Ethical considerations
Study Protocol has been approved from Institutional Ethics Committee (Ref No: IEC/NP-122/2012 & RP-14/2012).
Design of study
Case control study design.
Patients and methods
Patients will be eligible if they meet all the inclusion criteria and none of the exclusion criteria.
Selection of cases and controls
Inclusion and exclusion criteria for cases
Inclusion criteria for cases
a) Diagnosis of stroke as defined by World Health Organization, b). NCCT-Head consistent with ischemic stroke, c). Stroke onset within three years before the recruitment, d). Age 18–85 years (both sexes), e). Willingness to provide written informed consent by self or legal representative, f). Should be resident of North India (residing for last one year or longer), g). Be ’North Indian’. A North Indian defined in consultation with Department of Paediatric (Genetics) AIIMS and Genetics Department of the Institute of Genomics and Integrative Biology (IGIB) as having all of the following:
(i) The subject’s birth place should be in North India, (ii) Their ancestor (2 generation) should be North Indian, (iii) Subjects must know any North Indian language which include any language spoken in North India.
For this study, North India included the states of Himachal Pradesh, Bihar, Delhi, Punjab, Uttar Pradesh, Madhya Pradesh, Uttrakhand, Jharkhand, Rajasthan, Jammu and Kashmir and Haryana.
Exclusion criteria for cases
a). Stroke associated with pregnancy, b). Stroke associated with surgery, c). Unwillingness to provide written informed consent (by self or legal representative).
Inclusion and exclusion criteria for controls
Inclusion criteria for controls
a). Age (−5 to + 5) and sex matched, b). Controls have not had prior stroke by questionnaire for Stroke-free Status (QVSS) [23], c). Spouse or friends but not a relative (by blood), d). Age 18–85 years (both sexes), e). Should be Resident of North India (residing for last one year or longer), f). Willingness to provide written informed consent by self or legal representative, g). No evidence of any serious brain disorders, h). Be ’North Indian’. (North Indian criteria is same as above).
Exclusion criteria for controls
a). Unwillingness to provide written informed consent (by self or legal representative), b). Pregnancy, c). Subjects with any serious brain disorder.
Definition of variables
Definitions of variables were modified from the study [24] and are as follows: Hypertension: Subjects will be considered to have hypertension if they either have the diagnosis of hypertension or treated for hypertension before the stroke or reference date, In addition, if a control will have no recorded blood pressure before the reference date but diastolic pressure of 95 mm Hg or more or a systolic pressure of 160 mm Hg or more on two or more occasions during the study evaluation, he or she will be considered to have hypertension. Diabetes: if a subject will have the diagnosis documented by a physician on the medical record or if fasting blood sugar level will be >126 mg/dl, Dyslipidemia: if they either will have the diagnosis of dyslipidemia or treated for dyslipidemia. Angina pectoris: chest discomfort or pain that described as heavy, tight, constricting, crushing, pressing, or squeezing. Smoker: Person will be defined as regular smoker if a person smoking ≥1 cigarettes daily, Biris, Cigar for proceeding>3 months. Body Mass Index (BMI): BMI will be calculated by weight in kilograms divided by the square of height in meters. Family history of Stroke: A positive family history of stroke will be considered if a subject’s first-degree relative (parent or sibling) had a stroke. Myocardial Infarction: The diagnosis will be based on clinical history of acute myocardial infarction; Migraine: subject will be considered to have a history of migraine if patients have a prescription for specific antimigraine therapy or diagnosis of migraine and a prescription for a potential antimigraine medication or analgesics in the absence of any other explanatory diagnosis within 1 years before the index date; Transient Ischemic Attack: TIA will be defined as subjects with focal neurologic symptoms relating to focal cerebral, brain stem, or retinal ischemia with abrupt onset and complete resolution within 24 hours. Economic status: the economic status of the subjects will be assessed based on the ownership of different commodities in house hold, mainly two wheeler, refrigerator, computer or car. The economic status will be classified into two classes: Low – not possessing any of the four, High: possessing either two- wheeler or refrigerator or computer or car. Physical activity will be determined on the basis of job profile of the subjects in which Sedentary (mostly sitting e.g. shopkeeper, clerk; Moderate physical activity (involves walking e.g. salesman, nurses, house work etc.); Heavy physical work (carrying, lifting e.g. labourer, coolie).
