Arq Bras Cardiol abc Arquivos Brasileiros de Cardiologia Arq. Bras. Cardiol. 0066-782X 1678-4170 Sociedade Brasileira de Cardiologia - SBC CWWMxxJSbcGg3YqJN4n3rgp S0066-782X2018000100016 10.5935/abc.20170177 Original Article Atherosclerosis/Endothelium/Vascular Association of Multiple Genetic Variants with the Extension and Severity of Coronary Artery Disease Fischer Simone Cristina Pinto Matheus Pinto Simone Pires Lins Lívia Campos do Amaral Silva Bianco Henrique Tria Monteiro Carlos Manoel de Castro Pinheiro Luiz Fernando Muniz Fonseca Francisco Antonio Helfenstein Izar Maria Cristina de Oliveira Universidade Federal de São Paulo São Paulo SP Brazil Universidade Federal de São Paulo (UNIFESP), São Paulo, SP - Brazil Universidade Federal de São Paulo Mailing Address: Maria Cristina de Oliveira Izar Alameda das Dracenas, 290. Postal Code 06539-240, Alphaville 5, Santana de Parnaíba, SP - Brazil. E-mail: mcoizar@cardiol.br, mcoizar@terra.com.br

Author contributions

Conception and design of the research: Fischer SCPM, Fonseca FAH, Izar MCO;Acquisition of data: Fischer SCPM, Pinto SP, Lins LCAS, Monteiro CMC, Pinheiro LFM, Izar MCO; Analysis and interpretation of the data and Critical revision of the manuscript for intellectual content: Fischer SCPM, Pinto SP, Lins LCAS, Bianco HT, Monteiro CMC, Pinheiro LFM, Fonseca FAH, Izar MCO; Statistical analysis: Bianco HT, Monteiro CMC, Pinheiro LFM, Fonseca FAH, Izar MCO; Obtaining financing: Izar MCO; Writing of the manuscript: Fischer SCPM, Fonseca FAH, Izar MCO.

Potential Conflict of Interest

No potential conflict of interest relevant to this article was reported.

 01 02 2018 01 2018 110 1 16 23 13 05 2017 11 07 2017 02 08 2017 This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background:

Metabolic syndrome (MS) is a condition that, when associated with ischemic heart disease and cardiovascular events, can be influenced by genetic variants and determine more severe coronary atherosclerosis.

 Objectives:

To examine the contribution of genetic polymorphisms to the extension and severity of coronary disease in subjects with MS and recent acute coronary syndrome (ACS).

 Methods:

Patients (n = 116, 68% males) aged 56 (9) years, with criteria for MS, were prospectively enrolled to the study during the hospitalization period after an ACS. Clinical and laboratory parameters, high-sensitivity C-reactive protein, thiobarbituric acid reactive substances, adiponectin, endothelial function, and the Gensini score were assessed. Polymorphisms of 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 analysed by polymerase chain reaction (PCR) technique, followed by the identification of restriction fragment length polymorphisms (RFLP, and a genetic score was calculated. Parametric and non-parametric tests were used, as appropriate. Significance was set at p < 0.05.

 Results:

Polymorphisms of PON-1, MTHFR and ENOS were not in the Hardy-Weinberg equilibrium. The DD genotype of LPL was associated with higher severity and greater extension of coronary lesions. Genetic score tended to be higher in patients with Gensini score < P50 (13.7 ± 1.5 vs. 13.0 ± 1.6, p = 0.066), with an inverse correlation between genetic and Gensini scores (R = -0.194, p = 0.078).

 Conclusions:

The LPL polymorphism contributed to the severity of coronary disease in patients with MS and recent ACS. Combined polymorphisms were associated with the extension of coronary disease, and the lower the genetic score the more severe the disease.

 Keywords: Coronary Artery Disease / genetic Polymorphism, Genetic Metabolic Syndrome Sedentary Lifestyle FAPESP 2004/00325-8 Sources of Funding his study was funded by FAPESP, process nº 2004/00325-8. Introduction

Ischemic heart disease and stroke account for the majority of deaths in the world.1 With progressive urbanization, adoption of a sedentary lifestyle and better access to packaged food, increasing incidence of obesity and overweight has been observed in the population,2 accompanied by an increase in metabolic disorders and cardiovascular risks. The concept of metabolic syndrome (MS) was first described by Reaven, as a relationship between insulin resistance, arterial hypertension, lipid abnormalities and visceral obesity.3,4

MS has been associated with higher rates of fatal and non-fatal cardiovascular events.5,6 Its high prevalence in acute coronary syndrome (ACS) has also been associated with greater anatomical obstruction.7 Our group showed that patients with MS and ACS had lower insulin sensitivity, severe coronary artery disease (CAD) associated with high levels of C-reactive protein (CRP) and low IgG antibody titers to oxidized low-density lipoprotein,8-10 associated with greater extension of CAD.11

Some studies have explored the association of genetic variants with cardiovascular outcomes.12-16Large Mendelian randomization studies have allowed the understanding of the effects of some clinical parameters throughout life, such as LDL-c, HDL-c and triglycerides on cardiovascular risk, as well as the protective effect of polymorphisms associated with lower systolic blood pressure.17-19 Although hypothetical, the effects of polymorphisms have been associated with the extension and severity of CAD.20-22

Smaller studies, systematic review and meta-analyses have proposed genetic variants associated with CAD, as well as genetic scores to identify individuals at higher cardiovascular risk,23-25 since many genetic variants with only modest individual effect may have a larger impact when a greater number of genetic polymorphism is evaluated. Therefore, the use of genetic scores that evaluate the additional effect of each of these genetic variants may be relevant in the early identification of individuals at higher cardiovascular risk, who may benefit from differential attention.23-25

We chose polymorphisms related to lipid metabolism, lipoprotein oxidation, blood pressure changes and vasoreactivity to determine their possible association with coronary atherosclerosis severity in patients with MS and recent ACS. Studies on genetic polymorphisms in MS patients who developed ACS are poorly described in the literature, which reinforces the importance of the present study. We examined the contribution of combined and isolated genetic polymorphisms of potential importance in cardiovascular disease, by a genetic score, to the extension and severity of coronary obstructive disease in patients at very high cardiovascular risk.26

Methods Subjects

A total of 116 consecutive patients of both sexes, mean age 56 ± 9 years, with 3 or more criteria for MS according to the NCEPIII4 were prospectively assessed during hospitalization for ACS (acute myocardial or unstable angina).

Inclusion criteria:

Men and women aged from 30 to 75 years;

Understanding and agreement in giving written consent.

Two or more ACS criteria including chest pain, increased enzyme levels and electrocardiographic changes;

Three or more MS criteria,4

LDL-c < 130 mg/dL, HDL-c < 40 mg/dL in the first 24 hours of hospitalization;

Exclusion criteria:

Use of hypolipidemic agents in the last 30 days;

Uncontrolled hypothyroidism (TSH > 8.0 µU/mL);

Infections, inflammatory diseases, active liver or kidney diseases;

Pregnant women, patients selected for surgeries, including myocardial revascularization in the next six weeks or patients who underwent surgeries during hospitalization.

Ethical aspects

The study protocol was approved by the Research Ethics Committee and the study was conducted according to the Helsinki Declaration. All patients signed the informed consent form before participating in any study procedure.

 Study design

This was a prospective study that included hospitalized patients after ACS. Genetic polymorphisms related to lipid and lipoprotein metabolism, oxidative stress, endothelial function and blood pressure were evaluated.

 Clinical evaluation

Clinical evaluation and blood collection for laboratory measurements were performed in the first three days of hospital discharge. Demographic data, risk factors, medical history, clinical examination, anthropometric data (weight, height, waist circumference), systolic and diastolic blood pressure were obtained.27,28

Blood collection for laboratory tests

Blood was collected after 12 hours of fasting and laboratory tests were performed at the Associação Fundo de Apoio à Psicobiologia (AFIP - Psychobiology Support Fund Association). Genetic tests were carried out at the Molecular Biology Laboratory of the Division of Lipids, Atherosclerosis and Vascular Biology.

 Biochemical tests

Lipid profile (total cholesterol, LDL-c, HDL-c, triglycerides) was analyzed by automated method (Ópera, Bayer, Germany); glucose levels were determined by colorimetric reaction (ADVIA 1650 Chemistry System, USA), glycated hemoglobin (HbA1-c) was determined by high performance liquid chromatography (HPLC, Tosoh A1c 2,2 plus, USA). Mean insulin at -15min, -5min and 0min was assessed by direct chemiluminescence method (ADVIA Centaur, USA) with values expressed as µU/mL. Apolipoproteins A1, B and Lp(a) were analyzed by nephelometry (R100 analyzer, Behringer). Adiponectin was determined by ELISA (enzyme-linked immunosorbent assay), using the Humam Adiponectin/Acrp30 Immunoassay kit - Quantiquine, R&D Systems, and the BioTek ELx800 Absorbance Microplate Reader, with values expressed as ng/mL.

Albuminuria was assessed in 12-hour overnight urine samples, and measured by immunoturbidimetric method (ADVIA 1650, Chemistry System, USA), with results expressed as mg/L.

Plasma oxidative stress was analyzed by TBARS (Thiobarbituric acid reactive substances) test, as described by Ohkawa et al.,29 and measured using a spectrophotometer (Genesys 2, Spectronic); the results were expressed as nanomoles of malondialdehyde (MDA) per milliliter of plasma (hmoles/mL plasma).

