Bio-Repository of DNA in stroke (BRAINS): A study protocol

Evidence shows that stroke incidence rates in developing South Asian countries have increased by more than 100% during the last four decades, while they have decreased by 42% in developed European countries over the same time period [1]. Over a four-decade period, stroke incidence rates increased from 52 per 100,000 person-years (1970-1979) to 117 per 100,000 person-years (2000-2008) and at this rate stroke is expected to become one of the major causes of death in South Asia [1]. While much of this increase may be attributed to changing lifestyles, a high percentage of all strokes occur without any obvious aetiological cause suggesting additional factors that contribute to the stroke risk variability between ethnic groups. The ethnic differences may arise due to varying lifestyles, differences in medical treatment or the inherent genetic makeup.

Dissecting the genetic causes of a complex disorder such as stroke and estimating their relative attributable risks, necessitates a large study cohort in-order to provide sufficient power to the analysis. Herein, we describe a study protocol of two ongoing prospective, multicenter recruitment studies, BRAINS-UK and BRAINS-South Asia which aims to recruit 1500 highly characterised European Caucasian stroke patients from the United Kingdom and 1500 South Asians stroke patients from UK, India and Sri Lanka respectively from participating centres across the three countries. Both case control cohorts will contribute to a combined hypothesis-free genome wide association study (GWAS) approach in identifying novel gene markers of stroke in European Caucasians and South Asians from UK, India and Sri Lanka. BRAINS will be invaluable for future candidate-gene, GWAS and whole genome sequencing studies to identify the genetic associations of all forms of cerebrovascular disease including transient ischemic attacks (TIA), ischemic and hemorrhagic strokes. We aim to gain a much better understanding of the genetic differences between South Asians and the European Caucasian population. BRAINS uses similar definitions of stroke and stroke classification as the successfully completed candidate gene based ISGS and GWAS based SWISS studies [2, 3].

South Asians from the Indian sub-continent form the second largest ethnic group in the world and make up some 20% of the world's population whereas Caucasian Europeans constitute about 13%. Both ancestral populations have a genetically stratified structure [4, 5] which has led to the wide belief that genetic underpinnings of disease in ethnic populations differ. This is supported by population-based and case-control studies that have demonstrated significant differences in incidence and prevalence in stroke between ethnic groups that are only partly accounted for by differences in environment [6–10]. A number of modifiable risk factors have also been shown to account for the differences between the two populations including age, sex, hypertension, diabetes and prevalence of prior disease [11]. It is recognized that as South Asians migrate out of their native environments and expose themselves to new environmental factors such as diet, their chances of developing vascular disease increase greater than would be expected in the host population [11]. These, along with other epidemiological and molecular observations[12] suggest that genetic effects on stroke may depend upon ethnic background. As the burden of stroke is expected to greatly increase in India over the next decade it is important to characterize these genetic factors, and to compare them with those in European populations. Deciphering the molecular pathways underlying stroke in different populations offers the promise of therapies tailored to genetic (or racial) background, similar to the recognition of racial dependency of anti-hypertension treatment [13, 14].

The diagnosis of stroke can be complex with a large number of clinical mimics and no known reliable blood biomarkers or definitive genes to predict the risk of developing stroke or incidence of disease. While the majority of symptomatic strokes can be attributed to modifiable risk factors such as hypertension, age and prior disease [15], these factors do not explain why some individuals are more susceptible to environmental determinants compared to others with the same given risk factors. A 4-fold increase in the risk of stroke in progeny of stroke patients [16, 17], clustering of ischemic stroke in families [18] and occurrence of over 10% of haemorrhagic strokes in patients with a prior family history of the same condition [19] lend support to genetic factors being involved in causation of common, 'acquired' strokes, in addition to rarer familial stroke syndromes such as sickle cell disease and CADASIL which have been seen disproportionately in certain racial groups.

