The study was conducted within the framework of the Rotterdam Study, an ongoing prospective, population-based cohort study on determinants of a number of chronic diseases. The Rotterdam Study has been described in detail elsewhere . In brief, all inhabitants of Ommoord, a district of Rotterdam in the Netherlands, who were 55 years or over, were invited to participate in this study. Of all 10275 eligible individuals, 7983 agreed to participate (78%). Written informed consent was obtained from all participants and the Medical Ethics Committee of the Erasmus Medical Center approved the study.
The baseline examinations took place from 1990–1993. Participants were visited at home for an interview. Information on current health status, medical history, use of medication, and smoking status were obtained during the interview. The interview was followed by two visits at the research center for blood sampling and further examinations.
At baseline, participants were asked whether they have ever experienced a heart attack. A 12-lead electrocardiogram (ECG) was stored digitally and analyzed by using the Modular ECG Analysis System (MEANS). Myocardial infarction found on ECG was based on criteria partly derived from the Minnesota code. A history of myocardial infarction was considered present in case of a self-report of myocardial infarction confirmed by ECG or additional clinical information, or the presence of an ECG characteristic of prior myocardial infarction [12, 13].
Genomic DNA was extracted from leucocytes following standard procedures. Participants were genotyped for rs10757274 and rs10757278. Genotypes were determined in our study population in 2-ng genomic DNA by use of pre-designed TaqMan SNP genotyping assay (Assay ID C__26505812_10 and C__11841860_10, respectively; Applied Biosystems, Foster City, CA). Reactions were performed with the Taqman Prism 7900 HT 384 wells format.
Follow up for clinical events started at baseline and follow up examinations were carried out periodically in 1995–1996, 1997–1999, and 2002–2004. Participants were continuously monitored for fatal and nonfatal cardiovascular events through automated linkage with files from general practitioners and pharmacies working in the study district of Ommoord. In addition, all medical records of the participants under the care of general practitioners outside the study area were checked annually. Two research physicians independently coded all reported events according to the International Classification of Diseases, 10th edition (ICD-10). Codes on which the research physicians disagreed were discussed to reach consensus. Finally, a medical expert in cardiovascular disease, whose judgment was considered final, reviewed all events. Information on vital status was obtained regularly from municipal health authorities in Rotterdam. For the present study, follow up data were available until October 1, 2005.
Incident coronary heart disease and myocardial infarction
To identifying incident myocardial infarction and coronary heart disease, we collected information from baseline (1990 – 1993) until January 1, 2005. Fatal or non-fatal MI reported by general practitioners in the research area, letters from medical specialists and discharge reports for hospitalized patients were the sources of information used. Two research physicians coded the events independently and in case of disagreement the consensus was made ina separate session. Finally a specialist whose judgment was considered final verified the coding. We defined incident MI as fatal or non fatal MI (ICD-10 code I21). Incident CHD was defined as fatal or nonfatal myocardial infarction (ICD-10 code I21), coronary artery bypass grafting (CABG), and percutaneous transluminal coronary angioplasty (PTCA).
Population for analysis
The SNPs, rs10757274 and rs10757278 were genotyped in 6251 and 6265 out of 7129 participants who visited the research center at baseline.
Genotype frequencies were tested for Hardy-Weinberg equilibrium with a chi-square test using The Hardy-Weinberg package for R http://www.r-project.org. To compare the baseline characteristics between healthy subjects and those who experienced CHD or MI, we used chi-square for categorical variables and ANOVA for continuous variables.
A Cox regression analysis was used to assess the association of SNPs with incident CHD and MI. The proportional hazards assumption was validated by the use of a time-dependent variable to check increasing or decreasing trends in the hazard ratio (HR) over time. The basic model was adjusted for age and sex. The multivariate adjusted model was additionally adjusted for BMI, systolic and diastolic blood pressure, total cholesterol, HDL cholesterol, smoking, and diabetes.
To examine whether the effect of SNPs vary by the level of other risk factors we performed the analysis stratified by age, sex, family history of cardiovascular disease, HDL cholesterol, diabetes, hypertension, smoking, and history of CHD. For smoking, participants were categorized to never, former, and current smokers. For hypertension and diabetes, participants were categorized into those with and without the condition. History of CHD was defined as a history of MI, percutaneous transluminal coronary angioplasty (PTCA) or coronary artery bypass grafting (CABG). For other risk factors, the population was divided into two equal subgroups by use of the median. All statistical analyses were performed with the use of SAS, version 8.