 Vasoreactivity

Endothelium-dependent (FMD, flow-mediated dilation) and endothelium-independent (EIR, endothelium-independent relaxation) vasodilation tests were performed in the morning, after a 12-hour fast by an experient ultrasound technician according to the International Brachial Artery Reactivity Task Force guidelines.30 An ultrasound system (Sonos 5500; Hewlett-Packard-Phillips, Palo Alto, CA) was used, equipped with a vascular software for two-dimensional imaging, color and spectral Doppler, internal electrocardiogram (ECG) monitor and a linear array transducer (with a frequency of 7.5-12.0 MHz). Image acquisition, FMD and EIR measures with isosorbide dinitrate (5mg; sublingual) were performed. Percentage variations in vessel diameter were calculated for 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.

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

 Statistical analysis

Sample size was estimated for comparisons between proportions, considering the polymorphism dominant model, an a error of 5%, a b 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.

Demographic and clinical characteristics of participants
Variable n = 116
Male (%) 74 (68)
Age (years) 56 ± 9
Hypertension (%) 104 (90)
Smoking (%) 35 (30)
Diabetes mellitus (%) 36 (31)
AMI (%) 55 (47)
Unstable angina (%) 61 (53)
Stroke (%) 9 (8)
PVD (%) 12 (10)
BMI (Kg/m2) 30.2 ± 4.7
Waist circumference (cm) 104.4 ± 10.8
Systolic arterial pressure (mmHg) 132 ± 24
Diastolic arterial pressure (mmHg) 86 ± 16
Heart rate (bpm) 68 ± 13
Gensini score (au) 21 (0-36)
Gensini ≥ median (%) 42 (51)
FMD (%) 13.7 ± 8.6
EIR (%) 14.9 (7.3-18.8)

Categorical variables expressed as N (%); numerical variables expressed as mean ± standard deviation or median and interquartile ranges. PVD: peripheral vascular disease; FMD: flow-mediated dilation; AMI: acute myocardial infarction; BMI: body mass index, EIR: endothelium‑independent relaxation; AU: arbitrary units.

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 allele and genotypic frequencies for the polimorphisms of PON-1, MTHFR, ENOS, ECA, AT1R, APOC3 and LPL genes
Gene Allele frequency Genotypic frequency (%) p-value (Hardy Weinberg)
PON-1 Q R QQ QR RR
0.63 0.37 36 (31) 76 (65) 4 (4) 0.0004
MTHFR C T CC CT TT
0.61 0.39 35 (30) 72 (62) 9 (8) 0.0131
ENOS G T GG GT TT
0.43 0.57 10 (9) 80 (69) 26 (22) 0.0006
ACE I D II ID DD
0.33 0.67 17 (15) 42 (36) 57 (49) 0.1955
AT1R A C AA AC CC
0.75 0.25 70 (60) 35 (30) 11 (10) 0.2351
APOC3 S1 S2 S1S1 S1S2 S2S2
0.16 0.84 2 (2) 33 (28) 81 (70) 0.8310
LPL D N DD DN NN
0.34 0.66 17 (15) 45 (39) 54 (46) 0.3095

PON-1: paraoxonase-1; MTHFR: methylenotetrahydrofolate reductase; ENOS: endothelial nitric oxide synthase; ACE: angiotensin-converting enzyme; AT1R: angiotensin II type 1 receptor; APOC3: apolipoprotein C3; LPL: lipoprotein lipase. p < 0.05, chi-square test. Expected vs. observed genotypic frequencies were not in the Hardy‑Weinberg equilibrium in the studied population

 Distribution of clinical, demographic and laboratory characteristics

Results were analyzed by genetic polymorphism. The results are presented in details in electronic version in the Supplementary Tables 1-14 (access the link: http://www.arquivosonline.com.br/2018/english/11001/pdf/Tabelas_suplementares_Eng.pdf). For didactic purposes, data are presented by genotype, considering the R allele of PON-1, the T allele of MTHFR, the T allele of ENOS, the D allele of ACE, the C allele of AT1R, the S1 allele of APOC3 and the N allele of LPL as risk alleles.

 Summary of findings

For the PON-1 gene, hs-CRP (mg/dL) values were higher in the QQ genotype as compared with the QR/RR genotype [(12.8 (6.7-24.1) vs. 7.0 (4.8-16.5), p = 0.029] (Supplementary Tables 1 and 2), (access the link: http://www.arquivosonline.com.br/2018/english/11001/pdf/Tabelas_suplementares_Eng.pdf). For the MTHFR gene, HbA1-c (%) and adiponectin (ng/mL) levels were higher in the CT/TT genotype as compared with the CC genotype [6.0 (5.5-7.3) vs. 5.7 (5.2-6.6); p = 0.031 and 6.996 ± 5.032 vs. 4.990 ± 3.165; p = 0.015] (Supplementary Tables 3 and4), access the link: http://www.arquivosonline.com.br/2018/english/11001/pdf/Tabelas_suplementares_Eng.pdf. In addition, fasting glucose (mg/dL), HbA1c (%) and adiponectin levels (ng/mL) were higher in GT/TT vs. GG genotypes [(127 ± 48 vs. 106 ± 12, p = 0,001; 6,6 ± 1,9 vs. 5,9 ± 0,6, p = 0,028; 5010 (2688-10139) vs. 2148 (1912-3435), p = 0,011] (Supplementary Tables 5and6), (access the link: http://www.arquivosonline.com.br/2018/english/11001/pdf/Tabelas_suplementares_Eng.pdf). For the ACE gene, heart rate (bpm) was higher in the ID/DD genotype compared with the II genotype (71 ± 13 vs. 65 ± 11, p = 0.042 (Supplementary Tables 7 and 8), (access the link: http://www.arquivosonline.com.br/2018/english/11001/pdf/Tabelas_suplementares_Eng.pdf). Polymorphisms in the AT1R gene had no effect on clinical or laboratory variables (Supplementary Tables 9 and 10), (access the link: http://www.arquivosonline.com.br/2018/english/11001/pdf/Tabelas_suplementares_Eng.pdf).

For the APOC3 gene, FMD (%) was higher among S2S2 patients than in S1S1/S1S2 patients (14.7 ± 9.6 vs. 11.5 ± 5.2, p = 0.026) (Supplementary Tables 11 and 12), (access the link: http://www.arquivosonline.com.br/2018/english/11001/pdf/Tabelas_suplementares_Eng.pdf), and for the LPL gene, diastolic arterial pressure (mmHg) was lower in the DD genotype as compared with DN/NN (79 ± 15 vs. 87 ± 16, p = 0.043). In addition, a more severe degree of coronary atherosclerosis, evaluated by the Gensini score > median (%) value was found in patients with DD genotype compared with DN/NN (77% vs. 46%, p = 0.039) (Supplementary Tables 13 and 14) (access the link: http://www.arquivosonline.com.br/2018/english/11001/pdf/Tabelas_suplementares_Eng.pdf).

 Associations of genotypes with CAD extension and severity

A dominant model was assumed, as well as the score from 1 to 3 for the isolated genotypes - 1 for absence of risk allele; 2 for the presence of one risk allele; and 3 for the presence of 2 risk alleles in the same gene. Then, score 1 was assigned to the genotypes QQ of PON-1, CC of MTHFR, GG of ENOS, II of ACE, AA of AT1R, S2S2 of APOC3 and DD of LPL. The score 2 was assigned to the genotypes QR of PON-1, CT of MTHFR, GT of ENOS, ID of ACE, AC of AT1R, S1S2 of APOC3 and DN of LPL. The score 3 was assigned to the genotypes RR of PON-1, TT of MTHFR, TT of ENOS, DD of ACE, CC of AT1R, S1S1 of APOC3 and NN of LPL.

The sum of the values assigned to each gene (7-21) yielded a genetic score, which was evaluated in absolute values and also in relation with the median (above or below) value. Correlations between genetic and Gensini scores were performed in absolute values and in relation to the median. Results are described in Table 4 and Supplementary Table 15 (access the link: http://www.arquivosonline.com.br/2018/english/11001/pdf/Tabelas_suplementares_Eng.pdf). Both genetic and Gensini scores had a normal distribution.

Distribution of genetic score considering all polymorphisms of the PON-1, MTHFR, ENOS, ACE, AT1R), APOC3 and LPL genes
Genetic score
N 116
Mean 13.3
Median 13.5
Standard deviation 1.58
Minimum 10
Maximum 17
Asymmetry -0.263
Kurtosis -0.146
P25 12.0
P75 14.0

PON-1: paraoxonase-1; MTHFR: methylenotetrahydrofolate reductase; ENOS: endothelial nitric oxide synthase; ACE: angiotensin-converting enzyme; AT1R: angiotensin II type 1 receptor; APOC3: apolipoprotein C3; LPL: lipoprotein lipase.

Genetic score tended to be higher in patients with a Gensini score < 50th percentile (13.7 ± 1.5 vs. 13.0 ± 1.6; p = 0.066, Student’s t test for independent samples).

Gensini score was not different between genetic scores above and below the median (p50) [26 (0.00-44.00) vs. 18 (0.25-33.70), P=0.329]. In this population of patients with recent ACS and MS, a weak, inverse correlation was observed between the genetic and the Gensini scores (R = -0.194, p = 0.078, Pearson correlation coefficient).

