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Date: 01/06/2004

Ref: 04/MRE00/36

Division of Clinical Neuroscience

St George's Hospital Medical School

_____________________________________________________________________ ADDRESS: Clinical Neuroscience St George's Hospital Medical School Cranmer Terrace London SW17 0RE Department of Neurology: Telephone: Facsimile: Email: 020 8725 2735 020 8725 2950 [email protected]

UNIVERSITY OF LONDON

Hugh Markus, Professor of Neurology

STUDY PROTOCOL

Version 1 May 24 2004

A DNA RESOURCE FOR LACUNAR (SMALL VESSEL DISEASE) STROKE PROJECT AIM: To establish a DNA resource of carefully phenotyped patients of lacunar stroke, aged <= 70 years. BACKGROUND: Much evidence suggests genetic factors are important in stroke. Several single gene disorders are associated with stroke including diseases such as CADASIL. However, the vast majority of cases of stroke have a multi-factorial pathogenesis and genetic contributions to this are likely to be polygenic. Nevertheless genetic factors also appear to be important in this population, as supported by a number of lines of evidence. Firstly, twin studies show greater concordance rates in monozygotic compared with dizygotic twins. Secondly, most family history studies have found that a family history of stroke is a risk factor for stroke in case control studies. Thirdly, animal studies suggest that specific genes influence the risk of cerebral ischaemia. Fourthly, recently a linkage based approach on extended pedigrees in Iceland has reported an independent stroke risk gene (STKR1), and further studies have identified the underlying gene as phosphodiesterase 4D. Therefore a large body of evidence suggests genetic factors are important in stroke risk but the genes responsible remain to be determined. Small Vessel Disease (SVD) Stroke (Lacunar Stroke) Lacunar stroke accounts for a quarter of all ischaemic strokes. It results from ischaemia in the territory of the perforating arteries supplying the white matter, and deep grey matter nuclei, which results in small regions of infarctions (lacunes) which themselves result in typical clinical syndromes without `cortical' signs. Lacunar infarction can occur in isolation or can be accompanied by patchy or confluent changes in the periventricular white matter on CT or MRI. This appearance is referred to as leukoaraiosis. The pathogenesis of lacunar stroke is poorly understood. A small number of cases result from the autosomal dominant form of SVD, CADASIL, but screening studies suggest this is an uncommon cause in the general population. Recent evidence suggests that there may be a strong genetic component to SVD,

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Date: 01/06/2004

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unrelated to CADASIL. Firstly, family studies have shown that family histories are a particularly strong risk factor for lacunar stroke. For example a family history of stroke in a first degree relative at aged <=65 years was a risk factor for lacunar stroke at <=65 years with an odds ratio of 2.7. Secondly, twin studies looking at high signal white matter lesions on MRI (which appear to represent asymptomatic SVD) have shown a strong heritability of 71%. Previous Studies in Ischaemic Stroke and Lacunar Stroke Despite the importance of stroke there have been relatively few genetic studies of polygenic stroke in man compared with other complex diseases with similar heritability such as ischaemic heart disease. Those that have been performed have largely failed to produce replicable results. This is likely to be due to methodological problems including inadequate sample sizes, inadequate phenotyping of cases, poor matching of cases and controls, a failure to focus on younger stroke cases in whom the genetic component appears to be strongest, and a failure to replicate associations in a second population. Importantly, it has not always been appreciated that stroke is a hetrogenous syndrome caused by multiple pathologies which may have different risk factor profiles. For example CADASIL only predisposes to lacunar stroke and not other types of stroke. Therefore it may be appropriate to look for responsible genes in particular subtypes of stroke, such as lacunar stroke. We will only study Caucasian cases. This is because our modelling studies have demonstrated that we would not be able to recruit a sufficient sample size of other ethnic groups with young lacunar stroke. It is important not to mix ethnic groups as this can lead to bias due to population stratification. Therefore this project aims to collect a large DNA resource of young patients with well phenotyped lacunar stroke and appropriate drug-free controls.

METHODS

Overall Study Design We will collect the following: 1) 1100 well phenotyped cases of lacunar stroke aged <=70 years from specialist stroke centres throughout the UK. 2) 2000 stroke-free community controls from four sites in the UK. 3) DNA from cases will be screened for notch 3 mutations. A second sample will be sent to ECCAC for storage for future cell line production to allow unlimited supplies of DNA. 4) The resource will be made avaliable to researchers throughout the UK and will be managed by a resource management committee chaired by Professor Peter Fentham.

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Data Collection 1) Cases a) Centre Characteristics. All will have well developed stroke services, a stroke physician / neurologist / geriatrician with an interest in stroke and good access to investigational facilities, including MRI, as well as a track record for infrastructure necessary for clinical research projects. b) Subject Identification. In each participating centre patients seen in both the hospital inpatient acute stroke service and stroke outpatients will be reviewed. Patients aged <=70 years with SVD will be identified and the following inclusion and exclusion criteria will be applied: Inclusion Criteria 1) Age <=70 years 2) Clinical lacunar syndrome (hemiparesis, hemisensory deficit, sensorimotor deficit, ataxic hemiparesis, clumsy hand dysarthuria) or partial lacunar syndrome (e.g. pure motor stroke affecting face and arm or arm and leg). Patients with symptoms affecting only one body part (e.g. arm or leg only) will not be included. 3) Lacunar infarction on MRI scan (defined as subcortical infarct <=1.5cm in diameter). 4) Caucasian ethnicity. Exclusion Criteria 1) Stenosis >50% in the extracranial or intracranial cerebral vessels, or previous carotid endarterectomy. 2) Cardioembolic source of stroke, defined according to the TOAST criteria as high or moderate probability. 3) Cortical infarct on MRI. 4) Subcortical infarct >1.5cm diameter, as these can be caused by embolic mechanisms (striatocapsular infarcts). 5) Any other specific cause of stroke, e.g. lupus anticoagulant, cerebral vasculitis, dissection. 6) Ethnic groups other than Caucasian.

