Bio-Repository of DNA in Stroke (BRAINS): London

Supplementary Information

Immunochip Discovery Cohorts

Bio-Repository of DNA in Stroke (BRAINS): London

The Bio-Repository of DNA in Stroke (BRAINS) is an international study recruiting highly phenotyped patients with stroke. For the purposes of the current work all patients were Caucasians. Diagnosis of stroke was confirmed using positive imaging (MRI or CT) and ischemic stroke subtypes were assigned using TOAST criteria, based on clinical, imaging and risk factor data. The cohort has been described in detail elsewhere (Yadav S, Schanz R, Maheshwari A, et al. Bio-Repository of DNA in stroke (BRAINS): a study protocol. BMC Med Genet 2011; 12: 34).

Glasgow: Scotland

Patients with ischemic stroke attending the cerebrovascular service of the Western Infirmary, Glasgow, were recruited between 1990 and 2004 as part of an ongoing study of genetic and circulating biomarkers in stroke. All patients underwent brain imaging and extracranial carotid ultrasound in accordance with a standard clinical protocol. The study was approved by the West Ethics Committee.

Controls for the UK samples were drawn from shared WTCCC controls obtained from the 1958 Birth Cohort. This is a prospectively collected cohort of individuals born in 1958 (http://www.b58cgene.sgul.ac.uk/), and ascertained as part of the national child development study (http://www.cls.ioe.ac.uk/studies.asp?section=000100020003). Data from this cohort are available as a common control set for a number of genetic and epidemiological studies.

Leuven: Belgium

Cerebral ischemia, defined as a clinical stroke with imaging confirmation or a TIA with a new ischemic lesion on diffusion weighted MRI, who were admitted to the Stroke Unit of the University Hospitals in Leuven. All patients underwent brain imaging (MRI in 91% of patients, CT in the remainder) and a standardized protocol including carotid ultrasound or CT angiography and cardiac examination (echocardiography and Holter monitoring) in all patients. Control individuals were selected from the same population and were either spouses of patients with multiple sclerosis, amyotrophic lateral sclerosis or stroke.

Lund Stroke Register, Sweden

Lund Stroke Register (LSR) since 2001 continuously enrolls patients aged 18 and older with first-ever stroke, living in the primary uptake area of Skane University Hospital, Lund. The study is mainly hospital-based but has a good coverage of the whole geographical population.28 All included patients are examined with CT/MR or autopsy of the brain. When clinically indicated, the patients are examined with ultrasound imaging of carotid arteries, echocardiography, and angiography. In this study, first-ever ischemic stroke patients from LSR between 2001 and 2004 were included. All patients were assessed by a neurologically trained physician regarding stroke type. Informed consent was obtained from all individuals or when they were not able to respond from their next-of-kin. The study was approved by the Ethics Committee of Lund University. Biobank services were performed at Region Skane Competence Centre (RSKC Malmo), Malmo University Hospital, Malmo, Sweden.

Swedish Control Samples

Controls for the Lund cases were provided by the Swedish SLE network. Controls were healthy blood donors from the geographical areas of Uppsala, Stockholm and Lund. The studies were approved by the regional ethics boards and all subjects gave their informed consent to participate. Genotyping of the Swedish control samples was performed at the SNP&SEQ technology platform in Uppsala, Sweden (www.genotyping.se).

Munich: Germany

Cases were consecutive European Caucasians recruited from a single dedicated Stroke Unit at the Department of Neurology, Klinikum Gro§hadern, Ludwig-Maximilians-University, Munich. Ischemic stroke subtypes were determined according to TOAST criteria based on relevant clinical and imaging data.

German control samples

German healthy control individuals were obtained from the PopGen biobank [Krawczak et al., Community Genet 9:55-61, 2006]. Written, informed consent was obtained from all study participants and all protocols were approved by the institutional ethical review committee of the participating centre. The panel is a cross-sectional control cohort from the Kiel area in Northern Germany. More than 300 phenotypes were collected for the cohort and a 3-year follow has recently been completed. All data and biomaterials are accessible via the PopGen biobank. The Genotyping was part of the German National Genome Research Network (NGFN) GWAS initiative and performed by the Institute of Clinical Molecular Biology (Christian-Albrechts-University of Kiel).

Poland:Krakow

Patients were recruited in the stroke unit of the Jagiellonian University in Krakow, Poland (a single-center study). All stroke patients and controls were 18 years of age and were white. All patients had clinically relevant diagnostic workup performed, including brain imaging with computed tomography (CT) (100%) and/or magnetic resonance imaging (MRI) (8%) as well as ancillary diagnostic investigations including duplex ultrasonography of the carotid and vertebral arteries (85.2%), echocardiography (54.8%). MR-angiography, CT-angiography Holter monitoring, transesophageal echocardiography and blood tests for hypercoagulability were performed were indicated. Patients were classified into etiologic subtypes according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST).