 Discussion

The present study demonstrated that the genetic polymorphisms analyzed in patients with MS and recent ACS had a modest association with the severity of obstructive coronary disease. Only the DD genotype of D9N polymorphism of LPL was associated with higher prevalence of more severe coronary lesions. Analysis of the genetic score revealed that the combinations of the studied polymorphisms showed a trend of negative correlation with the anatomical extension of the coronary disease. This finding suggests that in these MS patients, coronary disease may be primarily associated with other mechanisms, with a strong environmental influence.

Many of the studied polymorphisms were associated with clinical and laboratory variables, such as heart frequency, diastolic arterial pressure, CRP, HbA1c and adiponectin.

A study involving six polymorphisms of LPL, including the D9N, showed that the severity of obstructive lesions, analyzed by the Gensini score, was associated with LPL haplotypes.32 Corsetti et al.33 showed an interaction of D9N polymorphism with Taq1B of CETP, which was a predictor of cardiovascular disease risk in women. In addition, LPL polymorphisms have been associated with increased concentrations of triglycerides;34 in our study, a trend towards higher values was found for the DD genotype as compared with the DN/NN genotype (p = 0.07), possibly due to the high prevalence of overweight/obesity and changes in glucose metabolism in these MS patients.

APOC3 polymorphism has an important role in the metabolism of triglyceride-rich lipoproteins and an influence on the development of CAD, particularly in MS and diabetes, with an association of haplotypes in the AI-C3-AIV gene cluster with coronary disease.35

Renin-angiotensin system genetic polymorphisms (I/D of ACE and AT1R) were not associated with CAD severity in our study. However, the ACE gene was previously associated with higher ACE levels in D/D patients with CAD,36 which was not confirmed in a larger sample.

In our study, the 192R allele of PON-1 was associated with higher CRP levels, which is a biomarker of cardiovascular outcomes. In a large, three-year follow-up study (n = 3668), arylesterase activity, but not paraoxonase levels or PON-1 polymorphism, was associated with cardiovascular outcomes.37

For the MTHFR C677T polymorphism, a meta-analysis (n = 6912) demonstrated its association with early CAD,38 with higher homocysteine levels in the presence of T allele.39 In our study, CT/TT individuals had higher HbA1c and adiponectin levels as compared with CC individuals.

ENOS polymorphisms are involved in endothelial dysfunction. The T allele of the G894T polymorphism showed lower levels of nitric oxide and an association with CAD.40 The variant allele -786T (promoter region) was associated with CAD in a meta-analysis.41

Genetic score was inversely associated with greater extension and higher severity of CAD. In this population with MS, environmental factors may have an impact on the modulation of the expression of genes related to lipid metabolism, lipoprotein oxidation, endothelial function and blood pressure, and hence on atherogenesis.

 Conclusion

The studied polymorphisms had a small contribution to the extension of CAD. Only LPL D9N polymorphism was associated with CAD extension in patients with MS and recent ACS. Analysis of combined genetic polymorphisms showed a weak association with CAD extension, and an inverse relationship of genetic score with CAD extension and severity.

Study limitations

The small sample size and the cross-sectional design may be considered limitations of our study. However, the exploratory aim of the study was to identify genetic polymorphisms that may be used in the identification of more severe atherosclerotic disease in this population of patients with MS and recent ACS. Its results contribute to the selection of genetic polymorphisms to be tested in prospective studies involving larger samples.

Sources of Funding

This study was funded by FAPESP, process nº 2004/00325-8.

Study Association

This article is part of the thesis of master submitted by Simone Cristina Pinto Matheus Fischer, from Universidade Federal de São Paulo.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of the Universidade Federal de São Paulo (CEP UNIFESP) under the protocol number CEP 0283/11. All the procedures in this study were in accordance with the 1975 Helsinki Declaration, updated in 2013. Informed consent was obtained from all participants included in the study.

