associated with any stroke, any ischemic stroke and ischemic stroke subtypes has significantly increased in recent genome-wide association studies, rare variant studies for stroke have thus far shown limited capacity for discovery of novel signals. In the UK Biobank, where whole-exome sequencing data are available for approximately 450,000 subjects with clinical outcome data, the power for locus discovery for stroke is low, mostly due to a small number of stroke events. Harnessing information on parental and/or sibling information has been shown to enhance the power for common variant discovery up to 63%. However, whether this approach likewise, increases the potential for rare variant discovery, remains unexplored. Design/Methods To address this, we employed the liability threshold model on family history (LT-FH) for stroke on 424,504 unrelated UK Biobank participants of European ancestry and compared it to the classical case-control phenotype. Results Starting with common variants, we identified 11 and 10 genome-wide significant loci for any stroke and any ischemic stroke, respectively, for the LT-FH phenotype, while the case-control phenotype revealed only one locus. Global genetic correlation with the gold standard GIGASTROKE data set was similar for the LT-FH and case-control phenotypes, whereas local genetic correlation at the 60 GIGASTROKE GWAS loci was significantly higher for the LT-FH phenotype compared to the case-control phenotype. Focusing on rare variants and established Mendelian stroke genes i.e., NOTCH3 and HTRA1, we identified a substantial power increase for discovery for the LT-FH phenotype. Performing whole-exome gene aggregation tests on both phenotype definitions, we identified 8 novel genes associated with any stroke (N = 3) or any ischemic stroke (N = 5). Conclusions Taken together, our results show that incorporating family history information substantially enhances the power for locus discovery, particularly for rare variant studies. Efforts to validate the rare variant signals in independent data sets are under way.

Contact Email: rainer.malik@med.uni-muenchen.de

6. Evaluation of circulating exosomal microRNAs in acute anterior circulation large vessel occlusion

S. Mainali MD ¹ , A. Hite PhD ² , P. Fadda, Pharm D ³ , D. Woo MD ⁴ , and Brad Worrall MD ⁵

Objective To evaluate the pattern of temporal evolution of circulating exosomal microRNAs (miRs) in acute anterior circulation large vessel occlusion(acLVO) stroke. Background We lack a reliable surrogate biomarker of stroke lesion age. Between unreliable history and wake-up strokes, over 3 million acute ischemic stroke (AIS) patients present with unknown stroke onset time. Recently, several miRs have been reported as AIS biomarkers but studies specific to acLVO stroke are lacking. It is unclear how miRs evolve over time and whether certain miRs or clusters correlate with lesion age. Design/Methods After IRB approval, acLVO patients aged >18 years were prospectively enrolled within 6 hours(h) of onset. Blood samples were collected at 4 time points from onset (0–6 h; 6–12 h; 12–24 h; and 5–7 days). Blood from healthy volunteers were collected once. We conducted a pilot study on 36 plasma sample (7 patients and 8 volunteers). After exosome and total RNA isolation, Nanostring assay was used for miR profiling. Counts were normalized and fold changes (FC) were calculated (FC > 1.5 considered significant). Results Using ANOVA, we identified 11 miRs with significant differential expression between any 2 time points (6 vs 24 hours, 6 vs 5–7 da, 12 vs 5–7da, 24 vs 5–7 da) and healthy controls. In particular, 3 miRs (miR 140-5p, mir 7-5p and mir 210-3p) were found to have significant differential expression within the 1st 6 hours of stroke compared to other time points and controls (Figure 1). Preclinical data have also shown association of these 3 miRs with hypoxia/ischemia in in vivo and in vitro models. Pathway enrichment analysis of mRNA targets of these miRs also demonstrate known links to stroke. Conclusions Expression profiles of circulating exosomal miRs in acLVO stroke demonstrate clear temporal evolution. Further analysis on a larger sample is needed to investigate the role of miR as surrogate stroke clock.

Figure 1. (Abstract 6) Evaluation of circulating exosomal microRNAs in acute anterior circulation large vessel occlusion.

Contact Email: shraddha.mainali@vcuhealth.org

7. GWAS of a CSF-based volumetric endophenotype finds variants implicated in cerebral edema after acute stroke

Q. Bui, MSc ¹ , Y. Sun, BS ¹ , J. Cárcel-Márquez, MS ² , L. Heitsch, MD ¹ , D. Strbian, MD, PhD ³ , A. Slowik, MD, PhD ⁴ , I. Fernandez-Cadenas ² , PhD; C. Cruchaga, PhD ¹ , J. Lee, MD ¹ , PhD, L. Ibanez, PhD ¹ , and R. Dhar, MD ¹ ; for the GENISIS group

Objective Identify genetic loci associated with cerebral edema after ischemic stroke using a quantitative endophenotype that measures severity of brain swelling. Background Cerebral edema represents accumulation of water with tissue swelling in the region of ischemic brain injury and can lead to neurologic deterioration and death after stroke. It results from a complex interplay of injury responses, including neuro-inflammation, disruption of the blood-brain barrier, and up-regulation of ion channels. Genetic research has been limited by lack of a phenotype that fully captures its formation in large cohorts. Here, we present a new measure of brain swelling applied as an endophenotype to perform the first GWAS for cerebral edema. Design/Methods Degree of displacement of CSF (ΔCSF) was measured between baseline and 24-hour follow-up CTs using an automated algorithm. This was applied as a quantitative dynamic endophenotype of brain edema to subjects in the GENISIS study. ΔCSF was available for 598 European ancestry individuals from 3 sites, exhibiting a normal distribution with broad range across subjects. Genetic analyses were performed using plink2 and adjusted for age, sex, 10 PCs, baseline NIHSS, baseline CSF volume, and TOAST subtype. Then gene-based tested was performed using MAGMA and eQTLs evaluated in Braineac. Results Heritability estimate for ΔCSF was 39% using GNOVA. We identified one genome-wide variant in the 12q12 region which is an eQTL for PRICKLE1 in the putamen (gene-based test, p = 0.037). PRICKLE1 has been implicated in the pharmacogenomic response to anti-hypertensive drugs in stroke patients and with (in dominant form) with inherited epilepsy with ataxia. An additional suggestive locus was found on chr 10, with variants in this region serving as eQTLs for CUBN in several brain regions. Conclusions We found 2 encouraging genetic signals in this first GWAS of cerebral edema, measured by an imaging-based phenotype of volumetric brain swelling. We identified PRICKLE1, a gene already implicated in stroke, as well as CUBN. These promising results will be expanded by increasing sample size from the GENISIS and other cohort studies and incorporating cross-ancestry analyses (Figure 2).

Figure 2. (Abstract 7) GWAS of a CSF-based volumetric endophenotype finds variants implicated in cerebral edema after acute stroke.

Contact Email: dharr@wustl.edu

8. Rare variant burden analysis identifies SERPINE1 as potential risk gene for intracranial aneurysms

M.K. Bakker, PhD ¹ , J.H. Veldink, MD, PhD ¹ , and Y.M. Ruigrok, MD, PhD ¹

Objective To identify rare intracranial aneurysms (IA) susceptibility variants and assess their clinical relevance in the general IA patient population. Background Rare damaging genetic variants have been identified in families with Mendelian inheritance of IA. Thus far, none of these variants were found to play a role in the general IA patient population. Here, we aimed to identify additional risk variants in known IA genes, and to identify new risk genes in a hypothesis-free manner. Design/Methods Rare variant burden analysis 442 IA patients and 166,674 controls of the UK Biobank. Variants were grouped into 3 bins of predicted impact and 3 minor allele frequency (MAF) bins and analyzed using Firth logistic regression. We selected 16 candidate genes linked to familial IA and also performed a hypothesis-free approach. Results Among candidate genes, PCNT was associated most strongly but nominally significantly with IA (MAF below 1%, at least low impact, odds ratio [OR] = 1.18, 95% confidence interval [95% CI] 1.05–1.30, p = 0.0044). This was driven mostly by exon 30 (OR = 1.47, 95% CI 1.15–1.82, p = 0.0028), an exon linked to Majewski's osteodysplastic dwarfism type II, which disease predisposes to IA. No obvious causal variants were found, so we will sequence exon 30 in 971 patients and 1,047 controls. In our hypothesis-free analysis SERPINE1 was most strongly associated with IA (OR = 6.54, 95% CI 3.31–11.5, p = 3.75e-6). Three rare damaging variants drove the signal and will be genotyped in 971 patients and 1,047 controls. SERPINE1 is involved in blood clot degradation and cell adhesion. It was linked to IA risk and outcome after subarachnoid hemorrhage in candidate gene studies. Conclusions Rare variant burden of PCNT exon 30 was most strongly associated with IA among known IA genes, while SERPINE1 has the strongest association among all genes. SERPINE1 is involved in thrombotic diseases. Our data suggest it may also play a role in IA risk. In the future we will investigate rare variant burden in 500,000 whole exomes from the UK Biobank that were recently released.

Contact Email: m.k.bakker-25@umcutrecht.nl

9. Mutations in Mendelian stroke genes is not rare in early-onset stroke patients

Hong-Kyun Park, MD, MSc ¹ , Keon-Joo Lee, MD, MSc ^2,3 , Stéphanie Debette, MD, PhD ⁴ , and Hee-Joon Bae, MD, PhD ²

Objective To investigate the prevalence of clinically relevant genetic variants (CRGV) in Mendelian stroke genes and explore the relationships between variants and the clinical and neuroimaging characteristics in a large, unselected, young stroke population. Background Conventional stroke risk factors do not cover all cause of stroke, and genetics may attribute the gap. Heritability of stroke might be high in the young-age stroke population. However, most genetic studies have been performed in highly selected patients with typical clinical or neuroimaging characteristics. Design/Methods From a prospective, nationwide, multicenter stroke registry, patients were enrolled when they had experienced their stroke or TIA at age of 55 years or younger. We identified CRGV in 15 genes which are known to be associated with stroke occurrence (GLA, NOTCH3, HTRA1, RNF213, ACVRL1, ENG, CBS, TREX1, ABCC6, COL4A1, FBN1, NF1, COL3A1, MT-TL1, and APP) using a customized, targeted next-generation sequencing panel. CRGV was defined as pathogenic or likely pathogenic variants according to the American College of Medical Genetics and Genomics guidelines. Results Among 1,033 patients (male, 70.7%; mean age 45.8 years), 131 (12.7%) had 28 CRGV (22 pathogenic and 6 likely pathogenic), most frequently in RNF213 (n = 59), followed by ABCC6 (n = 53) and NOTCH3 (n = 15). For ischemic stroke subtype, the frequency of CRGV was highest in other determined etiology (20.1%), followed by large artery atherosclerosis (13.6%). The frequency of CRGV differed between patients aged ≤35 years and those aged 51–55 years (17.1% vs 9.3%, p = 0.02). Only 27.1% and 26.7% of patients with RNF213 and NOTCH3 variants showed typical neuroimaging features of the corresponding disorders, respectively. We found variants of uncertain significance (VUS) in 15.4% patients. Conclusions CRGV in 15 Mendelian stroke genes may not be rare in the young stroke population. The majority of patients with CRGV did not have typical neuroimaging characteristics of the corresponding monogenic disorders. Clinical implications of having CRGV or VUS in young stroke patients should be evaluated.

Contact Email: braindocbae@gmail.com

10. Update on the second phase GISCOME GWAS: A genome-wide association meta-analysis of functional outcome after ischemic stroke

C. Lagging MD^1,2,*, M. Söderholm MD, PhD^3,4*, A. Pedersen MD, PhD^1,2, S. Klasson MSc¹, B. Andersson MSc¹, T.M. Stanne PhD¹, J.M. Maguire RN,PhD^5†, A.G. Lindgren MD, PhD^3,6,†, and C. Jern MD, PhD^1,2,†; on behalf of the International Stroke Genetics Consortium and GISCOME network.

*These authors contribute equally to this work. †These authors jointly supervise this work.