The control group included unrelated subjects taken from the population of southern Poland. Control subjects had no apparent neurological disease based on the findings in a structured questionnaire and a neurological examination. The study was approved by local research ethics committees and informed consent was obtained from all participants.

PROMISe Study (The Netherlands)

The PROMISe study has been described elsewhere in detail.1 We included patients with non-disabling cerebral ischemia of arterial origin, who were referred to the University Medical Center Utrecht, The Netherlands and were included in the SMART (Second Manifestations of Arterial disease) study,2 or the Utrecht Stroke Database (USDB). Patients with non-atherosclerotic causes of cerebral ischemia or with potential source of embolism in the heart were excluded from this study. We included 1125 patients enrolled into SMART or USDB between April 1994 and May 2009 for the current analysis. All cases were genotyped at the Genetic Laboratory at the Erasmus Medical Center in Rotterdam, The Netherlands. As controls, we included Dutch individuals genotyped separately from the cases (at the Department of Human Genetics, University Medical Center Groningen, The Netherlands), as described in a published study of celiac disease.3 Written informed consent was obtained from all subjects with approval from the ethics committee or institutional review board of all participating institutions.

1. Achterberg S, Kappelle LJ, Algra A. Prognostic modelling in ischaemic stroke study, additional value of genetic characteristics. Rationale and design. Eur Neurol 2008;59(5):243-52.

2. Simons PC, Algra A, van de Laak MF, Grobbee DE, van der Graaf Y. Second manifestations of ARTerial disease (SMART) study: rationale and design. Eur J Epidemiol 1999 October;15(9):773-81.

3. Trynka G, et al. Dense genotyping identifies and localizes multiple common and rare variant association signals in celiac disease. Nat Genet. 2011 Nov 6;43(12):1193-201.

Immunochip replication cohorts

Both the WTCCC2 and METASTROKE cohorts have been previously published, including full details of case and control cohorts. Readers are referred to the following publications for full details:

Bellenguez C et al. Genetic analysis identifies a new susceptibility locus in HDAC9 for large vessel ischemic stroke, and supports genetic heterogeneity across stroke subtypes. Nature Genetics 44, 328-333 (2012).

Traylor M et al. Genetic risk factors for ischaemic stroke and its subtypes (the METASTROKE collaboration): a meta-analysis of genome-wide association studies. Lancet Neurol 11, 951-962 (2012).

Interstroke

INTERSTROKE is an international, multicenter case-control study. Cases are patients with a first stroke within 72 h of hospital presentation in whom CT or MRI is performed. Proxy respondents are used for cases unable to communicate. Etiological and topographical stroke subtype is documented for all cases. Controls are hospital- and community-based, matched for gender, ethnicity and age (±5 years). A questionnaire (cases and controls) is used to acquire information on known and proposed risk factors for stroke. Cardiovascular (e.g. blood pressure) and anthropometric (e.g. waist-to-hip ratio) measurements are obtained at the time of interview. Nonfasting blood samples and random urine samples are obtained from cases and controls.

VISP

The VISP trial (P.I. James Toole, MD, Wake Forest University School of Medicine (WFU); R01 NS34447) was a multi-center, double-blind, randomized, controlled clinical trial that enrolled patients aged 35 or older with Homocysteine levels above the 25th percentile at screening and a non-disabling cerebral infarction (NDCI) within 120 days of randomization.34,35 NDCI was defined as an ischemic brain infarction not due to embolism from a cardiac source, characterized by the sudden onset of a neurological deficit. The deficit must have persisted for at least 24 hours, or if not, an infarction in the part of the brain corresponding to the symptoms must have been demonstrated by CT or MRI imaging. The trial was designed to determine if daily intake of a multivitamin tablet with high dose folic acid, vitamin B6 and vitamin B12 reduced recurrent cerebral infarction (1° endpoint), and nonfatal myocardial infarction (MI) or mortality (2° endpoints). Subjects were randomly assigned to receive daily doses of the high-dose formulation (n=1,827), containing 25mg pyridoxine (B6), 0.4mg cobalamin (B12), and 2.5mg folic acid; or the low-dose formulation (n=1,853), containing 200µg pyridoxine, 6µg cobalamin and 20µg folic acid. Enrollment in VISP began in August 1997, and was completed in December 2001, with 3,680 participants enrolled, from 55 clinic sites across the US and Canada and one site in Scotland.

Control data for comparison with VISP stroke cases were obtained through the database of genotypes and phenotypes (dbGAP) High Density SNP Association Analysis of Melanoma: Case-Control and Outcomes Investigation (phs000187.v1.p1; R01CA100264, 3P50CA093459, 5P50CA097007, 5R01ES011740, 5R01CA133996, HHSN268200782096C; PIs Christopher Amos, Qingyi Wei, Jeffrey E. Lee).

Definitions for Risk Factor Defined Analysis

Hypertension was defined as being treated for hypertension, systolic pressure >140mmHg or diastolic pressure >90mmHg. Diabetes mellitus was defined as presence or absence, hypercholesterolemia was defined as treated or Cholesterol > 5.2mmol/l, past history of symptomatic coronary artery disease or ischaemic heart disease was defined as yes or no; smoking status was defined as ever-never.