Matching criteria for control to case
Matching is required for case control study for the elimination of bias in comparison between cases and controls. It assures that no large imbalance between cases and controls occurs. Controls will be matched with gender and age (± 5 years of cases) in 1:1 ratio. As Spouses would have similar environmental exposure as cases spouse can be used as control for case control stroke genetics study [25]. We will use spouses of cases as a match for other cases for age and sex matching. If there would be any lag and unavailability of spouse we will recruit age and sex match control from relatives/patients attending neurology department for treatment other than stroke and fulfilling the inclusion criteria for recruitment of controls.
Stroke classification
We will use TOAST classification for the determination of stroke subtypes [10]. In TOAST classification stroke has five subtypes (i) Large vessel stroke (ii) Small vessel stroke (iii) Cardioembolic stroke (iv) Other determined aetiology (v) Undetermined aetiology.
Sample size
Sample size calculation for all gene polymorphism was based on the parameters of our meta-analysis of association of MTHFR polymorphism with stroke. In this meta-analysis result prevalence for TT variant genotype were 0.17 and 0.13 in cases and control respectively, Odds ratio for this polymorphism was 1.31 Assuming 80% power and 5% alpha, with one control per case, we obtained estimated minimum sample size 578 cases and 578 controls. 600 cases and 600 controls will be included in this study to compensate for any loss of sample.
Blood sample collection, processing, storage and genotyping
Four ml of blood sample will be collected in EDTA coated vial from all consenting participants in single-time venipuncture from antecubital vein. Samples will be used for Genomic DNA isolation from white blood cells by using phenol chloroform isolation method and extracted genomic DNA will be dissolved in 200-600 ml TE buffer depending upon the concentration of DNA and will be stored at -20°C. DNA will be isolated in weekly basis for isolation of good quality of DNA. Its quality will be checked first in 0.8% agarose gels. Quality of DNA in per μl will be checked in Nanodrop spectrophotometer. The purity of the DNA sample will be ascertained by calculating a 260/280 ratio. The ratio between 1.5-1.8 will be acceptable for PCR amplification. Genotyping will be done by the PCR – RFLP method. The PCR-RFLP results will be confirmed by direct sequencing of three samples of each genotype of all the chosen polymorphisms.
Data collection and data analysis
Data will be recorded in standardized data collection forms. The data will be managed and analyzed using statistical software SPSS version 17. T-test will be used for continuous variables. Chi Square tests and logistic regression techniques will be used when outcome variable are categorical (Present/Absent). Association between each risk factor of interest and stroke will be performed using a conditional logistic regression approach. Odds ratio (ORs) and corresponding confidence intervals will be calculated for each polymorphism. A multivariable conditional logistic regression analysis will be performed for adjustment of other covariates. The other associated risk factors will be treated as covariates in examining the associations with stroke. Significance in the final model will be defined as P<0.05. Phenotype-genotype and genotype-environment interaction will be analyzed using routine statistical methods. Haplotypes will be constructed from selected SNPs and its association with stroke will be estimated using regression techniques
Outcome measure
The primary outcome of the present study is to determine whether singly or in combination of any of selected polymorphisms are associated with stroke or its subtypes. Cases will be compared with controls to frequency and distribution of susceptible allele. Other outcome measure includes association of gene polymorphisms with different environmental exposures such as hypertension, smoking, diabetes, dyslipidemia etc. Data from association of genetic polymorphism with outcome of stroke is lacking. Determining the relationship of genetic variations with the stroke outcome will improve our understanding that how variations in the genes influence the stroke outcome. In present study we will assess the outcome of recruited patients at six months by telephone to assess the status of patients. One research worker will assess the Barthel Index and modified Ranking scale after the six months of stroke. Chronic stroke patient who will come after the six months of onset of stroke their six months Barthel index and modified Ranking scale will be assessed retrospectively.