Objective To discover common genetic variants associated with post-stroke functional outcome. Background Genetic factors likely account for part of the large inter-individual variability in recovery after ischemic stroke. The first genome-wide association study (GWAS) on functional outcome within the GISCOME network has been completed (n = 6,165). One variant, implicated in brain plasticity, reached genome-wide significance and several variants had suggestive association (p < 10⁻⁵) to outcome. Some of these variants were within or near genes with reported experimental relation to stroke volume and/or brain plasticity. Our results need replication, and a larger sample is needed in order to identify novel variants. We therefore initiated recruitment to a second GWAS within the GISCOME initiative. Design/Methods This study comprises the participants from the first GISCOME GWAS and newly included ischemic stroke subjects with data on modified Rankin Scale (mRS) score at ∼90 days after stroke. Associations between common single-nucleotide polymorphisms and stroke outcome (mRS 0–2 = good outcome vs mRS 3–6 = poor outcome) will be investigated in logistic regression analyses adjusted for age, sex and ancestry (model 1), with additional adjustment for initial stroke severity (baseline NIH Stroke Scale score) in model 2. GWA analyses will be performed in each cohort separately and subsequently meta-analyzed. The newly included cases will also participate as a replication cohort for the phase 1 GWAS, whereafter a joint meta-analysis of all cases will be performed. Results We have received summary statistics from 9 cohorts comprising 8,862 new adult stroke cases to be included. In addition, we await summary statistics on an additional 2,000–3,000 cases from international collaborators. We have also recently genotyped 3,000 additional cases to be included from our study sites in Gothenburg and Lund, Sweden. Conclusions In total, we estimate that approximately 14,000 new cases will be included in the second GISCOME GWAS, leading to a total sample size of ∼20,000 participants. We anticipate to complete the analyses by the end of 2022. The study will constitute the largest GWAS on stroke outcome to date.

Contact Email: cecilia.lagging@gu.se

11. Deep resequencing of the 1q22 locus in non-lobar intracerebral hemorrhage

L. Parodi, PhD ^1-3 , M.E. Comeau, MA ⁴ , G.J. Falcone, MD, ScD, MPH ⁵ , D. Woo, MD ⁶ , J. Rosand, MD, MSc ^1-3 , C.D. Langefeld, PhD ⁴ , and C.D. Anderson, MD, MMSc ^1-3,7

Objective To investigate the genetic architecture underlying ICH susceptibility at locus 1q22, we leverage high-depth sequencing and orthogonal methods. Background Genome-wide association studies have identified 1q22 as a susceptibility locus for non-lobar intracerebral hemorrhages (ICH). We performed targeted high-depth sequencing of 1q22 in ICH cases and controls to further characterize this locus and prioritize potential causal mechanisms. Design/Methods 95,000 base pairs spanning 1q22, including SEMA4A, SLC25A44 and PMF1/PMF1-BGLAP were sequenced in 1,055 ICH cases (534 lobar and 521 non-lobar) and 1,078 controls. Because 1q22 is a susceptibility locus for non-lobar ICH alone, non-lobar ICH patients were compared with lobar ICH patients and ICH-free controls pooled together, with sensitivity analyses comparing non-lobar ICH with only ICH-free controls. Analyses included: (1) Firth regression to assess the role of common variants, (2) z-score based RIFT analysis to assess the role of rare variants, (3) chromatin interaction using ChIA-PET data from ENCODE and Hi-C data (analyzed using Juicebox), and (4) multivariable Mendelian randomization of expression data from the eQTLGen Consortium to assess whether alteration in gene-specific expression relative to regionally co-expressed genes could be causally related to non-lobar ICH risk at 1q22. Results Common and rare variant analyses both prioritized variants in SEMA4A 3′-UTR and PMF1 intronic regions, overlapping with active promoter and enhancer regions regulating PMF1 expression based on ENCODE annotation. ChIA-PET data analysis highlighted the presence of long-range interactions between the same 2 SEMA4A-promoter and PMF1-enhancer regions. Hi-C data analysis clarified that the 1q22 locus is spatially organized in a single chromatin loop and that the harbored genes belong to the same Topologically Associating Domain (TAD), with potentially shared expression regulation. Mendelian randomization analyses revealed that only overexpression of PMF1, controlled for co-expression of other genes across the TAD, could be causally related to the higher risk of non-lobar ICH observed at 1q22 (p = 0.004). Conclusions Single variant and gene-based analyses of targeted sequencing data combined with orthogonal methods revealed potential causal mechanisms for established associations between variants at 1q22 and non-lobar ICH. Based on our findings, we hypothesize that alterations in promoter-enhancer interactions leading to PMF1 overexpression and dysregulation of polyamine catabolism may represent a causal pathogenetic mechanism in ICH.

Contact Email: lparodi@mgh.harvard.edu

12. Leveraging multi-ancestry and whole genome sequence data to improve our understanding of the genetic architecture of neurologic traits—applications from the NHLBI Trans-Omics for Precision Medicine (TOPMed) program

C. Sarnowski, PhD ¹ , L.M.P. Shade, BS ² , M. Fornage, PhD ¹ , J.B. Meigs, MD ^3,4 , S. Seshadri, MD ^5,6,7 , and A.C. Morrison, PhD ¹ ; on behalf of the TOPMed Neurocognitive & Diabetes working groups

Objective To better characterize genetic variations underlying neurologic traits by leveraging whole-genome sequencing (WGS) data in participants of diverse ancestry from the Trans-Omics for Precision Medicine (TOPMed) program. Background Genome-wide association studies (GWAS), conducted mainly in European (EA) participants, have identified common genetic variants with modest effect sizes for brain volumes, accepted endophenotypes of vascular brain injury, and few genetic loci for insulin resistance (IR), a major risk factor for stroke and dementia. Design/Methods (1) We performed WGS association analyses of hippocampal (HV), total brain, lateral ventricular (LVV), and intracranial (ICV) volumes in ∼8k participants (62% EA, 21% African-American (AA), 16% Hispanic/Latino (HA)). (2) We derived 3 ancestry-specific polygenic scores (PS-EA, PS-AA, PS-HA) based on UK Biobank reference panels and fasting insulin GWAS summary statistics, adjusted for body mass index. We generated a multi-ancestry PS (Multi-PS) by fitting a linear combination of the standardized PS-EA, PS-AA, and PS-HA that most accurately predicted IR in a validation set of ∼17k participants without diabetes (34% EA, 28% AA, 38% HA). We evaluated the association of the PSs with neurologic traits in a testing set of ∼14k participants (66%EA, 22%AA, 11% HA). Mixed-effect models were used for all analyses, adjusted for age, sex, study, and principal components, and accounting for relatedness and trait variance variability. Brain volume analyses were adjusted for ICV and excluded participants with dementia or stroke. Results We identified novel significant hits (p < 5 × 10⁻⁸) at 2q22 & 5q14 for LVV and at 1q32 & 13q14 for HV. The top 13q14 variant was common in AA (13%), less frequent in HA (1.4%), and rare in EA (0.1%), and had a consistent effect size across population groups (−0.27 to −0.34). The Multi-PS was strongly associated with IR (proportion of variance explained: 12%). The PS-EA was significantly (p < 0.002) associated with ICV (p = 7 × 10⁻⁷), and suggestively with LVV (p = 0.004). Conclusions By leveraging TOPMed multi-ancestry and WGS data, we identified new loci underlying brain volumes, including ancestry-specific associations, and confirmed the association of IR with brain volumes.

Contact Email: Chloe.Sarnowski@uth.tmc.edu

13. Genetic influence on stroke severity measured by baseline NIHSS: Results from the GENISIS Study

Y. Sun, BS ¹ , L. Heitsch, MD ² , J.M. Lee, MD, PhD ³ , C. Cruchaga, PhD ^1,3,4 , and L. Ibanez, PhD ^1,3,4 ; on behalf of the GENISIS Study Group

Objective To identify genetic loci related to the severity of acute ischemic stroke (AIS), defined as NIHSS measured within 6 hours after stroke onset. Background It is known that genetic factors influence stroke risk and recovery. However, little is known about its influences on initial stroke severity. We described the genetic architecture of stroke severity using baseline NIHSS obtained within 6 hours after stroke onset as an endophenotype. Design/Methods AIS patients (N = 5,361) with baseline NIHSS available within 6 hours after stroke onset or last known normal were selected from the multi-ancestry study GENISIS (European; African American, Asian, and Latino). For all patients, Hg38 imputed genotypes were available. Baseline NIHSS was normalized using the inverse normalization transformation. Association was adjusted by age, sex, 10 principal components, genotyping rounds, and TOAST classification. Genetic heritability was calculated in the European population using GCTA. Samples were analyzed by country of origin using Plink2 and then meta-analyzed by-ancestry and multi-ancestry using METAL and MANTRA. Results Within the European population, baseline heritability was 20.5% (p = 5.65 × 10⁻⁰⁴). The multi-ancestry meta-analysis (Figure 3) revealed one genome-wide significant locus in chr4 (LBF = 7.67) that seem to be driven by INPP4B which presents several eQTLs in Braineac portal (e.g., Substantia Nigra-p = 6.00 × 10⁻⁰⁴. A suggestive locus in chr1 (LBF = 5.43) reached significance in the gene-based analyses for GALNT2 (p = 0.03) which also had several eQTLs (e.g., Temporal Cortex-p = 6.60 × 10⁻⁰³). Conclusions We demonstrated that initial stroke severity is influenced by genetic factors. We identified GALNT2, a protein known to be involved in lipid levels and associated with stroke, and INPP4B, the transcript of which is known to be dysregulated after stroke. Thus, GALNT2 and INPP4B are plausible risk genes involved in stroke severity. We plan to perform Mendelian randomization and colocalization to infer causality.

Figure 3. (Abstract 13) Genetic influence on stroke severity measured by baseline NIHSS: Results from the GENISIS Study.

Contact Email: ibanezl@wustl.edu

14. Genome-wide mapping of WMH spatial patterns reveals region-specific association of WMH with blood pressure

C.L. Phuah, MD, MMSc ^1,3 , L. Ibanez, PhD ^1,2,3 , Y. Chen, DSc ¹ , C. Cruchaga, PhD ^2,3 , and J.M. Lee, MD, PhD ¹

Objective We sought to study the utility of leveraging WMH spatial heterogeneity in genetic studies of CSVD.Background White matter hyperintensities (WMH) are the most common imaging marker of cerebral small vessel disease (CSVD). WMH burden is the most used intermediate phenotype to understand the genetic architecture of CSVD. However, WMH have heterogeneous etiology and histopathology, where similar WMH burden have different impacts on disease and cognitive outcomes in patients with CSVD. We previously demonstrated that different CSVD subtypes are associated with distinct WMH spatial patterns on MRI. Here, we investigated the genetic determinants of these specific WMH spatial patterns. Design/Methods We performed a series of genome-wide association studies on 5 spatially-distinct WMH distribution patterns (deep frontal, periventricular, juxtacortical, parietal, and posterior) derived using a data-driven approach to phenotyping WMH subtypes from 3D WMH probability maps of 949 stroke-free individuals of European ancestry from the Alzheimer's Disease Neuroimaging Initiative database. Pattern-specific WMH burden relative to global burden were quantified for each WMH spatial pattern as continuous traits. Linear regression models were adjusted for age at imaging, sex, and 2 ancestry principal components. Results We identified a novel genome-wide significant locus, rs28756683 (MAF 0.26; p = 2.40 × 10⁻⁸) on chr7 associated with the deep frontal WMH spatial pattern – a WMH pattern related to arteriolosclerosis-related risk factors for CSVD (hypertension and diabetes mellitus). Expression quantitative trait loci mapping indicated that POLR1F, previously associated with blood pressure regulation in East Asians, was driving the association. No genome-wide significantly associated variants were identified for other WMH spatial patterns. Conclusions In this proof of principle study, we showed that data-driven WMH spatial patterns are unique intermediate phenotypes of WMH that may reduce WMH heterogeneity in global WMH burden measurements. Our results provided genetic evidence that suggests blood pressure regulation influencing the deep frontal WMH spatial pattern, concordant with its association with arteriolosclerosis. Future studies with larger sample sizes will improve detection of novel genes and pathways relevant in CSVD.

