ABC | Volume 114, Nº2, February 2020

Original Article Rocha et al. Association between periodontitis, polymorphisms and CAD Arq Bras Cardiol. 2020; 114(2):268-272 Methods The sample consisted of 80 patients (mean age 60.5 ± 10.5) who underwent diagnostic cardiac catheterization in the hemodynamic laboratory of the University Hospital of Universidade Federal de Santa Maria (HUSM) from September 1, 2010 to March 30, 2011 and who agreed to participate in the study by signing the informed consent form. Were excluded for the sample Patients who were smokers, diabetics, those with autoimmune diseases and those with fewer than two teeth present in one of the sextants, as well as those who did not agree to participate in the study. The study was approved by the Ethics and Research Committee of the São Leopoldo Mandic Dental Research Center on 11/26/2014, CAAE: 35879614.7.0000.5374. Periodontal examination was performed by the researcher, using the Community Periodontal Index (CPI) and Periodontal Insertion Loss Index (PIP), as recommended by the World Health Organization. 21 The use of this index was justified by the partial immobilization of the patient in bed and the short time in which it was available to the examiner. Patients with a CPI score of 3 or 4 and a PIP score above 1 were considered as having periodontitis. DNA was extracted from a saliva sample obtained through a mouthwash with 3% glucose solution for 1 minute, and then this material was deposited in capped, sterile test tubes and frozen at -20°C. 22 The diagnosis of CAD was performed through cardiac catheterization, via femoral or radial access, performed by the physicians of the hemodynamic laboratory of the Santa Maria University Hospital. Patients with a positive medical report for the presence of coronary stenosis were considered as having CAD. Genotyping DNA was extracted from saliva samples had using a genomic DNA extraction kit (Norgen Biotek Corp, Canada), and gene amplification was performed by the polymerase chain reaction using the following primer pairs for the +1444 PCR: forward 5 ‘- AGCTCGTTAACTATGCTGGGGCA-3’ and reverse 5 - CTTCTCAGCTCTTGCCTTATGAGT-3 ‘, with an annealing temperature of 60 °C and for IL-6 -174: forward 5’-AACCTAATTCTACCCCCCTTGG-3 and reverse 5’-TCAGAGGCAGCCGAAGAGTT-3’, with an annealing temperature of 59°C.17 The amplification was carried out using one cycle at 95°C for 3 min, 29 cycles at 95°C for 1 minute, with annealing temperature varying as cited, for 1 minute, 72°C for 1 minute and a of 72°C cycle for 10 minutes, performed in an automatic cycler. Polymerase chain reaction results were then digested by Sdul (Thermo Fischer Scientific, Massachusetts, USA) for +1444 PCR and HaeIII (Thermo Fischer Scientific, Massachusetts, USA) for IL-6 -174, using the specified amounts. The resulting fragments were identified by silver- stained 8% polyacrylamide gel electrophoresis. Genetic analyses were performed in July 2017 at the São Leopoldo Mandic molecular biology laboratory, Campinas, SP. Statistical analysis The variables were categorized as follows: CAD as present or absent, periodontitis as present or absent, age ≥ 60 years or ≤ 59 years, gender as male or female, ethnicity as white or non-white, overweight and obesity when BMI was ≥ 25 and ≤ 24 and polymorphisms by the presence or absence of the risk allele. The outcome was the presence or absence of CAD. A significance level of 5% (p < 0.05) was used. The sample was divided into two groups, according to the presence of CAD; case group with 52 patients (mean age of 62.8 ± 11.2) and control group with 28 patients (mean age of 56.2 ± 8.8). The distribution of the genotypes of the two SNPs was tested for the Hardy-Weinberg equilibrium by chi-square test (x2), the collected variables were crossed with the chi-square with odds ratio (OR) and 95% confidence interval (95%CI) The variables that showed statistical significance were submitted to adjustment through a binary logistic regression with CAD as outcome and 95% CI. All data collected were treated using the SPSS © version 25 software (IBM © Corp., New York, NY). Results Table 1 shows the association between the studied variables and the presence of coronary artery disease, and it was observed that the age categorized as the cut-off of the mean of all participants, 60 years (p = 0.035), male gender (p = 0.012) and patients with periodontitis (p = 0.013) were statistically related to the presence of CAD. Patients with the + 1444 C > T polymorphism, with the presence of risk allele T (p = 0.001), as well as those with the IL6 -174 G > C polymorphism, with risk allele C (p = 0.025), were associated with the presence of CAD. A binary logistic regression analysis was carried out, in which all the significant independent variables were included in the bivariate analyses in a direct way, with the objective of verifying which of themwould be predictors of the dependent variable, i.e., the presence of CAD. The result of this adjustment (Table 2) showed that the presence of CAD was still associated with age > 60 years (p = 0.029) and the presence of the PCR polymorphism +1444 C > T (p = 0.014). Discussion Periodontitis is a chronic, multifactorial inflammatory disease, resulting from a series of dysbiosis processes, activating the production of proteins and proinflammatory cytokines and signaling processes, thus, according to recent epidemiological, interventional and functional studies, establishing a causal association with the development of coronary artery disease. 10-12,16 The dichotomized age over 60 years and the male gender were also statistically associated with a higher probability of presenting CAD. It is noteworthy that age and male gender are already known risk factors for coronary artery disease and periodontitis, 23 so much so that many studies adjust for these factors only when analyzing atherosclerosis. However, 269