Candidate-gene case-control studies have identified multiple genetic polymorphisms associated with stroke. The most consistent associations found are: Methylenetetrahydrofolate Reductase (MTHFR), Factor V Leiden (FVL), Angiotensin-converting enzyme (ACE) and Prothrombin (PT)[20]. Some genes found to be risk factors for one subtype of stroke may be protective towards the other, such as Factor V Leiden a known prothrombotic risk factor for ischemic stroke but likely protective against haemorrhagic stroke [21]. Other studies have implicated genes MTHFR, ACE (I/D) and Apolipoprotein E (ApoE) e4 as risk factors for haemorrhagic stroke in European Caucasians whereas Factor V Leiden is shown to be protective [22]. In agreement with the European Caucasian population, these genes have also been implicated as risk factors for ischemic stroke in people of Asian ethnicity (Chinese, Korean and Japanese) [23]. Our group has also identified MTHFR, Factor V Leiden and Prothrombin as genetic risk factors of cerebral venous thrombosis (CVT) in Caucasians adults and children [24] as well as South Asians from India, Pakistan and Bangladesh. Collectively, the above studies help us understand the possible molecular mechanisms underlying stroke and provide us with our best hope for developing population specific drugs to combat this devastating disease.

Stroke is a major public health burden in the UK costing the National Health Service (NHS) about £7 billion per year in direct care costs, loss of productivity, disability and informal costs [40]. The disease ranks as the second largest cause of mortality and disability in the UK after heart disease and cancer [41, 42]. Stroke affects about 130,000 people in England and Wales alone with a higher incidence risk in people older than 75 years of age [43]. The majority (69%) of all strokes are ischemic whereas 19% are haemorrhagic in nature. Of the latter, 13% are classified as intracerebral haemorrhage and 6% subarachnoid haemorrhage. However, 12% of all strokes have an uncertain diagnosis [44]. Worldwide, stroke ranks as the third largest cause of death after ischemic heart disease and cancer and accounts for 3% of the world's disability burden [45]. The USA alone, reports 795,000 new and recurrent strokes per year [46]. India, China and Russia collectively account for the highest number of stroke cases in the world, with 80% of deaths occurring in 2005 in India from stroke alone [47]. It is estimated that by 2050, 80% of the global burden of new strokes of 15 million will occur in the low and middle income countries such as India[48, 49].

The BRAINS study is a dedicated genetic risk association study with well classified inclusion and exclusion criteria which allow recruitment of large numbers of stroke patients and controls. The detailed repository of information and DNA bank allows a unique opportunity to apply both candidate gene and whole-genome approaches to cerebrovascular disease. The latter aim will be realised by increasing availability of SNP databases and gene chips. The use of spouses as controls allows candidate genes hypotheses to be tested quickly using the method of allelic-association, while controlling for environmental variability. BRAINS allows both established and novel candidate genes (acting either independently or with other risk genes) to be detected, which in time, may facilitate identification of novel potential therapeutic targets. This is particularly important for a disease like stroke where there are few treatment options.

Sustainability and the development of a long-term collaboration is a major objective to this initiative and once the biobank is fully established it can be used as a permanent international genetic resource to spawn new ideas and answer important questions in stroke genetics, particularly those relating to genetic differences of effects as a result of differing ancestral origins. BRAINS is uniquely placed as the largest repository for South Asian stroke data consisting of samples from Northern, Central and Southern South Asia. The results from our study will contribute to ongoing GWAS studies in stroke in understanding the genetic and molecular underpinnings of stroke.

SY is a research postgraduate at Imperial College London and has co-written the final protocol. RS, and MSK are BRAINS-UK project managers in London. JS is a stroke specialist nurse and research postgraduate at Imperial College London who contributed to developing BRAINS. AM is the BRAINS-SA project manager in New Delhi. Professors KP and PShr are the New Delhi site investigators who contributed to developing the final local protocol. Dr RdS is the Sri Lankan site investigator. Dr. PB is a Neurologist who contributed to the TOAST classification of probands. Professor PF provides genomics expertise and Professor JK cardiovascular expertise. Dr. PSha is the principal investigator of BRAINS (UK and SA) and has developed the protocol from its inception to the final version.