Contact Email: cphuah@wustl.edu

15. Bayesian network analysis increases understanding of the interplay of intracranial aneurysm rupture risk factors: Preliminary results of a multicentric study

M. Delucchi ^1,4 , G.R. Spinner ¹ , P. Bijlenga ² , S. Morel ^2,3 , and S. Hirsch ¹

Objective To demonstrate the feasibility of a multicentre based, clinically accessible intracranial aneurysm (IA) rupture risk model that supports treatment decision-making in patients with unruptured IA. Background The risk of IA rupture depends on various risk factors. Weighted by the clinicians' experience, they influence treatment decision. We showed in a single centre study that Bayesian networks (BNs) provide insights into the mechanistic rupture risk factor relationships and have now extended to multiple centres. A better understanding of the complex system of IA rupture risk factors can guide treatment decisions. In addition, interpretability is a clear advantage of BNs, which is beneficial for clinical acceptance as a decision-making system. However, the generalization of such a decision support model requires accommodating diverse data sets. Design/Methods Data from 5 cohorts (Geneva, Nantes, Utrecht, Kuopio, UCL) were harmonized according to our recently published processing routine resulting in a pooled data set of n = 7,481 complete, patient-level records of 9 rupture risk factors: Patient's sex, age at diagnosis, familial disease history of IA, awareness of hypertension, smoking behaviors as well as IA multiplicity, location, size and rupture status. BNs show the dependence structure of the rupture risk factors. Modelled by de-confounding the variables, BNs reconstruct the graphical representation of the joint distribution of the data-generating structural model. The resulting BNs were compared to multivariate generalized logistic mixed effects models. Results The results from standard methods agreed with previous studies. BN models confirmed the findings from standard analysis methods and uncovered additional associations in the data. While regression models show significant correlations between all risk factors except for multiplicity, only IA characteristic risk factors are directly associated with IA rupture in the estimated dependence structure from BN models. Conclusions The graphical representation of the IA rupture risk factors in BNs facilitated the result understanding. The preliminary results of this multicentre study provide new hypotheses on the risk factor interdependencies.

Contact Email: matteo.delucchi@zhaw.ch

16. Inflammation-related proteins are associated with short-term functional outcome after ischemic stroke

A. Angerfors, MScEng ¹ , B. Andersson, PhD ² , S. Klasson, MScEng ¹ , C. Brännmark, MD, PhD ¹ , T.M. Stanne*, PhD ¹ , and C. Jern*, MD, PhD ^1,3

*Equal contribution

Objective To investigate plasma levels of a broad range of inflammation-related proteins in acute ischemic stroke patients for association with 3-month functional outcome (i.e., modified Rankin Scale, mRS). Background There is a large inter-interindividual variability in recovery after ischemic stroke and the acute-phase inflammatory response could contribute to this variation. Both C-reactive protein (CRP) and interleukin-6 (IL-6) are well established to be elevated during the acute phase of ischemic stroke and associate with poor functional outcome. However, circulating levels of a broader range of inflammatory proteins (e.g., other interleukins, chemokines, surface molecules and immune receptors) have not been studied in large clinical studies of ischemic stroke. Design/Methods Plasma levels of 65 inflammation-related proteins were measured using a proximity extension assay (PEA) technology during the acute phase in 600 consecutively recruited ischemic stroke patients from the Sahlgrenska Academy Study on Ischemic Stroke (SAHLSIS). Functional outcome was categorized as good (mRS 0–2) vs poor (mRS 3–6) and outcome associations with protein levels were analyzed by multivariable logistic regression analysis adjusted for clinical characteristics. Results In univariable analyses 40 proteins were associated at the suggestive significance level (p less than 0.05) and 20 of these were associated after correction for multiple testing (pFDR less than 0.05). We then adjusted for clinical factors and found that the results were largely unchanged. A total of 39 proteins were associated with outcome at the suggestive significance level, 18 of which remained after correction for multiple testing. Conclusions Using PEA technology, we confirmed previous associations between IL-6 and short-term outcome after ischemic stroke and demonstrate that other less well-studied inflammatory proteins may also have a role in stroke recovery and warrant further study.

Contact Email: annelie.angerfors@gu.se

17. Rare variant analysis uncovers potential association of VNN2 with stroke outcome

E. Alcaide-Consuegra ¹ , G. Escaramís ² , U. Lazcano ³ , M. Mola-Caminal ³ , C.D. Bruque ⁴ , C. Vives-Bauza ⁵ , J. Jiménez ³ , I. Fernández–Cadenas ⁶ , GeneStroke Consortium, S. Balcells ¹ , and R. Rabionet ¹

Objective This study aims to identify genetic contributions to the variability in stroke outcome by analyzing the effect of rare variants. Background Recently, some GWAS studies highlighted the involvement of common or low-frequency variants in PP1 and PATJ in functional outcome. However, the role of rare genetic variants in stroke recovery has not been investigated yet. Design/Methods A pilot study was performed sequencing 90 exomes of patients with extreme stroke outcomes (modified Rankin Scale (mRS) at 3 months 0–1 vs 3–5). The target regions and other candidate regions from the GWAS studies were sequenced by targeted next-generation sequencing in 702 stroke cases. After filtering for potentially damaging rare variants based on their CADD score, considering only those with a score over 20, we performed rare variant association analysis under an ordinal (mRS 0–6) and dichotomic (mRS 0–1 vs 3–5 and 0–1 vs 3–6) scenario. Tests were performed using BATI, a Bayesian-based rare variant association tool, and were adjusted for multiple testing. Relevant variants in candidate genes were further studied by protein structure and stability analysis using AlphaFold2 and FoldX. Results Our studies show enrichment of rare coding variants in VNN2 in patients with a better stroke outcome (∆DIC >10, equivalent to p-value <0.001). Six rare variants were predicted to significantly affect protein stability (∆∆G > 1.6 kcal/mol), meanwhile, another variant is located in the active site, possibly affecting the electrostatic surface. VNN2 acts in cell adhesion and migration of neutrophils. Conclusions We have identified the association of rare variants in VNN2 with a better stroke outcome. However, further functional experiments are needed to understand the effect of these variants on the expression and location of VNN2 and how they may lead to differences in recovery.

Contact Email: ealcaide@ub.edu

18. Analysis of age at onset of ischemic stroke identifies ApoE: possibly a result of selection bias

P.F. McArdle, PhD¹, J. von Berg, PhD² S.W. van der Laan, PhD², S.J. Kittner, MD¹, and B.D. Mitchell, PhD¹

Objective In a prior GWAS analysis we identified a variant associated with stroke age-at-onset (AAO). The objective of this study was to evaluate the potential for bias in AAO analysis that could arise from selective survival. Background Large genome-wide association studies (GWAS) employing case-control study designs are a powerful tool for identifying disease and trait susceptibility loci. As a complement to these studies, analysis of AAO have been proposed to identify further disease-associated loci. We recently performed a GWAS of stroke AAO in 10,857 cases from SiGN and identified among women a significant association of AAO with rs429358 - an exonic variant in APOE that encodes for the APOE-Є4 allele (p-value = 2.4 × 10⁻⁸). Design/Methods To evaluate whether this association could be attributable to selective survival across APOE alleles, we performed a series of simulations that included 3 different loci: 2 of which are associated with stroke with different parametric forms, and one which was not associated with stroke but with mortality. We simulated a population of individuals who were followed from birth until death based on sex-specific mortality rates. A traditional case control analysis was performed on the simulated data, and additionally, an AAO analysis was performed among the stroke cases only. Results The simulations suggested that a locus strongly associated with mortality, as the ApoE locus is, will have decreased allele frequencies at advancing age as individuals are more likely to have stroke resulting in an observed association. We further demonstrate that conventional case control analyses have greater power to detect stroke risk loci, many of which are not associated with age at onset amongst cases only. Conclusions We conclude that associations of loci with age at onset but not associated in case control studies are likely due to selection bias and should be scrutinized carefully. The association observed between ApoE and age at onset of stroke may be an artifact resulting from ApoE's association with lifespan.

Contact Email: pmcardle@som.umaryland.edu

19. Genome-wide study reveals novel sex-specific loci associated with lacunar stroke risk

J. Cárcel-Márquez, MSc ¹ , E. Muiño, PhD ¹ , C. Gallego-Fabrega, PhD ¹ , N. Cullell, MSc ^1,2 , M. Lledós, MSc ¹ , L. Lluciá-Carol, MSc ¹ , J.M. Martín-Campos, PhD ¹ , and Israel Fernández-Cadenas, PhD ¹ ; On behalf GeneStroke Consortium and International Stroke Genetics Consortium

Objective Study of the genetic complexity of lacunar stroke by sex stratification. Background Only 3 loci have been found associated with lacunar stroke risk in the European population, these do not explain the whole complexity of the disease. We sought to identify new associations and disease mechanisms in stratified by sex genome-wide association analyses. Design/Methods A total sample size of 5,817 individuals were genotyped and analyzed, 228 lacunar strokes confirmed by MRI and 5,589 non-stroke controls. Stringent quality controls were performed. Genetic analyses were performed with GCTA using an additive model and adjusting for age, sex and the first 10 genetic principal components, additionally, stratified by sex. Significant threshold was stablished at p < 5 × 10⁻⁸. Gene-based analysis were performed using MAGMA. Replication was explored in MEGASTROKE and GIGASTROKE cohorts for lacunar stroke in European population. In silico proteomic analysis was performed on whole blood tissue from the INTERVAL cohort (3,301 individuals) for the replicated variants. Results Two loci were significant and replicated in MEGASTROKE European lacunar analysis. One locus in chromosome 5 in both sexes analysis, rs59970332-T (p = 1.99 × 10⁻⁸; beta (standard error) = 0.08 (0.01)), prioritized gene CTNND2, with the strongest association in male analysis (p = 5.19 × 10⁻⁸). In female analysis, one locus in chromosome 8, rs146966463-C (p = 6.60 × 10⁻⁹; beta (standard error) = 0.08 (0.01)), prioritized gene FGFR1, not associated in male analysis (p > 0.05). Gene-ontology results revealed both sexes significant variant rs59970332-T is involved in modulating proteins associated with regulation of the immune response and cytokine production. In addition, female specific variant rs146966463-C regulating proteins involved in blood vessel remodeling and homophilic cell adhesion via plasma membrane adhesion molecules among other pathways. Conclusions We found 2 novel loci associated with lacunar stroke risk in the first stratified by sex analysis in this phenotype. We identified CTNND2 and FGFR1 as promising genes associated with lacunar stroke risk. Further analyses are warranted to understand the role of these 2 genes in the disease.

