ABC | Volume 111, Nº1, July 2018

Original Article Pereira et al. Genes and coronary artery disease risk Arq Bras Cardiol. 2018; 111(1):50-61 criteria, 3 or with CAD confirmed by coronary angiography with ≥ 1 coronary lesions of ≥ 70% stenosis in ≥ 1 major coronary artery or its primary branches. Absent or non-flow limiting atheroma was excluded from the analysis. The control group consisted of healthy volunteers, without symptoms or history of CAD, selected from the same population. All controls underwent clinical assessment of conventional cardiovascular risk factors, an electrocardiogram (ECG), and, in doubtful cases, an exercise stress test, a stress echocardiography or computerized tomography for calcium scoring. Cases and controls were matched for gender and age. Inclusion criteria comprised an age limit of 65 years and being a permanent resident to avoid genetic admixture. Principal Component Analysis (PCA) 4 was used for analysis of population stratification for possible genetic admixture and detection of significant genetic outliers (< 5%). 4 The study was approved by the Hospital ethics committee according to the Declaration of Helsinki and all patients provided written informed consent. Data collection Data was collected from all subjects in a standardized file comprising demographic, clinical characteristics and TRFs traditional risk factors (gender, age, level of exercise, smoking status, arterial hypertension, dyslipidemia, diabetes, family history of CAD, body mass index (BMI), heart rate and pulse wave velocity (PWV). “Smokers” referred to current smokers or subjects with less than 5 years of smoking cessation. 5 Essential hypertension was considered when patients, at the entry into this study, were already diagnosed and/or had been on antihypertensive medication for more than 3 months or newly diagnosed hypertensives with systolic blood pressure (SBP)/diastolic blood pressure (DBP) ≥ 140/90 mmHg measured on at least 3 occasions. 6 Dyslipidemia was defined for control population as low‑density lipoprotein (LDL) > 140 mg/dL, high-density lipoprotein (HDL) < 45 mg/dL for women and < 40 mg/dL for men, Triglycerides > 150 mg/dL and apolipoprotein (Apo) B > 100 mg/dL. For patients (at high risk) dyslipidemia was considered when LDL > 100, HDL < 45 mg/dL for women and < 40 mg/dL for men, triglycerides > 150 mg/dL, Apo B > 100 mg/dL and non-HDL (total cholesterol‑HDL) > 130 mg/dL. 7 Subjects were classified as having diabetes if they were taking oral anti-diabetic medication or insulin or if their fasting plasma glucose was higher than 7.0 mmol/L or 126 mg/dL. 8 Subjects were considered to have a family history of premature cardiovascular disease (CVD) if the father or brother had been diagnosed with CVD under the age of 55 or mother or sister under the age of 65. The definition of other TRFs was based on the standard criteria, as previously reported. 9,10 Biochemical analysis Blood samples were extracted after 12 hours’ fasting. Biochemical analyses were performed at the Central Laboratory of the Hospital, according to standard techniques. In order to determine total cholesterol, HDL, LDL, triglycerides and glucose, blood samples were placed in dry tubes, centrifuged half an hour later at 3,500 g and subsequently quantified by an enzymatic technique using an “AU 5400” (Beckman Coulter) autoanalyzer. Biochemical markers such as lipoprotein-a – Lp(a), (Apo B), and high-sensitivity C-reactive protein (hs-CRP) were quantified by immunoturbidimetry also using an “AU 5400” (Beckman Coulter) automatic system. Single Nucleotide Polymorphisms (SNP) selection Two parallel approaches were employed to identify SNPs for the GRS. In the first approach, we searched the National Human Genome Research Institute database, which included SNPs identified by means of GWAS and catalogued based on phenotype and/or trait. We searched for the keywords: “coronary artery disease”, “coronary disease”, “myocardial infarction” and “early myocardial infarction.” The second approach included SNPs that were identified through candidate gene approaches, included in a published GRS for CAD. Including criteria included genes described in previous studies with an Odds Ratio (OR) for CAD ≥ 1.1 and a minor allele frequency (MAF) > 5%. Genes with low Hardy-Weinberg equilibrium (p < 0.002) (after Bonferroni correction) were excluded. In total, 33 SNPs were selected according to their possible CAD-related function: association with cell cycle, cellular migration and inflammation (rs1333049 (9p21.3), rs4977574 (CDKN2B), rs618675 (GJA4), rs17228212 (SMAD3), rs17465637 (MIA3), rs12190287 (TCF21), rs3825807 (ADAMTS7), rs11556924 (ZC3HC1), rs12526453 (PHACTR1)); genes involved in pro-oxidative status (rs1801133 (MTHFR 677), rs1801131 (MTHFR 1298), rs705379 (PON 1), rs662 (PON 192), rs854560 (PON 55), rs6922269 (MTHFD1L); genes associated with modifiable risk factors such as lipids metabolism, hypertension and diabetes/obesity (rs3798220 (LPA), rs2114580 (PCSK9), rs20455 (KIF6), rs7412/rs429358 (APOE), rs964184 (ZNF259), rs599839 (PSRC1), rs5186 (AT1R), rs699 (AGT), rs4340 (ACE), rs4402960 (IGF2BP2), rs1326634 (SLC30A8), rs266729 (ADIPOQ), rs7903146 (TCF7L2), rs17782313 (MC4R), rs1801282 (PPARG), rs1884613 (HNF4A), rs8050136 (FTO) and rs1376251 (TAS2R 50)) (Supplementary Table 1). Genetic analyses Genetic analyses were performed at the Human Genetics Lab of the University of Madeira. Genomic DNA was extracted from 80 µl of peripheral blood using a standard phenol-chloroform method. A TaqMan allelic discrimination assay for genotyping was performed using labelled probes and primers pre-established by the supplier (TaqMan SNP Genotyping Assays, Applied Biosystems). All reactions were done on an Applied Biosystems 7300 Real Time PCR System and genotypes were determined using the 7300 System SDS Software (Applied Biosystems, Foster City, USA) without any prior knowledge of individual’s clinical data. Quality of genotyping techniques was controlled by the 51