ABC | Volume 110, Nº1, January 2018

Original Article Fischer et al Gene polymorphisms and coronary disease extension Arq Bras Cardiol. 2018; 110(1):16-23 Table 1 – Conditions for amplification and digestion of the studied polymorphisms Polymorphism Starters Annealing temperature Cycles Restriction enzyme PON-1 Forward : 5’-TATTGTTGCTGTGGGACCTGAG-3’ 61°C 35 Alw I Q192R Reverse : 5’-CACGCTAAACCCAAATACATCTC-3’ MTHFR Forward : 5’-GAAGCAGGGAGCTTTGAGG-3’ 63°C 32 Hinf I C677T Reverse : 5’-ACGATGGGGCAAGTGATG-3’ ENOS Forward : 5’-CTGGAGATGAAGGCAGGAGAC-3’ 56°C 35 Ban II G894T Reverse : 5’-CTCCATCCCACCCAGTCAATC-3’ ACE Forward : 5’-CTGGAGACCACTCCCATCCTTTCT-3’ 55°C* 32 - I/D Reverse : 5’-GTCTCGATCTCCTGACCTCGTG-3’ AT1R Forward : 5’-AATGCTTGTAGCCAAAGTCACCT-3’ 59°C 35 Dde I A1166C Reverse : 5’-GGCTTTGCTTTGTCTTGTTG-3’ LPL Forward : 5’-AAAATCAAGCAACCCTCAAG-3’ 57°C* 35 Taq I D9N Reverse : 5’-TAGGGCAAATTTACTTGCGA-3’ APOC3 Forward : 5’GGTGACCGATGGCTTCAGTT-3’ 58°C 30 Sst I SST I Reverse : 5’-CAGAAGTGGATAGAGCGCT-3’ * the touchdown PCR protocol was used for the ACE and LPL genes FMD and EIR determination, and expressed as percentage. Intra- and inter-observer variability were < 1% and 2%, respectively. Gensini score Gensini score was used to classify CAD severity determined by cineangiography. Gensini score is calculated taking into account the magnitude of the lesions and the myocardium at risk, assigning different ratings to different levels of obstruction in the segments affected. A 25%, 50%, 75%, 90%, 99% and 100% obstruction were given, respectively, the scores 1, 2, 4, 8, 16 and 32. The method also assigned a different score depending on the lesion location - left coronary trunk (5 points), proximal anterior descending artery (2.5 points), middle third of the anterior descending artery (1.5 point), distal anterior descending artery (1 point), second diagonal artery (0.5 point), proximal and distal right coronary artery and posterior descending branch (1 point). Gensini score results from the sum of individual scores given to each lesion, multiplying the stenosis severity by the lesion location; results were expressed as arbitrary units (AU). 11,31 Genetic study Polymorphisms of the genes paraoxonase-1 ( PON-1 ), methylenotetrahydrofolate reductase ( MTHFR ), endothelial nitric oxide synthase ( ENOS ), angiotensin-converting enzyme ( ACE ), angiotensin II type 1 receptor ( AT1R ), apolipoprotein C3 ( APOC3 ), lipoprotein lipase ( LPL ) were analyzed from total blood samples, collected with EDTA (ethylenediaminetetraacetic acid), using the polymerase chain reaction (PCR) technique, followed by the identification of restriction fragment length polymorphisms (RFLP), under conditions described in Table 1. Genetic score was calculated assuming the dominant model of the polymorphisms, which were considered as binary, dichotomous variables. Statistical analysis Sample size was estimated for comparisons between proportions, considering the polymorphism dominant model, an α error of 5%, a β power of 80%, a 95% confidence interval, and an expected proportion of 50%. The estimated sample size was of 90 participants. All analyses were performed using the SPSS 22.0 for Mac. Categorical variables were expressed as n (%) and compared between the genotypes using the Pearson’s chi-square test or the Fisher’s exact test, as appropriate. The chi-square test was used to analyze whether the distribution of observed and expected genotypic frequencies were in the Hardy–Weinberg equilibrium. Numerical variables were expressed as mean ± standard deviation or as median and interquartile range. The Kolmogorov – Smirnov test was used to evaluate whether the variables were normally distributed. For descriptive statistics, in case the variables were not normally distributed, the Student’s t test and the Mann Whitney test were used for unrelated samples. Pearson correlation was used to evaluate the correlations between the genetic score and the Gensini score. P‑values lower than 0.05 were considered statistically significant. Results A total of 116 patients were evaluated. Baseline characteristics of participants are described in Table 2. Table 3 presents the distribution of allele and genotypic frequencies of the studied polymorphisms. The PON-1, MTHFR and ENOS genes were not in the Hardy-Weinberg equilibrium in the study population. Distribution of clinical, demographic and laboratory characteristics Results were analyzed by genetic polymorphism. The results arepresented indetails in electronic version in the Supplementary 18