Contact Email: jara.carcel@gmail.com

20. Hemostatic factors are associated with the HT after r-tPA administration in the acute phase of Ischemic Stroke

C. Gallego-Fabrega, PhD ¹ , G. Temprano-Sagrera, MsC ² , J. Cárcel-Márquez, MsC ¹ , E. Muiño, MD, PhD ¹ , N. Cullell, MsC ^1,3 , M. Lledós, MsC ¹ , L. Llucià-Carol, MsC ¹ , P.S. de Vries, PhD ³ , I. Fernández-Cadenas, PhD ¹ *, and M. Sabater-Lleal, PhD ² ;, for the Cohorts for Heart and Aging Research in Genomic Epidemiology Consortium (CHARGE)

Objective To investigate the genetic relationship between 7 hemostatic factors and hemorrhagic transformation (HT) after r-tPA treatment in Stroke patients. Background Thrombolytic recombinant tissue-plasminogen activator (r-tPA) treatment presents a six-to seven-fold increased risk of intracerebral hemorrhage and has been associated with a worse patient's outcome and increased risk of 3-month mortality. However, genetic studies of HT have been sparse. Design/Methods Using existing Genome-wide association study (GWAS) summary data of European individuals, on risk of HT after r-tPA treatment, and on 7 hemostatic factors (factors VII, factor VIII, von Willebrand factor [VWF], factor XI, fibrinogen, plasminogen activator inhibitor-1, and tPA), we performed a Cross-trait linkage disequilibrium Score regression (LDSC), followed by a Multi-trait analysis (MTA) and colocalization, and finally a two-sample Mendelian Randomization (MR). Results The multi-trait analysis revealed 4 pleiotropic loci that regulate both, the levels of a specific hemostatic factor and the risk of suffering an HT after r-tPA treatment. Fibrinogen-HT MTA reveled an association of rs56026866 (PLXND1) (MTA-p.val = 8.80 × 10⁻¹⁰), rs1421067 (CHD9) (MTA-p.val = 1.81 × 10⁻¹⁴) with HT for the first time, and a novel loci rs34780449 near ROBO1 gene associated to both HT and to fibrinogen levels (MTA-p.val = 1.64 × 10⁻⁰⁸). VWF-HT MTA showed for the first time an association of and rs10942300 (ZNF366) (MTA-p.val = 1.81 × 10⁻¹⁴) with the risk of HT after r-tPA. The MR analysis suggest a potential causal relationship between circulating levels of factor XI and HT after r-tPA administration (Invers variance weighted: estimate [SE], 0.07 [−0.29 – 0.00]; OR 0.87 [0.75–1.00], raw p.val = 0.05, corrected q.val = 0.11). Conclusions We identified 4 pleiotropic traits between hemostatic factors and HT, one of them novel. As well as a nominal causal association between factor XI circulating levels and an increased risk of HT after r-tPA treatment.

Contact Email: cristina.gallego.fabrega@gmail.com

21. Genetic risk of BMI, blood pressure, and smoking is more strongly associated with early than late onset IS

K. Nguyen ¹ , H. Xu ¹ , B. Gaynor ¹ , J.W. Cole ² , R. Malik ³ , M. Dichgans ^3,4,5 , S.J. Kittner ² , and B.D. Mitchell ¹ ; on behalf of ISGC, SiGN, and the EOSC

Objective To utilize Polygenic Risk Scores (PRS) of conventional stroke risk factors to assess differences in association between early onset stroke (EOS, onset 18–59 years) and late onset stroke (LOS, onset >60 years). Background Ischemic Stroke (IS) is one of the leading causes of death and disability worldwide. Powerful methods, such as genome wide association studies, have been used to understand the genetic risk factors that contribute to the onset of the disease. This wealth of information can be leveraged to elucidate the impact of risk factors have on IS. Design/Methods Using large publicly available GWAS results, we calculate PRS for conventional stroke risk factors (BMI, hyperlipidemia, type 2 diabetes, blood pressure, and smoking initiation) in EOS cases (n = 6,728) and controls from the Early Onset Stroke Consortium and LOS cases (n = 9,272) and controls from the Stroke Genetics Network (SiGN) study. We then performed logistic regression of each PRS with EOS and LOS and then compared odds ratios for each PRS between EOS and LOS to determine if genetic risk for stroke risk factors differed between EOS and LOS. Results A BMI PRS was more strongly associated with EOS (OR = 1.11; p = 7.59E-13) than with LOS (OR = 1.00; p = 9.12E-01). PRS for both DBP and SBP were also more strongly associated with EOS (DBP OR = 1.19; p = 9.24E-31 and SBP OR = 1.20; p = 3.57E-33) than for LOS (DBP OR = 1.10; p = 1.69E-12 and SBP OR = 1.07; p = 4.78E-15). A smoking PRS was also more strongly associated with EOS (OR = 1.10; p = 1.42E-11) than with LOS (OR = 1.03; p = 1.01E-02). In contrast, there was little difference in the association between EOS and LOS of PRS for lipids and T2D. Conclusions The genetic component of BMI, blood pressure, and smoking initiation are more strongly associated in EOS, compared to LOS. This suggests that control of these risk factors is particularly important in young adults (Figure 4).

Figure 4. (Abstract 21) Genetic risk of BMI, blood pressure, and smoking is more strongly associated with early than late onset IS.

Contact Email: kevin.nguyen@som.umaryland.edu

22. Family-based whole exome sequencing in early onset ischemic stroke: rationale and design of the SECRETO Family Study

L. Tomppo, MD, PhD ¹ , N. Martinez-Majander, MD, MSc ¹ , M. Koskipirtti, BM ¹ , P. Jäkälä, MD, PhD ² , T. Sarkanen, MD, PhD ³ , J. Huhtakangas, MD, PhD ⁴ , N. Yesilot, MD ⁵ , I. Veen, BM ⁶ , A. Ilinca, MD, PhD ⁷ , A. Lindgren, MD, PhD ⁷ , J.W. Cole, MD, MS ⁸ , B.D. Mitchell PhD, MPH ⁸ , S. Kittner, MD, MPH ⁸ , and J. Putaala, MD, PhD ¹

Objective To identify rare coding variants associated with early onset ischemic stroke (IS) using family-based whole exome sequencing (WES) approach. Background Early onset IS has higher heritability and larger proportion of monogenic inheritance compared to older onset IS. By performing WES in multiple members of 2 families with early onset cryptogenic IS, we detected possibly pathogenic variants in genes not earlier associated with IS (Ilinca 2020, PMID: 32172663). We hypothesize that early onset IS patients more often carry rare variants with high penetrance and large effect sizes, and family-based WES aids their discovery. We now aim to enroll 50 families to study rare coding variations that underlie familial early onset IS and cardiovascular disease. Design/Methods SECRETO study is a European multicenter prospective case-control study, registered at ClinicalTrials.gov (NCT01934725). In this sub study, eligible probands have a neuroimaging positive IS at age <50 without obvious monogenic or non-genetic cause and at least one 1st degree relative with cerebrovascular event before age 60. Considering pleiotropic effects, the family members are screened for ischemic and hemorrhagic stroke, aortic and cerebral aneurysm, ischemic heart disease and venous thrombosis. We include at least 1 affected family member in addition, to proband and 2 healthy family members in each family. We will perform WES to recognize rare functional variants that segregate with the early onset IS or IS and other cardiovascular disease. Results As of August 2022, we have enrolled 179 participants in 34 families at 6 centers participating the SECRETO study. We anticipate initiating WES by Q4/2022 and to finalize all recruitment by Q4/2023. Conclusions Our approach is a complimentary to large scale sequencing efforts and takes advantage of careful phenotyping and characterization of families. To our knowledge, this is the largest family-based sequencing effort in early onset IS to date. Though not accounting for a large number of cases, rare variants may provide an important window into novel stroke mechanisms and new opportunities for stroke prevention.

Contact Email: liisa-maaria.tomppo@hus.fi

23. Sex-stratified transethnic GWAS meta-analysis of early onset ischemic stroke

L. Tomppo, MD, PhD ¹ , B. Gaynor, MS ² , J. Putaala, MD, PhD ¹ , B.D. Mitchell, PhD, MPH ² , and S.J. Kittner, MD, MPH ³ ; for the Early Onset Ischemic Stroke Consortium

Objective To study genetic sex specific effects in early onset ischemic stroke (IS). Background Early onset IS influences males and females unequally, but the etiologic background of the sex differences remains inadequately understood. We hypothesize that stratifying GWAS by sex provides an opportunity to 1) identify loci that shed light on mechanisms and pathways relevant to sex specific effects in IS and 2) discover novel IS loci. Design/Methods We performed a transethnic GWAS meta-analysis in 6,879 female IS cases and 11,264 male cases <60 years-old and >700,000 sex matched controls, from the Early Onset Stroke Genetics Consortium. Results We found genome-wide significant evidence for association (P < 5E-8) at a locus near NYAP2 for females and in PIAS4 for males in transethnic meta-analysis (Table 1). NYAP2 has previously shown suggestive evidence for association with early onset menopause. PIAS4, a candidate gene for migraine, has shown sex specific expression levels in migraine patients compared to controls. Additionally, 8 loci showed suggestive evidence (P < 1E-6) for association in transethnic or European ancestry meta-analysis in sex-dependent manner including MMP1-MMP3-MMP12 locus, a previously known stroke locus for males, and a novel stroke locus near TMX1, a transmembrane platelet protein, for females. The known early onset IS locus ABO did not show evidence for sex-specific association (p-value for homogeneity of odds ratios >0.05) despite suggestive association in the male stratified analysis. Conclusions We identified potential novel stroke loci and evidence for sex differences at other biologically relevant loci by stratifying GWAS by sex. Our findings require verification in large sample sizes and also evaluation in older onset IS. Sex-specific association analysis is a potentially useful tool for characterizing the genetic basis of early onset IS.

Table 1.

(Abstract 23) Sex-stratified transethnic GWAS meta-analysis of early onset ischemic stroke

Contact Email: liisa-maaria.tomppo@hus.fi

24. A New Approach to Stroke Recovery Phenotyping for Genome Wide Association Studies with Trajectory Profile Clustering

Chad Aldridge, DPT, MS-CR ¹ , Sanjukta Krishnagopal, PhD ² , Keith Lohse, PhD, PSTAT ³ , Fang-Chi Hsu, PhD ⁴ , Keith Keene, PhD ⁵ , Bradford Worrall, MD, MSc ^1,6 , and Robynne Braun, MD, PhD ⁷

Objective To define stroke recovery phenotypes for use in genome wide association studies (GWAS) by using a novel network science-based approach. Background Stroke recovery research entails tracking multiple domains of neurologic impairment over time that are measured on differing scales. This presents challenges for analytic approaches commonly used to study stroke genetics. Here, we present a novel approach using a network science-based method, Trajectory Profile Clustering (TPC), to define stroke recovery phenotypes for GWAS. Design/Methods We analyzed data from 3,679 patients in the VISP data set (Vitamin Intervention for Stroke Prevention) on 15 NIHSS impairment measures at 6 time points spanning 24 months post-stroke. The 4 identified TPC profiles (Figure 5, Panel A), were used as phenotypes for a subsequent GWAS on the 2,099 genotyped VISP patients. We used a multinomial generalized regression with TPC profiles as the response variable. The model adjusted for age, sex, treatment group, and the first 10 principal components. Results TOPMed imputation and strict quality control resulted in 6,392,745 SNPs. Although none of our loci reached genome wide significance, the GWAS identified multiple suggestive loci (p < 5 × 10⁻⁶) clustering within or near genes of biological relevance (Figure 1, Panel B): LINC02151 (non-coding RNA 11q23.3) is associated with lipid homeostasis, sphingomyelin levels, neurofibrillary tangles and coronary artery disease. SEC63P2 (RNA-seq 4p15.1) is associated with hippocampal volume and is expressed in choroid plexus during development. IDE (10q23.33) is associated with triglyceride levels, Type 2 diabetes and BMI, and in the present analysis also differentiated among the 4 TPC-derived phenotypes (p < 0.05). Conclusions Phenotypes defined using TPC, even in this limited sample, identified multiple suggestive genetic associations with plausible biological relevance for both stroke risk and stroke recovery.

Figure 5. (Abstract 24) New Approach to Stroke Recovery Phenotyping for Genome Wide Association Studies with Trajectory Profile Clustering.