Genotyped SNPs / Imputed SNPs / Exclusion based on info <0.3 / Exclusion based on MAF <0.01 / Available for Analysis
Belgium / 135,006 / 16,557,991 / 11,560,100 / 1,081,367 / 4,051,530
Germany / 123,920 / 16,795,032 / 12,486,052 / 831,497 / 3,601,403
Netherlands / 88,511 / 9,224,829 / 5,696,130 / 93,007 / 3,524,203
Poland / 130,732 / 16,722,066 / 11,612,683 / 1,069,671 / 4,170,444
Sweden / 133,679 / 16,789,789 / 11,935,797 / 1,004,710 / 3,982,961
UK / 131,030 / 16,792,341 / 12,332,145 / 915,039 / 3,676,187

Supplementary Table e-1: number of genotyped and imputed SNPs by cohort.

SNP / Chr / BP / RA / RAF / Unconditional Analysis
IC + WT / Unconditional Analysis
IC + WT + MS / Conditional analysis
IC + WT
OR (95% CI) / P / OR (95% CI) / P / OR (95% CI) / P
rs11065987 / 12 / 112,072,424 / T / 0.41 / 1.10 (1.08-1.12) / 2.78E-05 / 1.10 (1.08-1.12) / 1.06E-09 / 0.96 (0.93-0.99) / 0.14
rs642898 / 12 / 112,141,233 / A / 0.71 / 1.11 (1.09-1.14) / 2.11E-05 / 1.09 (1.07-1.10) / 4.82E-08 / 1.05 (1.00-1.12) / 0.36
rs2238151 / 12 / 112,211,833 / T / 0.66 / 1.13 (1.11-1.16) / 2.31E-07 / 1.10 (1.08-1.11) / 1.79E-09 / 1.31 (1.00-1.71) / 0.31
rs10744777 / 12 / 112,233,018 / T / 0.66 / 1.13 (1.11-1.16) / 1.75E-07 / 1.10 (1.08-1.11) / 1.24E-09 / - / -
rs4767293 / 12 / 112,463,296 / A / 0.66 / 1.13 (1.10-1.15) / 7.62E-07 / 1.09 (1.07-1.10) / 1.67E-08 / 1.02 (0.92-1.12) / 0.86
rs17696736 / 12 / 112,486,818 / T / 0.43 / 1.10 (1.08-1.12) / 3.61E-05 / 1.10 (1.09-1.12) / 6.06E-10 / 0.96 (0.93-0.99) / 0.14
rs17630235 / 12 / 112,591,686 / A / 0.41 / 1.09 (1.07-1.12) / 7.17E-05 / 1.09 (1.07-1.11) / 7.59E-09 / 0.97 (0.94-0.99) / 0.21
rs11066188 / 12 / 112,610,714 / A / 0.40 / 1.09 (1.07-1.12) / 8.76E-05 / 1.10 (1.08-1.11) / 7.61E-09 / 0.97 (0.94-0.99) / 0.22
rs11066301 / 12 / 112,871,372 / T / 0.42 / 1.10 (1.08-1.12) / 2.80E-05 / 1.10 (1.08-1.11) / 4.62E-09 / 0.96 (0.93-0.98) / 0.11
rs11066320 / 12 / 112,906,415 / A / 0.42 / 1.10 (1.07-1.12) / 4.72E-05 / 1.08 (1.06-1.10) / 1.28E-08 / 0.96 (0.93-0.99) / 0.14

Supplementary table e-3: Conditional analysis on rs10744777. SNPs are ordered by base-pair position. IC = Immunochip, WT = WTCCC2, MS = METASTROKE. Lack of individual level genotyping precluded METASTROKE conditional analysis. Chr = chromosome, BP = base pair position, RA = risk allele, RAF = risk allele frequency.

Supplementary Figure e-1A

Sup Figure e-1A: QQ plot for meta-analysis of all genotyped and imputed Immunochip SNPs filtered for heterogeneity and missingness. λ = 1.165 λ1000 = 1.036.

Supplementary figure e-1B

Sup Figure e-1B: QQ plot for meta-analysis of all the ‘stroke’ subset of Immunochip SNPs (n=3402 SNPs) filtered for heterogeneity and missingness. λ = 1.252 λ1000 = 1.055.

Supplementary Figure e-1C

Sup Figure e-1C: QQ plot for meta-analysis of all the ‘reading and writing’ subset of Immunochip SNPs (n=2726 SNPs) filtered for heterogeneity and missingness. λ = 1.300 λ1000 = 1.066.

Supplementary figure e-2: SNAP plot of 12q24.12 showing A) association with all ischaemic stroke data from the Immunochip, WTCCC2 and METASTROKE cohorts, and B) the same region after conditional analysis based on rs10744777.