c) Data to be Collected on Index Cases Data on cases and controls will be collected on a standardised proforma to ensure consistency of data across centres and allow comparability of risk factor data collected in cases and controls. The following data will be collected on cases: 1) Cardiovascular Risk Factors: these will include age, gender, past history of hypertension and treatment with anti-hypertensive drugs, diabetes mellitus, smoking history, body mass index, socioeconomic status, alcohol history, ischaemic heart disease, peripheral vascular disease, atrial fibrillation, and CABG/coronary angioplasty. 2) Routine investigations will include FBC, ESR, U&E, glucose, cholesterol, electrocardiogram and imaging of the extracerebral carotid arteries (ultrasound, MRA or CT angiography) in all patients. All patients will

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receive brain imaging with MRI. This is much more sensitive than CT for identifying lacunar infarcts and also allows the degree of leukoaraiosis to be graded. 3) Full Family History. In all cases a full family history will be taken to include both parents, sibs and children. We will determine whether individuals are alive, have ever suffered stroke or other cardiovascular disease, the age of onset of such disease, and also the country of birth of both parents. 4) All MRIs and clinical histories will be reviewed centrally to allow confirmation of phenotype and also grade the degree of any associated leukoaraiosis. 2) Controls a) Control Identification Unrelated controls will be obtained by random sampling from general practice lists from four regions throughout the UK, covering regions from which the largest, or significant, numbers of cases have been recruited. Sampling will be stratified for age and gender to obtain similar distributions to that of the cases. Only Caucasians will be studied for the reasons above. b) Data to be Collected on the Controls: The same cardiovascular risk factors and full family history will be collected on controls as in cases. All controls will have body mass index and blood pressure measured. Blood will be taken for FBC, cholesterol, HDL, LDL, and glucose. 3) Blood Sampling For all cases and controls 20ml blood in EDTA and 10ml of both serum and plasma will be taken. 10ml blood for DNA will be sent directly to ECCAC for storage for subsequent cell line production. 10ml of blood in EDTA for DNA, and serum samples will be sent to Clinical Neuroscience at St. George's Hospital Medical School. Blood for plasma and serum will be immediately centrifuged, separated, and stored at -700c. At three monthly intervals a co-ordinator will collect data and blood samples from centres. When samples arrive in the co-ordinating centre (St. George's), DNA will be extracted and assayed. The referring centre will be immediately notified if the sample is not adequate. Cases and controls will be coded by a number to ensure anonymity on the central database.

Resource Management On a day to day basis the project will be managed by the study administrator based at St. George's and the supervision of Professor Hugh Markus. The overall resource will be managed by a Resource Management Committee whose role will be to: 1) Ensure and demonstrate confidentiality of the data and sample resource. 2) Assess applications for the use of the sample resource from both investigators and outside collaborators.

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3) Ensure and demonstrate quality control in collection of samples, extraction and storage. The Committee will be composed of three members from the investigators including the principal investigator, two genetic or neuroscience researchers external to the collaborators, and an independent chairman (Professor Peter Fentham, Chairman of the Stroke Association R&D Committee). A validation subgroup (two members of the co-applicants) will review all cases (both clinical details and MRI scans) to: a) Confirm that they meet the inclusion criteria. b) Review the imaging ­ all scans will be graded for the presence, and degree, leukoaraiosis. Use of the Resource for Future Genetic Studies. The database will be a unique resource and will be available, in collaboration, to other UK groups. Individual collaborations will be judged on their academic merit by the Resource Management Committee, and external peer review will be utilised where additional expertise is required. Patient confidentiality will be assured at all times because the cases will be coded. A small cost per sample will be made to cover costs including distribution of the sample, production of cell lines if required, and continued maintenance of the resource beyond the period of the grant.

Research Governance Random checks will be made to assess reliability of data by visiting centres and requesting a random sample of the relevant clinical records and MRI scans.

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STUDY PERSONNEL Principal Investigator: Professor Hugh Markus, St. George's Hospital Medical School Dr Steve Bevan, St. George's Hospital Medical School Professor Pak Sham, Institute of Psychiatry Dr Charles Wolfe, Guy's, King's and St. Thomas' Medical School, London

Laboratory Genetics: Statistics: Epidemiology:

Participating Clinical Centres: Members of UK Stroke Genetics Group: Professor Jean-Claude Baron, Dr Liz Warburton -Addenbrookes Hospital, Cambridge Dr Martin Dennis, Dr Cathie Sudlow - Western General Hospital, Edinburgh Professor Kennedy Lees - Western Infirmary, Glasgow Dr Keith Muir - Institute of Neurological Sciences, Glasgow Dr John Bamford - Leeds Hospitals Professor John Potter - Glenfield Hospital, Leicester Dr Anil Sharma - Aintree Hospital, Liverpool Dr Charles Wolfe, Dr Anthony Rudd - Guy's & St. Thomas' Hospitals, London Professor Lalit Kalra - King's College Hospital, London Professor Hugh Markus - St. George's Hospital, London Professor Martin Brown - UCL & Institute of Neurology, London Dr Pippa Tyrell - Hope Hospital, Manchester Professor Gary Ford - Freeman Hospital, Newcastle Professor Philip Bath - City Hospital, Nottingham Dr Peter Rothwell - Radcliffe Hospitals, Oxford

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