Contact Email: robynne.braun@umm.edu

25. Genomic Risk Scores and Oral Contraceptive-Associated Stroke Risk: a Call for ISGC Collaboration

Forrest Lin ¹ , Brady Gaynor, MS ² , John W. Cole, MD, MS ¹ , Braxton D. Mitchell, PhD, MPH ² , Steven J. Kittner, MD, MPH ¹ , Jukka Putaala, MD, PhD ³ , and Liisa Tomppo, MD, PhD ³

Objective To evaluate whether a genomic risk score for ischemic stroke (IS) modifies oral contraceptive (OC)-associated IS risk. Background OCs are generally safe but vascular risk factors are known to increase OC-associated IS risk. If a genomic risk score could identify a subset of women with substantially increased risk of OC-associated IS, this could influence prescribing guidelines and reduce stroke events. Design/Methods In the Genetics of Early-Onset Stroke (GEOS) study, there were 340 premenopausal women (143 IS cases and 197 controls) with data on OC use within 30 days prior to index event for cases or prior to interview for controls. Using a previously validated genomic risk score (metaGRS) for IS based on 19 polygenic risk scores of vascular events and risk factors, we stratified our sample into tertiles of genomic risk and calculated the association between OC use and IS within each tertile. We tested if the association between OC use and IS depended on genomic risk of stroke (metaPRS) using logistic regression with an OC use*metaGRS interaction term. Results Among all women, OC use was significantly associated with IS (odds ratio = 2.4, p = 0.002). The odds ratio for IS associated with OC use decreased from 3.9 in the tertile with highest genomic risk of IS to 1.5 in the tertile with lowest genomic risk (Table 2). The formal test of interaction was consistent with our hypothesis (p < 0.07) that the genomic risk score modifies the association of OC use with IS. Conclusions The results of our exploratory analysis support the need for international collaboration to generate sufficient sample sizes to determine whether a genomic risk score could be clinically useful in reducing OC-associated IS.

Table 2.

(Abstract 25) Genomic Risk Scores and Oral Contraceptive-Associated Stroke Risk: a Call for ISGC Collaboration

Contact Email: skittner@som.umaryland.edu

26. Proteome-wide Mendelian Randomization Screen Identifies Circulating Protein Mediators of Early-Onset Ischemic Stroke

M. Chong ^1,2 , W. Zhu ³ , J.W. Cole ³ , S.J. Kittner ³ , B.D. Mitchell ³ , and G. Paré ^1,2 ; on behalf of the Stroke Genetic Network and Early Onset Stroke Consortium

Objective Discover blood proteins mediating risk of early-onset ischemic stroke (IS) via Mendelian Randomization (MR) analyses. Background About 10–15% of IS affect young adults (18–50 years) and, in contrast to declining rates in older individuals, IS incidence has risen in the young. Thus, new therapies tailored to early-onset IS are of urgent need. Design/Methods We conducted a proteome-wide MR screen of 653 blood proteins for susceptibility to early-onset IS (18–65 years) and its subtypes (cardioembolic stroke, large artery atherosclerosis, small artery occlusion, other stroke, and undetermined strokes) using our previous framework (Circulation, 2019). cis-pQTLs (LD r² < 0.01; p < 0.01) were derived from a meta-analysis of 5 pQTL GWAS (N < 20,509). Corresponding genetic effects on early and late-onset IS were derived from the Early-Onset Stroke Consortium (Ncase = 16,851; Ncontrol = 32,473) and Stroke Genetics Network (SiGN) GWAS (Ncase = 9,272; Ncontrol = 25,124), respectively. MR analyses were conducted using inverse variance weighted, weighted median, and MR-EGGER methods. Results Previously reported biomarkers were found to mediate risk of early-onset IS including ABO (ORall stroke = 1.10 per SD increase in genetically predicted levels; 95% CI 1.05–1.15; p = 2.8 × 10⁻⁵) and MMP12 (ORall stroke = 0.87; 95% CI 0.82–0.93; p = 2.1 × 10⁻⁵). In addition, at Bonferroni significance, we identified 2 new mediators of early IS: vascular endothelial growth factor 2 (KDR; ORsmall artery occlusion = 1.94; 95% CI 1.6–2.4; p = 2.1 × 10⁻⁹) and urokinase-type plasminogen activator (PLAU; ORundetermined stroke = 3.08; 95% CI 1.9–5.2; p = 1.3 × 10⁻⁵). Genetically predicted levels of these proteins were not significantly associated with late-onset IS (>65 years), and MR effect estimates differed significantly between early vs late-onset IS for KDR (I2 = 96; heterogeneity p = 9.7 × 10⁻⁷) and PLAU (I2 = 85; het. p = 0.01). Conclusions While targeting known protein mediators for IS at all ages may help reduce risk of early onset IS, there are likely additional distinct etiologic mechanisms precipitating premature IS that also may warrant specific therapeutic intervention.

Contact Email: skittner@som.umaryland.edu

27. Dissecting the downstream pathways driving the effects of IL-6 signaling on atheroprogression: a proteome-wide Mendelian randomization study

S. Prapiadou ^1,3-5 , R. Malik, MD, PhD ² , M. Dichgans, MD ² , J. Rosand, MD, MSc ^3-5 , C. Anderson, MD, MMSc ^3-6 , and M. Georgakis, MD, PhD ^2,3-5

Objective Leveraging large-scale proteogenomic data, we aimed to dissect the downstream causal proteomic mediators of the effects of IL-6 signaling on atheroprogression to detect novel drug targets with more specific effects. Background Genetic and experimental studies support a role of interleukin-6 (IL-6) signaling in atherosclerosis. However, pharmacologic IL-6 inhibition has been associated with impaired host immune response, thus highlighting the need for alternative immunotherapeutic targets. Design/Methods We investigated the effects of 26 genetic variants proxying IL-6 signaling inhibition on 3,622 plasma proteins in 3,301 individuals in a Mendelian Randomization (MR) framework. In subsequent mediation analyses, we explored potential causal mediators of IL-6 signaling on coronary artery disease (CAD), large artery atherosclerotic stroke (LAAS), and peripheral artery disease (PAD) in disease-specific consortia. We followed-up our findings in a population-based prospective cohort study. Results Following correction for multiple comparisons, we found significant effects of genetically proxied IL-6 signaling on 83 circulating proteins involved in immune response, cytokine production and regulation, and cell differentiation pathways. From these 83, the genetically predicted levels of 25 proteins were associated with the risk of cardiovascular outcomes. CXCL10, a chemokine involved in T cell recruitment and experimentally implicated in atheroprogression, showed consistent associations with CAD (beta [SE]: 0.146 [0.042], p-value = 0.001), LAAS (beta [SE]: 0.464 [0.181], p-value = 0.008) and PAD (beta [SE]: 0.204 [0.055], p-value = 0.0007). Mediation analyses revealed that a significant proportion of the effects of IL-6 signaling on cardiovascular disease endpoints (27.5% for CAD, 67.2% for LAAS, and 38.8% for PAD) were mediated by increases in CXCL10 levels. In an observational cohort, midlife measured circulating CXCL10 levels were associated with a higher risk of incident ischemic stroke and myocardial infarction (HR per-SD increment: 1.22, 95% CI: 1.06–1.40, p = 0.006). Conclusions Integrating genomic and proteomic data, we found a proteomic signature for IL-6 signaling, and mediators of its effect on cardiovascular disease. Our data consistently support CXCL10 as a significant mediator of atherosclerotic disease in 3 different vascular beds and as such might serve as a promising drug target for atheroprotection.

Contact Email: sprapiadou@mgh.harvard.edu

28. Early Blood Biomarkers of Neurologic Dysfunction Were Not Associated with Delirium in COVID-19 Patients

I. Snider ¹ , Y. Murtaza ¹ , J. Miller ¹ , G. Li ¹ , D. Woo-Kim ¹ , F. Kobaissy, PhD ¹ , K. Wang PhD ¹ , and A.N. Simpkins, MD, PhD, MSCR ¹

Objective Using the UF Health Shands and CTSI biorepository, we have phenotyped a cohort of patients with neurologic manifestations of COVID-19. We hypothesized that inflammatory CNS serum markers would be higher in COVID-19 patients with neurologic disease than compared to COVID-19 negative and control patients. Background Past studies on serum biomarkers in COVID-19 positive patients have found that CNS inflammation might contribute to neurologic disease. However, those at risk of COVID-19 encephalopathy is still difficult to predict. Design/Methods In our IRB approved study, we performed a retrospective chart review of consented COVID-19 positive patients admitted to UF Shands Hospital between March 31, 2019 and January 21, 2021. We obtained and analyzed descriptive data on our cohorts - including demographic information and comorbid disease and documentation of cognitive dysfunction/encephalopathy. We analyzed serum obtained early during the admission for biomarkers of CNS disease (neurofilament light polypeptide (NFL), glial fibrillary acidic protein (GFAP), and ubiquitin carboxy-terminal hydrolase L1 (UC-L1) and tau). Results Our cohort included 62 patients, 52% women, 26% Black, median age 46 (interquartile range 30–57), 37% admitted with COVID-19 pneumonia, 11% with sepsis, 37% with a history of prior neurologic disorder, and a total of 3 who had a stroke. Of the cohort, 58 had evaluable serum samples, 10 with cognitive deficits/encephalopathy documented. There was a trend for more Nfl1 in those without cognitive deficits, but no significance was found with nor with any other biomarker.

Conclusions Elevated blood biomarkers may need to be taken in context with the severity of COVID-19 infection. More biomarker studies, including GWAS, are needed to account for the heterogeneity the clinical manifestations of COVID-19 with and without cognitive dysfunction.

Contact Email: alexis.simpkins@neurology.ufl.edu

29. RNA Expression in Acute Ischemic Stroke with Asymptomatic COVID-19

Y.A. Murtaza ¹ , R.H. Shah ¹ , and A.N. Simpkins ¹

Objective Here, we describe a case of patient which RNA analysis was used to assess ischemic stroke etiology in the setting of asymptomatic COVID-19. Background COVID-19 has been associated with thrombotic events and stroke, mostly in patients with severe disease and risk factors for cardiovascular disease. However, it is still unclear how a COVID-19 infection contributes to cerebrovascular events in patients who are considered to have asymptomatic COVID-19. Design/Methods N/A Case report. Results A 92-year-old female with past medical history significant for hypertension, prior history of stroke, pacemaker placement, atrial fibrillation not on anticoagulation secondary to active gastrointestinal bleeding (GIB) and a pre-morbid modified Rankin score was 4 presented to the emergency department with aphasia, left gaze preference, right facial droop and right sided weakness that started 2.5 hours prior to presentation. Her NIHSS score was 15, imaging demonstrated a large left middle cerebral artery stroke from left M1 occlusion not amenable to revascularization or thrombolysis secondary to (GIB) and poor functional baseline. She was also found to be COVID-19 by RT-PCR testing, but did not have any respiratory symptoms, fever, or chest infiltrate. Her stroke work-up was unremarkable. Her infarct size was unchanged from admission. She was discharged back home with home rehabilitation on apixaban. A commercially available RNA stroke etiology test was sent to determine if RNA profile was different in the setting of the asymptomatic COVID-19 infection, and the stroke etiology was still determined to most likely be secondary to cardioembolism. Conclusions In this case, the asymptomatic COVID-19 infection did not alter RNA expression in whole blood enough to change the expected stroke etiology. More research is needed to determine if COVID-19 diagnosis is associated with stroke risk by causing decompensation or advancement of pre-morbid stroke risk factors, or alters biologic expression leads to inflammation or hypercoagulability in even if asymptomatic.