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Atherosclerosis. 2012;223(2):468-72. doi: 10.1016/j.atherosclerosis.2012.05.018. Novo S Peritore A Guarneri FP Corrado E Macaione F Evola S Metabolic syndrome (MetS) predicts cardio and cerebrovascular events in a twenty years follow-up A prospective study Atherosclerosis 2012 223 2 468 472 10.1016/j.atherosclerosis.2012.05.018 7 Sinha SK, Goel A, Madaan A, Thakur R, Krishna V, Singh K, et al. Prevalence of metabolic syndrome and its clinical and angiographic profile in patients with naive acute coronary syndrome in North Indian population. J Clin Med Res. 2016;8(9):667-73. doi: 10.14740/jocmr2655w. Sinha SK Goel A Madaan A Thakur R Krishna V Singh K Prevalence of metabolic syndrome and its clinical and angiographic profile in patients with naive acute coronary syndrome in North Indian population J Clin Med Res 2016 8 9 667 673 10.14740/jocmr2655w 8 Monteiro CM, Oliveira L, Izar MC, Helfenstein T, Santos AO, Fischer SM, et al. Early glucometabolic profile in patients with acute coronary syndromes and metabolic syndrome. Arq Bras Cardiol. 2009;92(2):89-99. doi: http://dx.doi.org/10.1590/S0066-782X2009000200004 Monteiro CM Oliveira L Izar MC Helfenstein T Santos AO Fischer SM Early glucometabolic profile in patients with acute coronary syndromes and metabolic syndrome Arq Bras Cardiol 2009 92 2 89 99 http://dx.doi.org/10.1590/S0066-782X2009000200004 9 Monteiro CM, Pinheiro LF, Izar MC, Barros SW, Vasco MB, Fischer SM, et al. Highly sensitive C-reactive protein and male gender are independently related to the severity of coronary disease in patients with metabolic syndrome and an acute coronary event. Braz J Med Biol Res. 2010;43(3):297-302. doi: http://dx.doi.org/10.1590/S0100-879X2010005000008. Monteiro CM Pinheiro LF Izar MC Barros SW Vasco MB Fischer SM Highly sensitive C-reactive protein and male gender are independently related to the severity of coronary disease in patients with metabolic syndrome and an acute coronary event Braz J Med Biol Res 2010 43 3 297 302 http://dx.doi.org/10.1590/S0100-879X2010005000008 10 Santos AO, Fonseca FA, Fischer SM, Monteiro CM, Brandão SA, Póvoa RM, et al. High circulating autoantibodies against human oxidized low-density lipoprotein are related to stable and lower titers to unstable clinical situation. Clin Chim Acta. 2009;406(1-2):113-8. doi: 10.1016/j.cca.2009.06.005. Santos AO Fonseca FA Fischer SM Monteiro CM Brandão SA Póvoa RM High circulating autoantibodies against human oxidized low-density lipoprotein are related to stable and lower titers to unstable clinical situation Clin Chim Acta 2009 406 1-2 113 118 10.1016/j.cca.2009.06.005 11 Izar MC, Fonseca HA, Pinheiro LF, Monteiro CM, Póvoa RM, Monteiro AM, et al. Adaptive immunity is related to coronary artery disease severity after acute coronary syndrome in subjects with metabolic syndrome. Diab Vasc Dis Res. 2013;10(1):32-9. doi: 10.1177/1479164112443374. Izar MC Fonseca HA Pinheiro LF Monteiro CM Póvoa RM Monteiro AM Adaptive immunity is related to coronary artery disease severity after acute coronary syndrome in subjects with metabolic syndrome Diab Vasc Dis Res 2013 10 1 32 39 10.1177/1479164112443374 12 Izar MC, Helfenstein T, Ihara SS, Relvas WG, Santos AO, Fischer SC, et al; GOLD Investigators. Association of lipoprotein lipase D9N polymorphism with myocardial infarction in type 2 diabetes: the genetics, outcomes, and lipids in type 2 diabetes (GOLD) study. Atherosclerosis. 2009;204(1):165-70. doi: 10.1016/j.atherosclerosis.2008.08.006. Izar MC Helfenstein T Ihara SS Relvas WG Santos AO Fischer SC GOLD Investigators Association of lipoprotein lipase D9N polymorphism with myocardial infarction in type 2 diabetes the genetics, outcomes, and lipids in type 2 diabetes (GOLD) study Atherosclerosis 2009 204 1 165 170 10.1016/j.atherosclerosis.2008.08.006 13 Kaur R, Matharoo K, Sharma R, Bhanwer AJ. C-reactive protein + 1059 G&gt;C polymorphism in type 2 diabetes and coronary artery disease patients. Meta Gene. 2013;1:82-92. doi: 10.1016/j.mgene.2013.10.012. Kaur R Matharoo K Sharma R Bhanwer AJ C-reactive protein + 1059 G&gt;C polymorphism in type 2 diabetes and coronary artery disease patients Meta Gene 2013 1 82 92 10.1016/j.mgene.2013.10.012 14 Song Z, Cao H, Qin L, Jiang Y. A case-control study between gene polymorphisms of polyunsaturated fatty acid metabolic rate-limiting enzymes and acute coronary syndrome in Chinese Han population. Biomed Res Int. 2013;2013:928178. doi: 10.1155/2013/928178. Song Z Cao H Qin L Jiang Y A case-control study between gene polymorphisms of polyunsaturated fatty acid metabolic rate-limiting enzymes and acute coronary syndrome in Chinese Han population Biomed Res Int 2013 2013 928178 10.1155/2013/928178 15 Palmer BR, Slow S, Ellis KL, Pilbrow AP, Skelton L, Frampton CM, et al. Genetic polymorphism rs6922269 in the MTHFD1L gene is associated with survival and baseline active vitamin B12 levels in post-acute coronary syndromes patients. PLoS One. 2014;9(3):e89029. doi: 10.1371/journal.pone.0089029. Palmer BR Slow S Ellis KL Pilbrow AP Skelton L Frampton CM Genetic polymorphism rs6922269 in the MTHFD1L gene is associated with survival and baseline active vitamin B12 levels in post-acute coronary syndromes patients PLoS One 2014 9 3 e89029 10.1371/journal.pone.0089029 16 Ellis KL, Zhou Y, Beshansky JR, Ainehsazan E, Selker HP, Cupples LA, et al. Genetic modifiers of response to glucose-insulin-potassium (GIK) infusion in acute coronary syndromes and associations with clinical outcomes in the IMMEDIATE trial. Pharmacogenomics J. 2015;15(6):488-95. doi: 10.1038/tpj.2015.10. Ellis KL Zhou Y Beshansky JR Ainehsazan E Selker HP Cupples LA Genetic modifiers of response to glucose-insulin-potassium (GIK) infusion in acute coronary syndromes and associations with clinical outcomes in the IMMEDIATE trial Pharmacogenomics J 2015 15 6 488 495 10.1038/tpj.2015.10 17 Ference BA. Mendelian randomization studies: using naturally randomized genetic data to fill evidence gaps. Curr Opin Lipidol. 2015;26(6):566-71. doi: 10.1097/MOL.0000000000000247. Ference BA Mendelian randomization studies using naturally randomized genetic data to fill evidence gaps Curr Opin Lipidol 2015 26 6 566 571 10.1097/MOL.0000000000000247 18 Holmes MV, Asselbergs FW, Palmer TM, Drenos F, Lanktree MB, Nelson CP, et al. Mendelian randomization of blood lipids for coronary heart disease. Eur Heart J. 2015;36(9):539-50. doi: 10.1093/eurheartj/eht571. Holmes MV Asselbergs FW Palmer TM Drenos F Lanktree MB Nelson CP Mendelian randomization of blood lipids for coronary heart disease Eur Heart J 2015 36 9 539 550 10.1093/eurheartj/eht571 19 Nordestgaard BG. Triglyceride-rich lipoproteins and atherosclerotic cardiovascular disease: New insights from epidemiology, genetics, and biology. Circ Res. 2016;118(4):547-63. doi: 10.1161/CIRCRESAHA.115.306249. Nordestgaard BG Triglyceride-rich lipoproteins and atherosclerotic cardiovascular disease New insights from epidemiology, genetics, and biology Circ Res 2016 118 4 547 563 10.1161/CIRCRESAHA.115.306249 20 Jia EZ, Chen ZH, An FH, Li LH, Li-Li, Guo CY, et al. Relationship of renin-angiotensin-aldosterone system polymorphisms and phenotypes to mortality in Chinese coronary atherosclerosis patients. Sci Rep. 2014;4:4600. doi: 10.1038/srep04600. Jia EZ Chen ZH An FH Li LH Li-Li Guo CY Relationship of renin-angiotensin-aldosterone system polymorphisms and phenotypes to mortality in Chinese coronary atherosclerosis patients Sci Rep 2014 4 4600 10.1038/srep04600 21 Osadnik T, Strzelczyk JK, Lekston A, Regula R, Bujak K, Fronczek M, et al. The association of functional polymorphisms in genes encoding growth factors for endothelial cells and smooth muscle cells with the severity of coronary artery disease. BMC Cardiovasc Disord. 2016;16(1):218. doi: 10.1186/s12872-016-0402-4. Osadnik T Strzelczyk JK Lekston A Regula R Bujak K Fronczek M The association of functional polymorphisms in genes encoding growth factors for endothelial cells and smooth muscle cells with the severity of coronary artery disease BMC Cardiovasc Disord 2016 16 1 218 10.1186/s12872-016-0402-4 22 Yang M, Zhu M, Tang L, Zhu H, Lu Y, Xu B, et al. Polymorphisms of TGFb-1 and TGFBR2 in relation to coronary artery disease in a Chinese population. Clin Biochem. 2016;49(12):873-8. doi: 10.1016/j.clinbiochem.2016.05.022. Yang M Zhu M Tang L Zhu H Lu Y Xu B Polymorphisms of TGFb-1 and TGFBR2 in relation to coronary artery disease in a Chinese population Clin Biochem 2016 49 12 873 10.1016/j.clinbiochem.2016.05.022 23 Anderson JL, Horne BD, Camp NJ, Muhlestein JB, Hopkins PN, Cannon-Albright LA, et al. Joint effects of common genetic variants from multiple genes and pathways on the risk of premature coronary artery disease. Am Heart J. 2010;160(2):250-256.e3. doi: 10.1016/j.ahj.2010.05.031. Anderson JL Horne BD Camp NJ Muhlestein JB Hopkins PN Cannon-Albright LA Joint effects of common genetic variants from multiple genes and pathways on the risk of premature coronary artery disease Am Heart J 2010 160 2 250 256 10.1016/j.ahj.2010.05.031 24 Brautbar A, Pompeii LA, Dehghan A, Ngwa JS, Nambi V, Virani SS, et al. A genetic risk score based on direct associations with coronary heart disease improves coronary heart disease risk prediction in the Atherosclerosis Risk in Communities (ARIC), but not in the Rotterdam and Framingham Offspring, Studies. Atherosclerosis. 2012;223(2):421-6. doi: 10.1016/j.atherosclerosis.2012.05.035. Brautbar A Pompeii LA Dehghan A Ngwa JS Nambi V Virani SS A genetic risk score based on direct associations with coronary heart disease improves coronary heart disease risk prediction in the Atherosclerosis Risk in Communities (ARIC), but not in the Rotterdam and Framingham Offspring, Studies Atherosclerosis 2012 223 2 421 426 10.1016/j.atherosclerosis.2012.05.035 25 Shah S, Casas JP, Gaunt TR, Cooper J, Drenos F, Zabaneh D, et al. Influence of common genetic variation on blood lipid levels, cardiovascular risk, and coronary events in two British prospective cohort studies. Eur Heart J. 2013;34(13):972-81. doi: 10.1093/eurheartj/ehs243. Shah S Casas JP Gaunt TR Cooper J Drenos F Zabaneh D Influence of common genetic variation on blood lipid levels, cardiovascular risk, and coronary events in two British prospective cohort studies Eur Heart J 2013 34 13 972 981 10.1093/eurheartj/ehs243 26 Faludi AA, Izar MC, Saraiva JF, Chacra AP, Bianco HT, Afiune Neto A, et al. Atualização da Diretriz Brasileira de dislipidemias e prevenção da aterosclerose - 2017. Arq Bras Cardiol 2017; 109(2Supl.1):1-76. doi: 10.5935/abc.20170121. Faludi AA Izar MC Saraiva JF Chacra AP Bianco HT Afiune A Neto Atualização da Diretriz Brasileira de dislipidemias e prevenção da aterosclerose - 2017 Arq Bras Cardiol 2017 109 2 Supl.1 1 76 10.5935/abc.20170121. 27 Sposito AC, Caramelli B, Fonseca FA, Bertolami MC, Afiune Neto A, Souza AD, et al; Sociedade Brasileira de Cardiologia. [IV Brazilian Guideline for Dyslipidemia and Atherosclerosis prevention: Department of Atherosclerosis of Brazilian Society of Cardiology]. Arq Bras Cardiol. 2007;88 Suppl 1:2-19. doi: http://dx.doi.org/10.1590/S0066-782X2007000700002. Sposito AC Caramelli B Fonseca FA Bertolami MC Afiune A Neto Souza AD Sociedade Brasileira de Cardiologia IV Brazilian Guideline for Dyslipidemia and Atherosclerosis prevention: Department of Atherosclerosis of Brazilian Society of Cardiology Arq Bras Cardiol 2007 88 Suppl 1 2 19 http://dx.doi.org/10.1590/S0066-782X2007000700002 28 Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL Jr, et al; Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. National Heart, Lung, and Blood Institute.; National High Blood Pressure Education Program Coordinating Committee. Seventh report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Hypertension. 2003;42(6):1206-52. doi: 10.1161/01.HYP.0000107251.49515.c2. Chobanian AV Bakris GL Black HR Cushman WC Green LA Izzo JL Jr Joint National Committee on Prevention.Detection.Evaluation.and Treatment of High Blood Pressure National Heart, Lung, and Blood Institute ; National High Blood Pressure Education Program Coordinating Committee. Seventh report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure Hypertension 2003 42 6 1206 1252 10.1161/01.HYP.0000107251.49515.c2 29 Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979;95(2):351-8. PMID: 36810. Ohkawa H Ohishi N Yagi K Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction Anal Biochem 1979 95 2 351 358 36810 30 Corretti MC, Anderson TJ, Benjamin EJ, Celermajer D, Charbonneau F, Creager MA, et al; International Brachial Artery Reactivity Task Force. Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force. J Am Coll Cardiol. 2002;39(2):257-65. PMID: 11788217. Erratum in: J Am Coll Cardiol. 2002;39(6):1082. Corretti MC Anderson TJ Benjamin EJ Celermajer D Charbonneau F Creager MA International Brachial Artery Reactivity Task Force Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force J Am Coll Cardiol 2002 39 2 257 265 11788217 Erratum in: J Am Coll Cardiol. 2002;39(6):1082 31 Gensini GG. A more meaningful scoring system for determining the severity of coronary heart disease. Am J Cardiol. 1983;51(3):606. PMID: 6823874. Gensini GG A more meaningful scoring system for determining the severity of coronary heart disease Am J Cardiol 1983 51 3 606 6823874 32 Rebhi L, Kchok K, Omezzine A, Kacem S, Rejeb J, Ben HadjMbarek I, et al. Six lipoprotein lipase gene polymorphisms, lipid profile and coronary stenosis in a Tunisian population. Mol Biol Rep. 2012;39(11):9893-901. doi: 10.1007/s11033-012-1856-9. Rebhi L Kchok K Omezzine A Kacem S Rejeb J Ben HadjMbarek I Six lipoprotein lipase gene polymorphisms, lipid profile and coronary stenosis in a Tunisian population Mol Biol Rep 2012 39 11 9893 9901 10.1007/s11033-012-1856-9 33 Corsetti JP, Gansevoort RT, Navis G, Sparks CE, Dullaart RP. LPL polymorphism (D9N) predicts cardiovascular disease risk directly and through interaction with CETP polymorphism (TaqIB) in women with high HDL cholesterol and CRP. Atherosclerosis. 2011;214(2):373-6. doi: 10.1016/j.atherosclerosis.2010.11.029. Corsetti JP Gansevoort RT Navis G Sparks CE Dullaart RP LPL polymorphism (D9N) predicts cardiovascular disease risk directly and through interaction with CETP polymorphism (TaqIB) in women with high HDL cholesterol and CRP Atherosclerosis 2011 214 2 373 376 10.1016/j.atherosclerosis.2010.11.029 34 Ariza MJ, Sánchez-Chaparro MA, Barón FJ, Hornos AM, Calvo-Bonacho E, Rioja J, et al. Additive effects of LPL, APOA5 and APOE variant combinations on triglyceride levels and hypertriglyceridemia: results of the ICARIA genetic sub-study. BMC Med Genet. 2010 Apr 29;11:66. doi: 10.1186/1471-2350-11-66. Ariza MJ Sánchez-Chaparro MA Barón FJ Hornos AM Calvo-Bonacho E Rioja J Additive effects of LPL, APOA5 and APOE variant combinations on triglyceride levels and hypertriglyceridemia results of the ICARIA genetic sub-study BMC Med Genet 2010 11 66 10.1186/1471-2350-11-66 35 Rigoli L, Raimondo G, Di Benedetto A, Romano G, Porcellini A, Campo S, et al. Apolipoprotein AI-CIII-AIV genetic polymorphisms and coronary heart disease in type 2 diabetes mellitus. Acta Diabetol. 1995;32(4):251-6. PMID: 8750764. Rigoli L Raimondo G Di Benedetto A Romano G Porcellini A Campo S Apolipoprotein AI-CIII-AIV genetic polymorphisms and coronary heart disease in type 2 diabetes mellitus Acta Diabetol 1995 32 4 251 256 8750764 36 Sahin S, Ceyhan K, Benli I, Ozyurt H, Naseri E, Tumuklu MM, et al. Traditional risk factors and angiotensin-converting enzyme insertion/deletion gene polymorphism in coronary artery disease. Genet Mol Res. 2015;14(1):2063-8. doi: 10.4238/2015.March.20.16. Sahin S Ceyhan K Benli I Ozyurt H Naseri E Tumuklu MM Traditional risk factors and angiotensin-converting enzyme insertion/deletion gene polymorphism in coronary artery disease Genet Mol Res 2015 14 1 2063 2068 10.4238/2015.March.20.16 37 Tang WH, Hartiala J, Fan Y, Wu Y, Stewart AF, Erdmann J, et al; CARDIoGRAM Consortium. Clinical and genetic association of serum paraoxonase and arylesterase activities with cardiovascular risk. Arterioscler Thromb Vasc Biol. 2012;32(11):2803-12. doi: 10.1161/ATVBAHA.112.253930. Tang WH Hartiala J Fan Y Wu Y Stewart AF Erdmann J CARDIoGRAM Consortium Clinical and genetic association of serum paraoxonase and arylesterase activities with cardiovascular risk Arterioscler Thromb Vasc Biol 2012 32 11 2803 2812 10.1161/ATVBAHA.112.253930 38 Hou X, Chen X, Shi J. Genetic polymorphism of MTHFR C677T and premature coronary artery disease susceptibility: a meta-analysis. Gene. 2015;565(1):39-44. doi: 10.1016/j.gene.2015.03.062. Hou X Chen X Shi J Genetic polymorphism of MTHFR C677T and premature coronary artery disease susceptibility a meta-analysis Gene 2015 565 1 39 44 10.1016/j.gene.2015.03.062 39 Helfenstein T, Fonseca FA, Relvas WG, Santos AO, Dabela ML, Matheus SC, et al. Prevalence of myocardial infarction is related to hyperhomocysteinemia but not influenced by C677T methylenetetrahydrofolate reductase and A2756G methionine synthase polymorphisms in diabetic and non-diabetic subjects. Clin Chim Acta. 2005;355(1-2):165-72. doi: 10.1016/j.cccn.2004.12.002. Helfenstein T Fonseca FA Relvas WG Santos AO Dabela ML Matheus SC Prevalence of myocardial infarction is related to hyperhomocysteinemia but not influenced by C677T methylenetetrahydrofolate reductase and A2756G methionine synthase polymorphisms in diabetic and non-diabetic subjects Clin Chim Acta 2005 355 1-2 165 172 10.1016/j.cccn.2004.12.002 40 Mahmoodi K, Nasehi L, Karami E, Soltanpour MS. Association of nitric oxide levels and endothelial nitric oxide synthase G894T polymorphism with coronary artery disease in the Iranian population. Vasc Specialist Int. 2016;32(3):105-12. doi: 10.5758/vsi.2016.32.3.105. Mahmoodi K Nasehi L Karami E Soltanpour MS Association of nitric oxide levels and endothelial nitric oxide synthase G894T polymorphism with coronary artery disease in the Iranian population Vasc Specialist Int 2016 32 3 105 112 10.5758/vsi.2016.32.3.105 41 Liu D, Jiang Z, Dai L, Zhang X, Yan C, Han Y. Association between the - 786T&gt;C 1polymorphism in the promoter region of endothelial nitric oxide synthase (eNOS) and risk of coronary artery disease: a systematic review and meta-analysis. Gene. 2014;545(1):175-83. doi: 10.1016/j.gene.2013.09.099. Liu D Jiang Z Dai L Zhang X Yan C Han Y Association between the - 786T&gt;C 1polymorphism in the promoter region of endothelial nitric oxide synthase (eNOS) and risk of coronary artery disease a systematic review and meta-analysis Gene 2014 545 1 175 183 10.1016/j.gene.2013.09.099 Artigo Original Aterosclerose/Endotélio/Vascular Associação de Múltiplas Variantes Genéticas com a Extensão e Gravidade da Doença Coronária Fischer Simone Cristina Pinto Matheus 2 Pinto Simone Pires 2 Lins Lívia Campos do Amaral Silva 2 Bianco Henrique Tria 2 Monteiro Carlos Manoel de Castro 2 Pinheiro Luiz Fernando Muniz 2 Fonseca Francisco Antonio Helfenstein 2 Izar Maria Cristina de Oliveira 2 Universidade Federal de São Paulo (UNIFESP), São Paulo, SP - Brasil Correspondência: Maria Cristina de Oliveira Izar Alameda das Dracenas, 290. CEP 06539-240, Alphaville 5, Santana de Parnaíba, SP - Brasil E-mail: mcoizar@cardiol.br, mcoizar@terra.com.br