Contact Email: alexis.simpkins@neurology.ufl.edu

30. Interplay between Chronic Kidney disease, Hypertension, and Stroke: Insights from a Multivariable Mendelian Randomization Analysis

D.M. Kelly ^1,2 , M.K. Georgakis ^2,3,4 , N. Franceschini ⁵ , D. Blacker ⁶ , A. Viswanathan ¹ , and C.D. Anderson ^2,4,7

Objective We leveraged large-scale genetic data to explore causal relationships between CKD, hypertension and cerebrovascular disease phenotypes. Background Chronic kidney disease (CKD) increases the risk of stroke, but the extent through which this association is mediated by hypertension is unknown. Methods We used data from genome-wide association studies (GWAS) of European ancestry to identify genetic proxies for kidney function (CKD diagnosis, estimated glomerular filtration rate [eGFR], and urinary albumin-to-creatinine ratio [UACR]), systolic blood pressure (SBP), and cerebrovascular disease (ischaemic stroke and its subtypes, and intracerebral haemorrhage [ICH). We then conducted univariable, multivariable and mediation Mendelian randomization (MR) analyses to investigate the effect of kidney function on stroke risk and the proportion of this effect mediated through hypertension. Results Univariable Mendelian randomization revealed associations between genetically determined lower eGFR and risk of all stroke (OR per 1-log decrement in eGFR, 1.77; 95% CI 1.31–2.40; p < 0.001), ischaemic stroke (OR, 1.81; 95% CI 1.31–2.51; p < 0.001), and most strongly with large artery stroke (LAS) (OR, 3.00; 95% CI 1.33–6.75; p = 0.008). These associations remained significant in the multivariable MR analysis, controlling for SBP (OR, 1.98; 95% CI 1.39–2.82; p < 0.001 for AS; OR, 2.16; 95% CI 1.48–3.17; p < 0.001 for IS; OR, 4.35; 95% CI 1.84–10.27; p = 0.001 for LAS). with only a small proportion of the total effects mediated by SBP (10.5%, 6.6% and 7.8%, respectively). Total, direct and indirect effect estimates were similar across a number of sensitivity analyses. Conclusion Our results demonstrate an independent causal effect of impaired kidney function, as assessed by decreased eGFR, on stroke risk, particularly LAS, even when controlled for SBP. Targeted prevention of kidney disease could lower atherosclerotic stroke risk independent of hypertension.

Contact Email: dearbhlakelly2@gmail.com

31. Genomic Risk Scores and Oral Contraceptive-Associated Ischemic Stroke Risk: A Call for ISGC Collaboration

F. Lin ¹ , B. Gaynor MS ² , J.W. Cole MD, MS ¹ , B.D. Mitchell PhD, MPH ² , S.J. Kittner, MD, MPH ¹ , J. Putaala, MD, PhD ³ , and L. Tomppo, MD, PhD ³

Objective To evaluate whether a genomic risk score for ischemic stroke (IS) modifies oral contraceptive (OC)-associated IS risk. Background OCs are generally safe but vascular risk factors are known to increase OC-associated IS risk. If a genomic risk score could identify a subset of women with substantially increased risk of OC-associated IS, this could influence prescribing guidelines and reduce stroke events. Design/Methods In the Genetics of Early-Onset Stroke (GEOS) study, there were 340 premenopausal women (143 IS cases and 197 controls) with data on OC use within 30 days prior to index event for cases or prior to interview for controls. Using a previously validated genomic risk score (metaGRS) for IS based on 19 polygenic risk scores of vascular events and risk factors, we stratified our sample into tertiles of genomic risk and calculated the association between OC use and IS within each tertile. We tested if the association between OC use and IS depended on genomic risk of stroke (metaPRS) using logistic regression with an OC use*metaGRS interaction term. Results Among all women, OC use was significantly associated with IS (odds ratio = 2.4, p = 0.002). The odds ratio for IS associated with OC use decreased from 3.9 in the tertile with highest genomic risk of IS to 1.5 in the tertile with lowest genomic risk (Table 3). The formal test of interaction was consistent with our hypothesis (p < 0.07) that the genomic risk score modifies the association of OC use with IS. Conclusions The results of our exploratory analysis support the need for international collaboration to generate sufficient sample sizes to determine whether a genomic risk score could be clinically useful in reducing OC-associated IS.

Table 3.

(Abstract 31) Odds Ratio for Oral Contraceptive Use Stratified by Genetic Risk Score

Tertile based on metaGRS	OR for OC use (95% CI)	# Of OC users/# of cases	# Of OC users/# of controls
Upper Tertile	3.9 (1.3–11.8)	13/51	5/62
Middle Tertile	2.9 (1.0–8.0)	12/47	7/66
Lower Tertile	1.5 (0.6–3.9)	11/45	12/69
All participants	2.4 (1.4–4.3)	36/143	24/197

CI = confidence interval; OC = oral contraceptive; OR = odds ratio.

Contact Email: SKittner@som.umaryland.edu

32. Serum NOTCH3 extracellular domain is significantly decreased in patients with CADASIL

Hyesung Kim, PhD ¹ , and Jay Chol Choi, MD ²

Objectives To measure serum levels of NOTCH3 extracellular domain (N3ECD), transforming growth factor-β (TGF-β), latent TGF-β binding protein 1 (LTBP-1) and compare those serum levels of patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) to healthy control and as well as to sporadic ischemic stroke patients. Background Blood biomarkers can have many valuable applications in CADASIL such as screening CADASIL, monitoring disease progression, predicting prognosis, and surrogate endpoints in clinical trials. Previous research suggested N3ECD, TGF-β, LTBP-1 as potential blood biomarkers in CADASIL. In this study, we measured serum levels of N3ECD, TGF-β, LTBP-1 and compared those serum levels of patients with CADASIL to healthy control and as well as to sporadic ischemic stroke patients. Design/Methods The study participants consisted of 64 patients with genetically proven CADASIL patients (Jeju CADASIL cohort), age- and sex-matched 64 healthy individuals and same number of patients with sporadic ischemic stroke. Because the Jeju CADASIL cohort consists of predominant p.Arg544Cys variant, we enrolled additional 19 CADASIL patients from other institutions with various pathogenic variants for validation. Blood levels of potential biomarkers were determined in serum using a sandwich ELISA (ELISA). Receiver operating characteristic (ROC) curve was used to assess the accuracy of the blood biomarkers in discriminating between CADASIL from healthy participants or sporadic stroke patients. Results Serum N3ECD level was significantly decreased in the Jeju CADASIL cohort (mean age: 66 years, men: 47%), compared to healthy participants and sporadic stroke controls (19.5 ± 10.4 ng/mL vs 78.9 ± 36.3 ng/mL vs 74.2 ± 0.43.5 ng/mL, p < 0.001). The validation cohort had no significant difference in serum N3ECD level from that of the Jeju CADASIL cohort, but still showed a significantly decreased level compared with control subjects. The area under ROC curve was 0.989 (95% Confidence Interval [CI], 0.997–1.000) for CADASIL vs healthy controls and 0.933 (95% CI 0.888–0.978) for CADASIL vs sporadic stroke with a cut-off value of 35.0 ng/mL. Serum levels of TGF-β, LTBP-1 did not differ significantly between the groups. Conclusions Serum N3ECD showed high sensitivity and reasonable specificity in differentiating CADASIL from healthy control and as well as sporadic stroke. Unlike other blood biomarkers for cerebral small blood vessel, serum N3ECD measurement is specific for CADASIL and can be used as a screening test for CADASIL.

Contact Email: jaychoi@jejunu.ac.kr

33. A high-throughput copy number variation genotyping strategy

J.L. Qiao ¹ , R. Levinson ² , B. Chen ³ , P. Erhart ¹ , A.G. Lindgren ⁴ , J.W. Cole ⁵ , and C. Grond-Ginsbach ¹ ; for the CNV and Stroke (CaNVAS) Study Group

Objective To accelerate array-based copy number variation (CNV) genotyping and improve its accuracy. Background Current CNV algorithms are labor-intensive and time-consuming, with high false-positive rate. We here present an alternative strategy by using clusterplots to visualize selected CNVs in whole study populations. Method Protein-coding CNV regions from previous CNV studies were analyzed. The median signal intensities for all SNPs within each CNV-region and in a flanking region were calculated. Signal intensities were normalized by subtracting flanking signals from CNV signals. All SNPs in a CNV region were classified as homozygous, heterozygous di-allelic or heterozygous tri-allelic. Clusterplots were generated and validated in the South London Ethnicity and Stroke Study (SLESS) (n = 1,587) and the Health Retirement Study (HRS) (n = 13,254). Findings were compared with findings of conventional methods (PennCNV analysis and visual inspection). Results Speed: Clusterplot analysis of 1000 CNV regions in SLESS took 504 hours (hrs). PennCNV analysis and visual inspection of 48,234 CNV findings required 1,608 hours. Accuracy: Normalization significantly reduced the rate of false-positive CNV-findings. Clusterplots identified 97,8% of the minor alleles of CNV-region Chr4: 165.86–165.88 mb, but conventional methods only 69,5%. Moreover, 4-copy genotypes were correctly recognized only with clusterplots. Robustness: During clusterplot analysis, 0.18% of the samples were excluded due to insufficient quality, whereas 9% of the samples were excluded after PennCNV analysis. Clusterplots could also be used for CNV analysis of sex chromosomes. Conclusion The new clusterplot strategy allows rapid and accurate CNV genotyping in large populations (Figure 6).

Figure 6. (Abstract 33) A high-throughput copy number variation genotyping strategy.

Contact Email: caspar.ginsbach@gmail.com

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

J.W. Cole ¹ , J.L. Qiao ² , O.J. Adebajo ³ , T. Coker ³ , T. Adigun ³ , O.M. Akpa ³ , R. Akinyemi ³ , S. Bell ⁴ , B. Chen ⁵ , J. Jimenez-Conde ⁶ , U.L. Dobao ⁶ , I Fernandez ⁷ , M. Fornage ⁸ , C. Gallego-Fabrega ⁷ , L. Heitsch ⁹ , L. Ibanez ⁹ , C. Jern ¹⁰ , J. Jiménez-Balado ⁶ , M. Krawczak ¹¹ , H.S. Markus ⁴ , O. Melander ^12,13 , M. Owolabi ³ , K. Schlicht ¹¹ , M. Soderholm ^13,14 , V. Srinivasasainagendra ¹⁵ , C. Soriano-Tarraga ⁶ , M. Stenman ¹² , H. Tiwari ¹⁵ , K. Nguyen ¹⁶ , K. Ryan ¹⁶ , B. Gaynor ¹⁶ , T. O'Connor ¹⁶ , O.C. Stine ¹⁶ , S.J. Kittner ¹ , P. McArdle ¹⁶ , B.D. Mitchell ¹⁶ , H. Xu ¹⁶ , A. Lindgren ¹² , and C. Grond-Ginsbach ² ; for the CNV and Stroke (CaNVAS) Study Group

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

Contact Email: jcole@som.umaryland.edu

35. Global vs de novo Copy Number Variation

C. Grond-Ginsbach ¹ , J.L. Qiao ¹ , R.T. Levinson ² , B. Chen ³ , S.T. Engelter ⁴ , P. Erhart ¹ , A.G. Lindgren ⁵ , K. Schlicht ⁶ , and J.W. Cole ⁷ ; for the CNV and Stroke (CaNVAS) Study Group

Objective To explore very rare and unique copy number variation (CNV) in different populations. Background The mutation rate of CNV is 100∼1,000 times higher than of single nucleotide polymorphisms (SNPs). De novo and recent CNVs are likely to be rare and population specific. Time may have been too short for complete eradication of deleterious variants by purifying Darwinian selection. Methods CNVs were identified across multiple Caucasian and African American populations of the CaNVAS (CNV And Stroke) study. CNVs were classified as global (observed in many different populations), ethnicity-specific, or even specific for a single CaNVAS site. Rare CNV findings were studied pairwise by haplotype analysis to assess the length of the shared haplotypes. Results In a preliminary analysis of 25,372 individuals from 12 CaNVAS sites, we identified CNV in 1,023 different protein-coding loci across the genome. In a pilot study of all protein coding CNV regions of chromosome 17 (n = 46), one CNV had minor allele frequency (MAF) > 0.01 and 14 further CNV had MAF >0.0001. The remaining 31 (67%) CNVs had extremely rare or singular minor alleles (MAF <0.0001). Site-specific rare findings showed long (>5 Mb) identical genomic regions around the variants, whereas shared haplotypes of global findings were significantly shorter (<1.5 Mb). Conclusion On the level of the population, very rare protein coding CNVs are common. Very rare and unique CNVs may be recent and pathogenic. CNV is not just large-sized and possibly affecting multiple protein-coding genes, CNV is also highly dynamic. The possible impact of very rare CNV on stroke risk and stroke outcome will be analyzed within and across the study populations.

Contact Email: caspar.ginsbach@gmail.com

36. Update on the second phase GISCOME GWAS: A genome-wide association meta-analysis of functional outcome after ischemic stroke

C. Lagging, MD ^1,2 *, M. Söderholm, MD, PhD ^3,4 *, A. Pedersen, MD, PhD ^1,2 , S. Klasson, MSc ¹ , B. Andersson, MSc ¹ , T.M. Stanne, PhD ¹ , J.M. Maguire, RN, PhD ⁵ †, A.G. Lindgren, MD, PhD ^3,6 †, and C. Jern, MD, PhD ^1,2 †; on behalf of the International Stroke Genetics Consortium and GISCOME network

*These authors contribute equally to this work. †These authors jointly supervised this work.

Objective To discover common genetic variants associated with post-stroke functional outcome. Background Genetic factors likely account for part of the large inter-individual variability in recovery after ischemic stroke. The first genome-wide association study (GWAS) on functional outcome within the GISCOME network has been completed (n = 6,165). One variant, implicated in brain plasticity, reached genome-wide significance and several variants had suggestive association (p < 10⁻⁵) to outcome. Some of these variants were within or near genes with reported experimental relation to stroke volume and/or brain plasticity. Our results need replication, and a larger sample is needed in order to identify novel variants. We therefore initiated recruitment to a second GWAS within the GISCOME initiative. Methods This study comprises the participants from the first GISCOME GWAS and newly included ischemic stroke subjects with data on modified Rankin Scale (mRS) score at ∼90 days after stroke. Associations between common single-nucleotide polymorphisms and stroke outcome (mRS 0–2 = good outcome vs mRS 3–6 = poor outcome) will be investigated in logistic regression analyses adjusted for age, sex and ancestry (model 1), with additional adjustment for initial stroke severity (baseline NIH Stroke Scale score) in model 2. GWA analyses will be performed in each cohort separately and subsequently meta-analyzed. The newly included cases will also participate as a replication cohort for the phase 1 GWAS, whereafter a joint meta-analysis of all cases will be performed. Results We have received summary statistics from 9 cohorts comprising 8,862 new adult stroke cases to be included. In addition, we await summary statistics on an additional 2,000–3,000 cases from international collaborators. We have also recently genotyped 3,000 additional cases to be included from our study sites in Gothenburg and Lund, Sweden. Conclusions In total, we estimate that approximately 14,000 new cases will be included in the second GISCOME GWAS, leading to a total sample size of ∼20,000 participants. We anticipate completing the analyses by the end of 2022. The study will constitute the largest GWAS on stroke outcome to date.

Contact Email: Arne.G.Lindgren@skane.se

37. Genome-wide association study of vasospasm and delayed cerebral ischemia in aneurysmal subarachnoid hemorrhage

E. Cuadrado-Godia ¹ , I. Hostettler ² , J. Jiménez-Balado ¹ , Y.M. Ruigrok ³ , M.K. Bakker ³ , R.J. Behnam ⁴ , M. Niemelä´s ⁴ , S. Morel ⁵ , P. Bijlenga ⁵ , P. Constant dit Beaufils ⁶ , R. Bourcier ⁷ , D. Werring ² , and J. Jiménez-Conde ¹

Objective To identify genetic variants associated with vasospasm and/or delayed cerebral ischemia (DCI) in aSAH, through a GWAS. Background After the initial bleeding severity, DCI is an important predictor for poor outcome in aneurysmal subarachnoid hemorrhage (aSAH) patients and is mainly caused by arterial vasospasm but can also occur without it. The genetic contribution to vasospasm and DCI has previously been investigated in 2 genome-wide association studies (GWAS) which failed in identifying genetic variants, which could have been due to low sample size in both studies (245 and 190 cases). Methods Multicentric cohort study within the ISGC will be conducted under the lead of researchers from UCL Queen Square Institute of Neurology (UK) and Institut Hospital del Mar d’Investigacions Biomèdiques (Spain). Inclusion criteria are as follows: aSAH with available data on vasospasm and DCI and individual level whole-genome genotype data. Samples with single nucleotide polymorphisms (SNPs) with a call-rate lower than 95%, outlier heterozygosity rates, gender mismatch or high relatedness will be removed. We will use principal components (PC) to adjust for population structure in subsequent analyses. Single nucleotide polymorphisms deviating from Hardy-Weinberg equilibrium (p < 1 × 10⁻⁵)or ambiguous, monomorphic or duplicated SNPs will be removed. We will use Michigan sever and HRC r1.1 2016 (GRCh37/hg19) as the reference genome for imputation. After imputation, SNPs with R2 < 0.6 and minor allele frequency <1% will be excluded. Two logistic regression models will be built for vasospasm and DCI separately, adjusting for sex, age, most significant PC, and the world federation of neurosurgeons grading scale. Results About 2000 cases of aSAH have already been included. Data transfer agreements and Quality Control analyses are currently under way. Conclusions With our study we aim to identify an underlying genetic predisposition for vasospasm and DCI that may help to better understand its pathophysiology and potentially find new targets for drug development to prevent this complication in aSAH patients.

Contact Email: jjimenez@imim.es

38. Enhancement of Stroke REhabilitation with Levodopa (ESTREL)—PRECISION—a genetic study nested within a large randomised clinical trial in stroke rehabilitation

C. Traenka ^1,2,* , S.T. Engelter ^1,2,* , A. Zietz ^1,2,* , A. Polymeris ^1,2 , K. Wiesner ^1,2 , F. Ravanelli ^1,2 , W. Kamerbeek ¹ , V. Altersberger ^1,2 , J. Kaufmann ^1,2 , H. Gensicke ^1,2 , and P.A. Lyrer ² ; Caspar Grond-Ginsbach ¹

*Equal contribution.

Objective to test whether Single Nucleotide Polymorphisms or Copy Number Variation of dopaminergic pathways modify functional recovery in stroke patients with or without levodopa enhanced rehabilitation. Background ESTREL is a randomized, placebo-controlled trial investigating whether Levodopa given in addition, to rehabilitative therapies is associated with a patient-relevant enhancement of motor recovery after acute stroke. Inter-individual variation in drug response as genetic polymorphisms modify pharmacokinetics with a potential clinical impact on (1) stroke recovery and (2) response to the assumed pharmacologic enhancement of levodopa. Methods ESTREL-PRECISION is a nested proof-of-principle study within the ESTREL clinical trial. It enables to combine pharmacogenetic profiles with functional outcome measures in well phenotyped stroke patients who received pre-specified standardized rehabilitation therapy. Sample size: n = 500; genetic analyses: from whole blood sample of each participant DNA extraction and genomic profiling on high density SNP-microarrays (Infinium Omni 2.5 Exome-8 v1.3) will be performed The normalized microarray data will be analysed in 2 different ways: (1) Extraction of relevant individual SNPs and relevant haplotypes that are known to modify the Levodopa response in patients with Parkinson's disease and therefore are assumed to modify the effect of Levodopa on stroke rehabilitation. (2) A genome-wide study of CNVs and assessment of global genomic imbalance in each patient as well as specific genomic imbalance affecting genes associated with dopaminergic pathways will be performed. Results Currently blood samples from 245 patients have been collected. First data are expected in 2024. Conclusion ESTREL-PRECISION provides the opportunity to comprehensively study the impact of individual patient-level genetic modifiers on stroke recovery. In addition, ESTREL-PRECISION data might be of interest for a better understanding of stroke recovery mechanisms in future pooled analyses.

Contact Email: Stefan.engelter@felix.platter.ch; Stefan.engelter@usb.ch

39. Genetics and Epigenetics of Post—Stroke Vascular Cognitive Impairment and Dementia Among Indigenous Africans

R. Akinyemi, MBBS, PhD^1,2, M. Coker, MSc², H.K. Tiwari, PhD³, V. Srinivasasainagendra, MSc³, O. Akpa, PhD⁴, B. Adewale, MBBS², H. Markus, MD⁵, I. Fernandez, PhD⁶⁺, R. Kalaria, PhD⁷⁺, and M. Owolabi, MBBS, MD^1,2+

⁺Equal contributions.

Objective To unravel the contributions of genetic and epigenetic factors to cognitive outcomes among indigenous African stroke survivors. Background Cognitive impairment and dementia (CID) attributable to vascular brain disease is an escalating public health problem. Stroke, a leading cause of vascular CID has the highest burden and worse outcomes (including cognitive dysfunction) among people of African ancestry. About 50% of indigenous African stroke survivors exhibited cognitive dysfunction in previous studies. The reasons for the susceptibility to poorer cognitive outcomes likely involve genetic, epigenetic and non - genetic mechanisms. Methods 1,166 stroke survivors in the SIREN cohort underwent cognitive assessment at 3 months after stroke using the Community Screening Instrument for Dementia (CSID), the Montreal Cognitive Assessment (MoCA and selected tools from the Vascular Neuropsychological Battery (V-NB). Subjects were cognitively categorized into normal and impaired using the VasCog Criteria. DNA genotyping was performed using the novel 2.5M H3Africa array chip and acquired GWAS data is being explored for variants associated with impairment. In EpiCogFAST, the epigenetics study, we explore epigenetic variants associated with impairment among Nigerian African and UK Caucasian stroke survivors from EWAS data acquired using the Human Methylation EPIC array chip from blood - derived DNA from the CogFAST Nigeria and SIREN studies (20 case - control pairs) and the CogFAST- Newcastle Study (20 case - control pairs). Analysis will be implemented using the R software with Bioconductor packages. Differentially methylated CpG (DMCs) sites will be analyzed using the nonparametric Mann–Whitney U test for independent samples and multivariable generalized linear analyses adjusting for principal components and DNAm potential covariates. Results Among 1,166 West African stroke survivors in the SIREN study, 48% were cognitively impaired at 3 months after stroke. Factors independently associated with post-stroke cognitive impairment include low educational attainment, higher stroke severity, male gender, low intake of green leafy vegetable, diabetes mellitus and older age while a higher cognitive and social lifestyle score (CSLS) was protective. Global and cortical atrophy on neuroimaging were also associated with cognitive impairment in the cohort. GWAS and EWAS findings are being awaited. Conclusion An integrative approach involving the exploration of sociodemographic, lifestyles, clinical, imaging, genetic and epigenetic determinants of vascular cognitive dysfunction in diverse populations including indigenous Africans will provide useful insights into pathways, processes and networks, potential identification and characterization of novel biomarkers for prevention, risk profiling, detection, diagnosis, prognosis and treatment of vascular cognitive disorders in a precision/personalized medicine framework.

Contact Email: rufusakinyemi@yahoo.com

40. Establishing a population - based cohort for longitudinal evaluation of cardiovascular health, cognition and frailty among older Africans: The Vascular heAlth, fraiLty and cognItion in Ageing Nigerians sTudy (VALIANT)

R. Akinyemi, MBBS, PhD ^1,2 , J. Yaria, MBBS ¹ , F. Popoola, MBBS ³ , J. Akinyemi, PhD ⁴ , T. Fagbemi, MBBS ¹ , T Makanjuola, MBBS ¹ , C. Udeh-Momoh, PhD ⁵ , M. Owolabi, MBBS MD ^1,2 , R. Romero-Ortuno, MD, PhD ⁶ , R. Kalaria, PhD ⁷ , A. Ogunniyi, MBChB, MD ¹ , and B. Lawlor, MD ⁶

Objective To establish a population-based cohort of older adults (50+ years) and investigate the association of cardiovascular health (CVH), genetic, epigenetic and biomarkers with cognition and frailty longitudinally. Background Globally, the proportion of global burden of disease that is attributable to brain health disorders is expected to rise partly because of the projected increase in the proportion of older adults. This rise will be steeper in low- and middle- income countries. It is not fully understood why individuals of African ancestry are more prone to vascular cognitive disorders and frailty. Methods VALIANT is a population-based cohort study involving the Ibadan North local government area (LGA) with a population of 352,270. One thousand participants selected by multistage, stratified cluster random sampling method are sampled from this population. The participants are interviewed to elicit information on selected sociodemographic characteristics, lifestyles, dietary intake, physical activity, and diagnosis and treatment history of cardiometabolic factors using standard definitions. Measurements include height, weight, abdominal and hip circumference, blood pressure (sitting and standing) following standard procedures. Community Screening Instrument for Dementia (CSID), the Montreal Cognitive Assessment (MoCA) and the Vascular Neuropsychological Battery (V-NB). Cognitive assessment is performed by experienced interviewers. Final cognitive categorization is based on the VASCOG and the DSM V criteria. Determination of frailty profile is done using the SHARE Frailty Instrument (SHARE-FI). Blood sample and fractions (serum, plasma, red cell concentrates, buffy coat) and DNA are stored for future omics analysis. Ethical approval was obtained from the University of Ibadan/University College Hospital Health Research Ethics Committee. Conclusion VALIANT will be the first detailed and rigorous epidemiologic study of association between cardiovascular health and cognition and frailty in Nigeria using a longitudinal design. Secondly, this study on a long term offers opportunity to study the genetics, epigenetics, biomarkers and intermediate phenotypes of vascular brain disease and thus contribute to understanding the neurobiology and mechanisms of vascular cognitive brain disorders and frailty in a population of African ancestry.