Contribuição dos autores

Concepção e desenho da pesquisa: Fischer SCPM, Fonseca FAH, Izar MCO; Obtenção de dados: Fischer SCPM, Pinto SP, Lins LCAS, Monteiro CMC, Pinheiro LFM, Izar MCO; Análise e interpretação dos dados e Revisão crítica do manuscrito quanto ao conteúdo intelectual importante: Fischer SCPM, Pinto SP, Lins LCAS, Bianco HT, Monteiro CMC, Pinheiro LFM, Fonseca FAH, Izar MCO; Análise estatística: Bianco HT, Monteiro CMC, Pinheiro LFM, Fonseca FAH, Izar MCO; Obtenção de financiamento: Izar MCO; Redação do manuscrito: Fischer SCPM, Fonseca FAH, Izar MCO.

Potencial conflito de interesses

Declaro não haver conflito de interesses pertinentes.

 Resumo Fundamento:

A síndrome metabólica (SM) é condição que, associada à doença coronária e a eventos cardiovasculares, pode ser influenciada por variantes genéticas, determinando maior gravidade da aterosclerose coronária.

 Objetivos:

Avaliar a contribuição de polimorfismos genéticos para a extensão da doença coronária em indivíduos com SM e recente síndrome coronária aguda (SCA).

 Métodos:

Pacientes (n = 116, 68% homens) com 56 (9) anos e critérios para SM foram prospectivamente selecionados no período de hospitalização após uma SCA. Parâmetros clínicos e laboratoriais, proteína C-reativa ultrassensível, substâncias reativas ao ácido tiobarbitúrico, adiponectina, função endothelial e o escore de Gensini foram analisados. Os polimorfismos da paraoxonase-1 (PON-1), metilenotetrahidrofolato redutase (MTHFR), óxido nítrico sintase endotelial (ENOS), enzima conversora da angiotensina (ECA), receptor tipo 1 da angiotensina II (AT1R), apolipoproteína C3 (APOC3), lipoproteína lipase (LPL) foram analisados por reação em cadeia da polimerase (PCR) seguida da identificação dos polimorfismos no comprimento do fragmento de restrição (RFLP), criando-se um escore genético. Testes paramétricos e não-paramétricos foram utilizados, conforme apropriado, com p < 0,05 considerado como significativo.