Contact Email: rufusakinyemi@yahoo.com

41. Copy Number Variation and Stroke risk and Outcome Study (CaNVAS): Phenotype characteristics

M. Stenman ¹ , P. McArdle ² , B. Gaynor ² , J.L. Qiao ³ , B. Cheng ⁴ , A. Olayinka ⁵ , M. Owolabi ⁵ , R. Akinyemi ⁵ , M. Coker ⁵ , V. Srinivasasainagendra ⁶ , H. Tiwari ⁶ , C. Grond-Ginsbach ³ , J.W. Cole ⁷ , and A.G. Lindgren ¹ ; for the CNV and Stroke (CaNVAS) Study Group

Objective To describe phenotype characteristics in a large sample of ischemic stroke and control subjects with available genotyping information. Background Copy number variations (CNVs) have been insufficiently examined regarding stroke risk and outcome. Design/Methods The international multicenter CaNVAS study assembles detailed phenotype and genotype information in a large number of ischemic stroke and control subjects. We here present the phenotype information we have collected and collated in CaNVAS to date. Results 15,163 ischemic stroke patients and 25,634 control subjects are included with additional subjects and phenotype information pending. Phenotype characteristics are presented in the Table 1. Conclusions CaNVAS aims to include subjects with different ancestries, pathogenetic mechanisms and outcome measures for ischemic stroke. This hitherto is the largest study on CNV and ischemic stroke.

Table 4.

(Abstract 41) Panel A: CaNVAS Cohort Table

Study site	Cases	Controls	Outcome mRS 30/90 d	Race (Y/N)	Cardiovascular risk factors available in 50% or more (Pat/Ctrl)
					Hypertension	AF	CAD	DM	Chol	Smok
Barcelona	2,323	0	0/1769	Y/-	Y/-	N/-	Y/-	N/-	Y/-	Y/-
BASICMAR	2083	0	170/1,408	Y/-	Y/-	N/-	Y/-	N/-	Y/Y	N/-
CADISP	1,223	0	0/1,128	Y/-	Y/-	N/-	N/-	Y/-	Y/-	Y/-
GASROS	470	0	211/0	Y/-	Y/-	Y/-	Y/-	Y/-	N/-	Y/-
GCNKSS	499	0	331/0	Y/-	Y/-	Y/-	Y/-	Y/-	N/-	Y/-
GEOS	PENDING	—	—	—	—	—	—	—	—	—
HRS	0	15,488	-/-	-/Y	-/Y	-/N	-/Y	-/Y	-/N	-/N
KRAKOW	952	776	952/0	Y/Y	Y/Y	N/N	Y/Y	Y/Y	N/N	Y/Y
LEUVEN	482	468	0/467	Y/Y	Y/Y	Y/Y	Y/Y	Y/Y	Y/Y	Y/Y
LSR	1,585	773	0/967	N/N	Y/Y	Y/Y	N/N	Y/Y	Y/Y	Y/Y
MDC	1,534	4,923	0/798	N/N	Y/Y	Y/Y	Y/Y	Y/Y	Y/Y	Y/Y
SAHLSIS	1,066	600	0/860	N/N	Y/Y	Y/Y	Y/Y	Y/Y	Y/Y	Y/Y
SLESS	808*	868	0/0	Y/Y	N/N	N/N	N/N	N/N	N/N	N/N
SIREN	1,683	1738	—	—	—	—	—	—	—	—
WUSTL	455	0	0/92	Y/-	Y/-	Y/-	Y/-	Y/-	N/-	Y/-
TOTAL	15,163	25,634	2,462/6,691

Panel B: CaNVAS Phenotype table
Characteristics	Ischemic stroke cases (n = 13,480**)	Controls (n = 23,896**)	Missing data cases/controls
Gender, female, n	6,259	13,251	6/0
Age, years, median (IQR)	69 (57–79)	67 (58–75)	3/0
Race, n	—	—	4,440/7,189
Black or African American, n	1,143	1705	—
White, n	7,485	13,836	—
Other, n	412	1,166	—
Ethnicity = Hispanic/Latino (only for White), n	34	2,190	4,462/7,128
First/recurrent stroke, n	9,156/2,700	N/A	1,624/NA
TOAST/CCS***	—	—	2,193/NA
Large artery atherosclerosis, n	1715	N/A	—
Cardioembolic, n	3,814	N/A	—
Small vessel disease, n	1,237	N/A	—
Other known etiology, n	1,424	N/A	—
Undetermined, n	3,097	N/A	—
Cardiovascular risk factors	-	—	—
Hypertension, n	8,679	12,974	862/1735
Atrial fibrillation, n	1,124	73	6,624/17,597
Coronary artery disease, n	1,548	4,028	5,673/2,561
Diabetes mellitus, n	1740	3,887	5,490/1,521
Hypercholesterolemia, n	3,864	4,608	4,058/17,327
Smoker current/former, n	2,498/1895	3,359/7,413	5,034/3,193
NIHSS****, n, (median (IQR))	9,605, (5 (2–11))	N/A	3,875/NA
NIHSS taken at day, median (IQR)	0 (0–1)	N/A	—
mRS (30 d), n, (median (IQR))	1,664, (2 (1–4))	N/A	11018/NA
mRS taken at day 30, median (IQR)	30, (30-30)	N/A	—
mRS (90 d), n, (median (IQR))	7,489, (2 (1–3))	N/A	6,789/NA
mRS, dichotomized 0–2/3-6	4,361/2,330	N/A	6,789/NA
mRS taken at day 90, median (IQR)	90 (90–95)	N/A	—

*Approximately 80 cases have had ICH and not ischemic stroke. **Excluding SIREN-samples. ***TOAST if available, otherwise CCS. ****First NIHSS assessment after stroke onset up to one week after stroke onset.

BASICMAR = Hospital Del Mar, Barcelona; CADISP = Cervical Artery Dissection and Ischemic Stroke Patients; GASROS = The Massachusetts General Hospital Stroke Genetics Group; GCNKSS = Greater Cincinnati/Northern Kentucky Stroke Study; GEOS = The Genetics of Early Onset Stroke; HRS = Health and Retirement Study; LSR = Lund Stroke Register; MDC = Malmö Diet and Cancer Study; SAHLIS = Sahlgrenska Academy Study on Ischaemic Stroke; SLESS = South London Ethnicity and Stroke Study; SIREN = The Stroke Investigative Research and Educational Network; WUSTL = Washington University St. Louis.

Contact Email: arne.lindgren@med.lu.se

42. A genome wide association study of outcome after aneurysmal subarachnoid haemorrhage: Discovery Analysis

B. Gaastra ^1,2 , S. Alexander ³ , M.K. Bakker ⁴ , H. Bhagat ⁵ , P. Bijlenga ⁶ , S.L. Blackburn ⁷ , M.K. Collins ⁸ , S. Doré ⁹ , C.J. Griessenauer ^10,11 , P. Hendrix ^10,12 , E.P. Hong ¹³ , I.C. Hostettler ^14,15 , H. Houlden ¹⁴ , K. Iihara ¹⁶ , J.P. Jeon ¹³ , B.J. Kim ¹³ , J. Li ¹⁰ , S. Morel ^6,17 , P. Nyquist ¹⁸ , D. Ren ³ , Y.M. Ruigrok ⁴ , D. Werring ¹⁴ , W. Tapper ¹ , I. Galea ¹ , and D. Bulters ²

Objective The overall aim of this study is to perform a two-stage (discovery and validation) genome-wide association meta-analysis to identify genetic variation associated with clinical outcome after aneurysmal subarachnoid haemorrhage (aSAH). Here we report completion of the of the discovery stage with preliminary results and aim to raise awareness of the study to recruit further samples for validation. Background Genetic variation has been associated with clinical outcome after aSAH in a number of candidate gene studies, however, no genome-wide association studies have been performed to date. Understanding genetic determinants of outcome will provide valuable insight into the pathophysiology of outcome following aSAH with the potential to improve prognostic modelling and target novel strategies to improve outcome after aSAH. Methods This discovery analysis consisted of an individual patient level data genome wide meta-analysis of multiple international cohorts. Logistic regression was used to test the association between genetic variants and dichotomised clinical outcome, adjusting for age and genetic ancestry. The primary outcome was the modified Rankin Scale (mRS) or Glasgow Outcome Scale (GOS) dichotomised into good (mRS 0–2, GOS 4–5) and poor (mRS 3–6, GOS 1–3). Results A total of 2,489 patients from 7 international cohorts were included in this discovery analysis. We report 157 independent genetic loci harbouring 756 genetic variants associated with outcome after aSAH (p < 1 × 10⁻⁴) for validation in the second stage of this study. A single genetic variant (rs12949158) reached genome wide significance (p = 4.29 × 10⁻⁸). This variant is located in an intronic region of SPNS2, implicating sphingosine-1-phosphate (S1P) signalling in outcome after aSAH. Conclusions In conclusion we identify 157 independent genetic loci for validation and implicate S1P signalling in outcome after aSAH. If validated, these findings will give significant insight into the pathophysiology of outcome after aSAH with potential therapeutic implications. An international effort is ongoing to recruit samples for validation.

Contact Email: ben.gaastra@gmail.com

43. Updated Stroke Gene Panels: Rapid evolution of knowledge on monogenic causes of Stroke

A. Ilinca¹, A. Puschmann¹, J. Putaala², F.E. de Leeuw³, J.W. Cole⁴, S.J. Kittner⁴, Ulf Kristoffersson⁵, and A.G. Lindgren¹

Objective We aimed to create an updated panel with genes related to monogenic stroke, by using similar systematic methods for data compilation as in our previous Stroke Gene Panel (SGP). We also aimed to investigate how fast knowledge develops regarding the level of evidence of monogenic conditions related to stroke and cerebrovascular disease. Background The present results update our previous Stroke Gene Panel (SGP) publication which was based on a literature search from August 2017. Methods Online Mendelian Inheritance in Man and PubMed were searched. We divided detected genes into 2 SGP groups: SGP1, genes reported in at least one person with stroke and associated one or more clinical subgroups: large artery atherosclerotic disease, large artery nonatherosclerotic conditions (tortuosity, dolichoectasia, aneurysm, non-atherosclerotic dissection or occlusion), cerebral small vessel disease, cardioembolic sources (arrhythmia, heart defect, cardiomyopathy), coagulation dysfunctions (venous thrombosis, arterial thrombosis, bleeding tendency), intracerebral hemorrhage, vascular malformations (cavernoma, arteriovenous malformations), and metabolic disorders; and SGP2, genes related to diseases that may cause stroke. Results We identified a total of 168 SGP1 genes—of which 70 have been validated for clinical practice according to our criteria—and 72 SGP2 genes. Nine genes were removed because conflicting evidence has emerged since the first SGP version. Conclusions The number of genes detected increased from 168 to 240 during a 4.5-year period, reflecting a dynamic evolution and the need for regular updates of a SGP for research and clinical use.

Contact Email: andreea.ilinca@med.lu.se