 Resultados:

Os polimorfismos da PON-1, MTHFR e ENOS não estavam em equilíbrio de Hardy-Weinberg. O genótipo DD da LPL associou-se com lesões coronarianas mais graves e extensas. O escore genético tendeu a ser maior nos pacientes com escore de Gensini < P50 (13,7 ± 1,5 vs. 13,0 ± 1,6, p = 0,066), com correlação inversa entre o escore genético e o de Gensini (R = -0,194, p = 0,078).

 Conclusões:

O polimorfismo da LPL associou-se à maior gravidade da doença coronária em pacientes com SM e SCA. Combinação de polimorfismos genéticos associou-se à extensão da doença coronariana, sendo a doença mais grave quanto menor o escore genético.

 Palavras-chave: Doencça Arterial Coronariana / genética Polimorfismo Genético Síndrome Metabólica Estilo de Vida Sedentário FAPESP 2004/00325-8 Fontes de financiamento O presente estudo foi financiado pela FAPESP, processo nº 2004/00325-8. Introdução

A doença isquêmica do coração e o acidente vascular cerebral são responsáveis pela maior parte das mortes em todo o mundo.1 Com a progressiva urbanização, adoção de estilo de vida sedentário e maior acesso a alimentos industrializados, a população humana apresentou crescimento das taxas de obesidade e sobrepeso,2 determinando aumento de distúrbios metabólicos e maior risco cardiovascular. O conceito de síndrome metabólica (SM) foi inicialmente descrito por Reaven, relacionando resistência insulínica, hipertensão arterial, anormalidades lipídicas e obesidade visceral.3,4

A SM tem sido associada com maior taxa de eventos cardiovasculares fatais e não fatais.5,6 Sua alta prevalência na síndrome coronariana aguda (SCA) também se associa a maior grau de obstrução anatômica.7 Nosso grupo demonstrou que pacientes com SM e SCA apresentavam menor sensibilidade à insulina, maior gravidade da doença arterial coronariana (DAC) associada a níveis de proteína C-reativa (PCR) elevados e menores títulos de anticorpos IgG anti-LDL oxidada,8-10 associados à maior extensão anatômica da DAC.11

Alguns estudos têm explorado a associação de variantes genéticas com desfechos cardiovasculares.12-16 Grandes estudos de randomização Mendeliana têm possibilitado maior compreensão dos efeitos de alguns parâmetros clínicos ao longo da vida, como níveis de LDL-c, HDL-c e triglicérides sobre risco cardiovascular, bem como o efeito protetor de polimorfismos associados a menores valores da pressão arterial sistólica.17-19 Embora com implicações apenas geradoras de hipóteses, a contribuição de vários polimorfismos também tem sido associada com a extensão e gravidade da DAC.20-22

A partir de estudos menores, revisões sistemáticas e metanálises, variantes genéticas associadas com a DAC têm sido identificadas, bem como escores genéticos têm sido propostos para identificação de indivíduos sob maior risco cardiovascular,23-25 uma vez que uma série de variantes genéticas, cujo efeito individual no risco cardiovascular é modesto, pode determinar um maior impacto quando um número maior de polimorfismos genéticos é avaliado. Assim, a utilização de escores genéticos, que avaliem o possível efeito aditivo de cada uma dessas variantes genéticas analisadas pode ser relevante na identificação precoce de indivíduos com maior risco cardiovascular, em quem uma atenção diferenciada possa ser implementada.23-25

Escolhemos polimorfismos relacionados ao metabolismo lipídico, oxidação de lipoproteínas, alterações da pressão arterial e vasorreatividade para determinar sua possível associação com a gravidade da aterosclerose coronária em portadores de SM e recente SCA. Estudos de polimorfismos genéticos em portadores de SM e que desenvolvem SCA são pobremente descritos na literatura, justificando-se a importância da realização deste estudo. Assim, examinamos, de maneira isolada e combinada, a contribuição de polimorfismos genéticos de potencial importância na doença cardiovascularpor meio de um escore genético, na extensão e na gravidade da doença obstrutiva coronariana em pacientes de muito alto risco cardiovascular.26

Métodos Casuística

Foram avaliados prospectivamente 116 pacientes consecutivos de ambos os sexos, com idade de 56 ± 9 anos e três ou mais critérios para SM com base na NCEP III,4 no período de hospitalização devido à SCA (infarto agudo do miocárdio ou angina instável).

Critérios de inclusão:

Homens e mulheres com idade entre 30 a 75 anos;

Compreensão e concordância em dar o consentimento livre e esclarecido por escrito.

Pelo menos dois de três critérios de SCA, incluindo dor no peito, elevação enzimática e alteração eletrocardiográfica;

Possuírem pelo menos três critérios para SM;4

LDL-c < 130 mg/dL, HDL- c < 40 mg/dL nas primeiras 24 horas da internação;

Critérios de exclusão:

Uso de hipolipemiantes nos últimos 30 dias;

Hipotiroidismo não controlado (TSH > 8,0 µU/mL);

Infecção, doenças inflamatórias, doença hepática ou renal ativa;

Gestantes ou pacientes com programação de cirurgias, incluindo revascularização miocárdica nas próximas seis semanas ou que tenha ocorrido durante a internação.

Aspectos éticos

O protocolo do estudo foi aprovado pelo Comitê de ética em Pesquisa da Universidade Federal de São Paulo (CEP UNIFESP) sob o número de protocolo CEP 0283/11 e o estudo foi conduzido em consonância à Declaração de Helsinki. Todos os pacientes assinaram o termo de consentimento livre e esclarecido antes de qualquer procedimento do estudo.

 Desenho do estudo

Estudo prospectivo que incluiu pacientes na fase hospitalar após uma SCA. Foram avaliados polimorfismos genéticos relacionados ao metabolismo dos lípides e lipoproteínas, estresse oxidativo, função endotelial e pressão arterial.

 Avaliação clínica

A avaliação clínica e a coleta de sangue para as dosagens laboratoriais foram realizadas dentro dos três primeiros dias após a alta hospitalar. Foram obtidos dados demográficos, fatores de risco, história médica, exame clínico, com dados antropométricos (peso, estatura e medida da circunferência da cintura), medidas da pressão arterial sistólica e diastólica.27,28

Coleta de sangue para dosagens laboratoriais

O sangue foi obtido em jejum de 12 horas e os exames laboratoriais foram realizados no Laboratório AFIP (Associação Fundo de Apoio à Psicobiologia). Os testes genéticos foram realizados no Laboratório de Biologia Molecular do Setor de Lípides, Aterosclerose e Biologia Vascular.

 Exames bioquímicos

O perfil lipídico (colesterol total, LDL-c, HDL-c, triglicérides) foi analisado por método automatizado (ópera, Bayer, Alemanha), a glicemia por reação colorimétrica (ADVIA 1650 Chemistry System, EUA), e a hemoglobina glicada (HbA1c) por cromatografia líquida de alto desempenho (HPLC, em aparelho Tosoh A1c 2,2 plus, EUA). A média da insulina em jejum nos tempos -15, -5 e 0 minutos, foi analisada por método de quimiluminescência direta (ADVIA Centaur, EUA), com valores expressos em µU/mL. As apolipoproteínas AI, B, Lp(a) foram analisadas por nefelometria (Array 360 da Beckmann), a proteína C-reativa ultra-sensível (PCR-us) pela técnica de imunoensaio por nefelometria (R100 analyzer da Behringer). A adiponectina foi analisada por ELISA (enzyme-linked immunosorbent assay), utilizando kit Human Adiponectin/Acrp30 Immunoassay - Quantiquine da R&D Systems, em leitora de ELISA Biotech ELX 800, com valores expressos em ng/mL.

A dosagem da albuminuria foi analisada em amostras de urina noturna (12h) por imunoturbidimetria (ADVIA 1650, Chemistry System, EUA), com resultados expressos em mg/L.

O estresse oxidativo do plasma foi analisado pela técnica dos TBARS (substâncias reativas ao ácido tiobarbitúrico, do inglês Thiobarbituric acid reactive substances), descrito por Ohkawa et al.,29 com leitura em espectrofotômetro (Genesys 2, Spectronic), e resultados expressos em nanomoles de malondialdeido (MDA) por mililitros de plasma (hmoles/mL plasma).

 Vasorreativbidade

O exame da vasodilatação dependente (FMD, flow-mediated dilation, ou dilatação fluxo-mediada) e independente do endotélio (RVEI, relaxamento vascular independente do endotélio) foi realizado pela manhã, após jejum de 12 horas, por um ultrassonografista experiente, conforme recomendações da Diretriz da Força Tarefa Internacional dos estudos de reatividade da artéria branquial.30 Utilizou-se um sistema de ultrassom (Sonos 5500; Hewlett-Packard-Phillips, Palo Alto, CA), equipado com software vascular para imagens bidimensionais, Doppler colorido e espectral, com monitoração interna do eletrocardiograma e transdutor linear (com frequência variando entre 7,5 e 12,0 MHz). A aquisição de imagens, a medida da FMD e do RVEI com dinitrato de isossorbida (5 mg; sublingual) foram obtidos. As variações percentuais no diâmetro do vaso foram calculadas para determinar a FMD e a RVEI, sendo expressas em %. Os coeficientes de variação intra- e inter-observador foram < 1 e 2%, respectivamente.

 Escore de Gensini

O escore de Gensini foi utilizado para avaliar a gravidade da doença coronariana pela cineangiocoronariografia. O escore de Gensini é calculado levando em consideração a magnitude das lesões e o miocárdio em risco, dando pontuações aos diferentes graus de obstrução e aos diversos segmentos acometidos. As lesões que causam obstrução de 25% recebem o escore de 1, 50%, 2, 75%, 4, 90%, 8, 99%, 16, 100%, 32. Em relação à localização: tronco da coronária esquerda: 5 pontos; artéria descendente anterior proximal: 2,5 pontos; artéria descendente anterior 1/3 médio 1,5 pontos; artéria descendente anterior distal 1 ponto; artéria primeira diagonal 1 ponto; artéria segunda diagonal 0,5 ponto; artéria circunflexa proximal 2,5 pontos; artéria circunflexa distal 1 ponto; primeiro e segundo ramos marginais da artéria circunflexa proximal 1 ponto e distal 0,5 ponto; artéria coronária direita proximal, distal e ramo descendente posterior 1 ponto. O escore de Gensini é o resultado da soma dos escores de cada lesão, multiplicando o grau de estenose pela localização da lesão, sendo expresso em unidades arbitrárias (UA).11,31

Estudo genético

Foram analisados os polimorfismos dos genes paraoxonase-1 (PON-1), metilenotetrahidrofolato redutase (MTHFR), óxido nítrico sintase endotelial (ENOS), enzima conversora da angiotensina (ECA), receptor tipo 1 da angiotensina II (AT1R), apolipoproteína C3 (APOC3), lipoproteína lipase (LPL) foram analisados a partir de amostras de sangue total colhido com EDTA (ácido etilenodiaminotetracético), por técnica da reação em cadeia da polimerase (Polymerase Chain Reaction, PCR), seguida da identificação dos polimorfismos no comprimento do fragmento de restrição (Restriction Fragment Length Polymorphisms, RFLP), nas condições descritas na Tabela 1. O escore genético foi calculado assumindo-se o modelo de dominância dos polimorfismos, considerados como variáveis dicotômicas, binárias.

Condições de amplificação e digestão dos polimorfismos estudados
Polimorfismo Iniciadores Temperatura de anelamento Ciclos Enzima de restrição
PON-1 Forward: 5'-TATTGTTGCTGTGGGACCTGAG-3' 61º C 35 Alw I
Q192R Reverse: 5'-CACGCTAAACCCAAATACATCTC-3'
MTHFR Forward: 5'-GAAGCAGGGAGCTTTGAGG-3' 63º C 32 Hinf I
C677T Reverse: 5'-ACGATGGGGCAAGTGATG-3'
ENOS Forward: 5'-CTGGAGATGAAGGCAGGAGAC-3' 56°C 35 Ban II
G894T Reverse: 5'-CTCCATCCCACCCAGTCAATC-3'
ECA 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'

para os genes ECA e LPL, foi usado o protocolo touchdown PCR.

 Análise estatística

O tamanho amostral foi estimado para comparações de proporções, considerando-se o modelo de dominância dos polimorfismos, um erro a de 5%, um poder b de 80%, um nível de confiança de 95%, e uma proporção esperada de 50%. Assim, o tamanho amostral estimado foi de 90 participantes. Todas as análises foram realizadas utilizando-se o programa SPSS 22.0 para Mac. As variáveis categóricas foram expressas como n (%) e comparadas entre os genótipos pelo teste do qui-quadrado de Pearson ou teste exato de Fisher, conforme apropriado. Para analisar se a distribuição das frequências genotípicas observadas e esperadas estava de acordo com o equilíbrio de Hardy-Weinberg, utilizou-se o teste do qui-quadrado de Pearson. As variáveis numéricas foram expressas como média ± desvio padrão (DP) ou mediana e intervalo interquartil (IQ). O teste de Kolmogorov - Smirnov foi utilizado para avaliar se as variáveis apresentavam distribuição normal. A estatística descritiva utilizou teste t de Student ou o teste de Mann-Whitney para amostras não relacionadas, caso as variáveis não apresentassem distribuição gaussiana. Utilizou-se o coeficiente de correlação de Pearson para avaliar as correlações entre o escore genético e o escore de Gensini. Considerou-se nível de significância para valores de p < 0,05.

 Resultados

Foram avaliados 116 pacientes, cujas características basais são apresentadas na Tabela 2.

Características demográficas e clínicas dos participantes
Variável n = 116
Sexo masculino (%) 74 (68)
Idade (anos) 56 ± 9
Hipertensão (%) 104 (90)
Tabagismo (%) 35 (30)
Diabetes mellitus (%) 36 (31)
IAM (%) 55 (47)
Angina instável (%) 61 (53)
AVC (%) 9 (8)
DVP (%) 12 (10)
IMC (Kg/m2) 30,2 ± 4,7
Circunferência da cintura (cm) 104,4 ± 10,8
Pressão arterial sistólica (mmHg) 132 ± 24
Pressão arterial diastólica (mmHg) 86 ± 16
Frequência cardíaca (bpm) 68 ± 13
Escore de Gensini (ua) 21 (0-36)
Gensini ≥ mediana (%) 42 (51)
FMD (%) 13,7 ± 8,6
RVEI (%) 14,9 (7,3-18,8)

Variáveis categóricas expressas como N (%); variáveis numéricas expressas como média ± desvio padrão, ou mediana e intervalos interquartis. AVC: acidente vascular cerebral; DVP: doença vascular obstrutiva periférica; FMD: dilatação mediada pelo fluxo; IAM: infarto do miocárdio; IMC: índice de massa corpórea; RVEI: relaxamento vascular independente do endotélio; UA: unidades arbitrárias.

A Tabela 3 apresenta a distribuição das frequências alélicas e genotípicas para os polimorfismos estudados. Os genes PON-1, MTHFR e ENOS não se encontravam em equilíbrio de Hardy-Weinberg na população estudada.

Distribuição das frequências alélicas e genotípicas para os polimorfismos dos genes que codificam a PON-1, MTHFR, ENOS, ECA, AT1R, APOC3 e LPL
Gene Frequência alélica Frequência genotípica (%) Valor de p (Hardy Weinberg)
PON-1 Q R QQ QR RR
0,63 0,37 36 (31) 76 (65) 4 (4) 0,0004
MTHFR C T CC CT TT
0,61 0,39 35 (30) 72 (62) 9 (8) 0,0131
ENOS G T GG GT TT
0,43 0,57 10 (9) 80 (69) 26 (22) 0,0006
ECA I D II ID DD
0,33 0,67 17 (15) 42 (36) 57 (49) 0,1955
AT1R A C AA AC CC
0,75 0,25 70 (60) 35 (30) 11 (10) 0,2351
APOC3 S1 S2 S1S1 S1S2 S2S2
0,16 0,84 2 (2) 33 (28) 81 (70) 0,8310
LPL D N DD DN NN
0,34 0,66 17 (15) 45 (39) 54 (46) 0,3095

PON-1: paraoxonase-1; MTHFR: metilenotetrahidrofolato redutase; ENOS: óxido nítrico sintase endotelial; ECA: enzima conversora da angiotensina; AT1R: receptor tipo 1 da angiotensina II; APOC3: apolipoproteína C3; LPL: lipoproteína lipase. p < 0,05, teste do qui-quadrado. Frequências genotípicas esperadas vs. observadas não estavam em equilíbrio de Hardy-Weinberg na população estudada.

 Distribuição das características clínicas, demográficas e laboratoriais

Os resultados foram analisados de acordo com os polimorfismos genéticos estudados. Os resultados detalhados são apresentados na versão eletrônica nas Tabelas Suplementares 1-14 (acesse o link: http://www.arquivosonline.com.br/2018/11001/pdf/Tabelas_suplementares_Port.pdf). Para fins didáticos, os dados são apresentados de acordo com o genótipo, considerando-se o alelo R da PON-1, o T da MTHFR, o T da ENOS, o D da ECA, o C do AT1R, o S1 da APOC3 e o N da LPL, como alelos de risco.

 Sumário dos achados

Para o gene PON-1, valores de PCR-us (mg/dL) foram maiores no genótipo QQ, comparado a QR/RR [12,8 (6,7-24,1) vs. 7,0 (4,8-16,5), p = 0,029] (Tabelas Suplementares 1 e 2) (acesse o link: http://www.arquivosonline.com.br/2018/11001/pdf/Tabelas_suplementares_Port.pdf). Para o gene MTHFR, valores de HbA1-c (%) e adiponectina (ng/mL) foram maiores no genótipo CT/TT comparado ao CC [6,0 (5,5-7,3) vs. 5,7 (5,2-6,6); p = 0,031 e 6996 ± 5032 vs. 4990 ± 3165; p = 0,015] (Tabelas Suplementares 3 e 4). Para o gene da ENOS, a prevalência de diabetes foi maior nos genótipos GT/TT comparada ao GG (34% vs. 0%; p = 0,026); o RVEI (%) tendeu a ser maior no genótipo GG vs. GT/TT [18,2 (15,2-24,0) vs. 14,0 (8,5-20,0); p = 0,050]. Além disso, a glicemia (mg/dL), a HbA1c (%) e os níveis de adiponectina (ng/mL) foram maiores nos genótipos GT/TT vs. GG [(127 ± 48 vs. 106 ± 12, p = 0,001; 6,6 ± 1,9 vs. 5,9 ± 0,6, p = 0,028; 5010 (2688-10139) vs. 2148 (1912-3435), p = 0,011] (Tabelas Suplementares 5 e 6) (acesse o link: http://www.arquivosonline.com.br/2018/11001/pdf/Tabelas_suplementares_Port.pdf). Para o gene ECA, a frequência cardíaca (bpm) foi maior entre os ID/DD comparada aos II (71 ± 13 vs. 65 ± 11, p = 0,042) (Tabelas Suplementares 7 e8) (acesse o link: http://www.arquivosonline.com.br/2018/11001/pdf/Tabelas_suplementares_Port.pdf). Os polimorfismos do gene AT1R não influenciaram variáveis clínicas ou laboratoriais (Tabelas Suplementares 9 e 10) (acesse o link: http://www.arquivosonline.com.br/2018/11001/pdf/Tabelas_suplementares_Port.pdf).

Para o gene APOC3, a FMD (%) foi maior entre os portadores do genótipo S2S2 comparado aos S1S1/S1S2 (14,7 ± 9,6 vs. 11,5 ± 5,2, p = 0,026) (Tabelas Suplementares 11 e 12) (acesse o link: http://www.arquivosonline.com.br/2018/11001/pdf/Tabelas_suplementares_Port.pdf) e para o gene LPL, a pressão arterial diastólica (mmHg) foi menor no genótipo DD comparado a DN/NN (79 ± 15 vs. 87 ± 16, p = 0,043). Observou-se ainda maior grau de aterosclerose coronária, avaliada pelo escore de Gensini > mediana (%) entre os portadores do genótipo DD comparado a DN/NN (77% vs. 46%, p = 0,039) (Tabelas Suplementares 13 e 14) (acesse o link: http://www.arquivosonline.com.br/2018/11001/pdf/Tabelas_suplementares_Port.pdf).

 Associações entre os genótipos estudados e a extensão e gravidade da DAC

Assumiu-se o modelo de dominância e uma pontuação de 1 a 3 para os genótipos isoladamente, com valores de 1 para a ausência do alelo de risco, 2, para a presença de um alelo de risco e 3, para a presença de 2 alelos de risco do mesmo gene. Assim, foi atribuído o valor 1 aos genótipos QQ da PON-1, CC da MTHFR, GG da ENOS, II da ECA, AA do AT1R, S2S2 da APOC3 e DD da LPL. Foi atribuído valor 2 para os genótipos QR da PON-1, CT da MTHFR, GT da ENOS, ID da ECA, AC do AT1R, S1S2 da APOC3 e DN da LPL. Foi atribuído valor 3 para os genótipos RR da PON-1, TT da MTHFR, TT da ENOS, DD da ECA, CC do AT1R, S1S1 da APOC3 e NN da LPL.

A soma dos valores atribuídos a cada gene (valores de 7 a 21) resultou em um escore genético. Esse escore foi avaliado em valores absolutos, e também em relação à mediana (acima ou abaixo). Foram feitas correlações entre os escores genético e de Gensini, quer de maneira absoluta ou categorizados pela mediana. Os resultados são apresentados na Tabela 4 e na Tabela Suplementar 15 (acesse o link: http://www.arquivosonline.com.br/2018/11001/pdf/Tabelas_suplementares_Port.pdf). Tanto o escore genético como o escore de Gensini apresentaram distribuição normal.

Distribuição do escore genético utilizando todos os polimorfismos dos genes da PON-1, MTHFR, ENOS, ECA, AT1R, APOC3 e LPL
Escore genético
N 116
Média 13,3
Mediana 13,5
Desvio padrão 1,58
Mínimo 10
Máximo 17
Assimetria -0,263
Curtose -0,146
P25 12,0
P75 14,0

PON-1: paraoxonase-1; MTHFR: metilenotetrahidrofolato redutase; ENOS: óxido nítrico sintase endotelial; ECA: enzima conversora da angiotensina; AT1R: receptor tipo 1 da angiotensina II; APOC3: apolipoproteína C3; LPL: lipoproteína lípase.

O escore genético teve tendência a ser maior nos pacientes com escore de Gensini < P50 (13,7 ± 1,5 vs. 13,0 ± 1,6, p = 0,066, teste t de Student para amostras independentes).

O escore de Gensini não diferiu entre os escores genéticos acima ou abaixo da mediana (P50) [26 (0,00-44,00) vs. 18 (0,25-33,70), p = 0,329]. Nessa população de pacientes com SCA recente e SM, houve fraca correlação inversa entre o escore genético e o de Gensini (R = -0,194, p = 0,078, coeficiente de correlação de Pearson).

 Discussão

O estudo demonstrou que os polimorfismos genéticos examinados, em pacientes com SM e recente SCA tiveram associação discreta com a gravidade da doença coronariana obstrutiva. Apenas o genótipo DD do polimorfismo D9N da LPL se associou, isoladamente, com maior prevalência de lesões coronarianas de maior gravidade. Quando analisamos o escore genético, a combinação dos polimorfismos estudados apresentou tendência de correlação negativa com a extensão anatômica da doença coronária, sugerindo que nesses pacientes com SM, a doença coronariana possa ser primariamente relacionada a outros mecanismos, com forte influência ambiental.

Vários dos polimorfismos estudados estiveram associados com variáveis clínicas ou laboratoriais, como frequência cardíaca, pressão arterial diastólica, PCR, HbA1c e adiponectina.

Estudo envolvendo seis polimorfismos da LPL, incluindo o D9N, demonstrou que a gravidade das lesões obstrutivas, avaliada pelo escore de Gensini, foi associada com haplótipos da LPL.32 Corsetti et al.33 demonstraram interação entre o polimorfismo D9N e o Taq1B da CETP, sendo preditor de subgrupo de mulheres de maior risco. Além disso, polimorfismos da LPL têm se associado a aumento nas concentrações de triglicérides;34 em nosso estudo, houve tendência para valores mais elevados para o genótipo DD vs. DN/NN (p = 0,07), possivelmente devido à alta prevalência de sobrepeso/obesidade, além de alterações no metabolismo glicêmico nesta população de pacientes com SM.

O polimorfismo da APOC3 tem papel relevante no metabolismo de lipoproteínas ricas em triglicérides, influenciando o desenvolvimento de DAC, sobretudo na SM e diabetes, havendo associação dos haplótipos no cluster gênico AI-C3-AIV com doença coronária.35

Os polimorfismos relacionados com o sistema renina angiotensina (I/D da ECA e AT1R) não se associaram com a gravidade da DAC em nosso estudo. No entanto, o gene ECA associou-se a maiores concentrações da ECA entre coronarianos nos indivíduos DD em um estudo prévio,36 o que não se confirmou com casuística maior.

Em nosso estudo, o alelo 192R da PON-1 associou-se a maiores níveis de PCR, biomarcador de risco de desfechos cardiovasculares. Em amplo estudo (n = 3668 indivíduos) com seguimento de três anos, apenas a atividade arilesterase, mas não os níveis de paraoxonase ou o polimorfismo da PON-1, apresentou relação com desfechos cardiovasculares.37

Para o polimorfismo C677T da MTHFR, metanálise (n = 6912) mostrou associação com DAC prematura,38 sendo os níveis de homocisteína maiores em presença do alelo T.39 Em nosso estudo, os indivíduos CT/TT apresentaram HbA1c e adiponectina maiores em relação aos portadores do genótipo CC.

Polimorfismos da ENOS são implicados na disfunção endotelial. O alelo T do polimorfismo G894T mostrou menores níveis de óxido nítrico e associação com DAC.40 Já a variante -786T (na região promotora), foi associada à DAC em metanálise.41

O escore genético se associou inversamente à maior extensão e gravidade da DAC. Sugere-se que, nesta população com SM, os fatores ambientais tenham um impacto na modulação da expressão de genes relacionados ao metabolismo lipídico, oxidação de lipoproteínas, função endotelial e níveis de pressão arterial, influenciando o processo de aterogênese.

 Conclusão

Os polimorfismos estudados tiveram pequena contribuição para a extensão da DAC. Apenas o polimorfismo D9N da LPL associou-se à extensão da DAC em pacientes com SM e recente SCA. Análise combinada de polimorfismos genéticos apresentou fraca associação com a extensão da doença coronariana, sendo o escore genético inversamente associado à extensão e gravidade da DAC.

Limitações do estudo

O tamanho amostral do estudo e seu desenho transversal podem ser limitações do nosso estudo. No entanto, o estudo teve objetivo exploratório de identificar polimorfismos genéticos que possam contribuir na identificação de maior gravidade da doença aterosclerótica nesta população de portadores de SM e recente SCA. Seus resultados contribuem para a escolha de polimorfismos genéticos a serem testados em estudos prospectivos envolvendo maior casuística.

Fontes de financiamento

O presente estudo foi financiado pela FAPESP, processo nº 2004/00325-8.

Vinculação acadêmica

Este artigo é parte de dissertação de Mestrado de Simone Cristina Pinto Matheus Fischer pela Universidade Federal de São Paulo.

Aprovação Ética e consentimento informado

Este estudo foi aprovado pelo Comitê de Ética da Universidade Federal de São Paulo (CEP UNIFESP) sob o número de protocolo CEP 0283/11. Todos os procedimentos envolvidos nesse estudo estão de acordo com a Declaração de Helsinki de 1975, atualizada em 2013. O consentimento informado foi obtido de todos os participantes incluídos no estudo.