SciELO - Scientific Electronic Library Online

 
vol.105 issue1Metabolic Syndrome and Importance of Associated Variables in Children and Adolescents in Guabiruba - SC, BrazilOne-year Mortality after an Acute Coronary Event and its Clinical Predictors: The ERICO Study author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Services on Demand

Journal

Article

  • text in Portuguese
  • text new page (beta)
  • English (pdf) | Portuguese (pdf)
  • Article in xml format
  • Article references
  • How to cite this article
  • SciELO Analytics
  • Curriculum ScienTI
  • Automatic translation

Indicators

Related links

Share


Arquivos Brasileiros de Cardiologia

Print version ISSN 0066-782XOn-line version ISSN 1678-4170

Arq. Bras. Cardiol. vol.105 no.1 São Paulo July 2015  Epub May 29, 2015

http://dx.doi.org/10.5935/abc.20150053 

Original Articles

p.Q192R SNP of PON1 seems not to be Associated with Carotid Atherosclerosis Risk Factors in an Asymptomatic and Normolipidemic Brazilian Population Sample

Daniel Zanetti Scherrer1 

Vanessa Helena de Souza Zago1 

Isabela Calanca Vieira1 

Eliane Soler Parra1 

Natália Baratella Panzoldo1 

Fernanda Alexandre1 

Rodrigo Secolin2 

Jamal Baracat3 

Eder Carlos Rocha Quintão4 

Eliana Cotta de Faria1 

1Universidade Estadual de Campinas, Faculdade de Ciências Médicas - Departamento de Patologia Clínica, São Paulo, SP - Brazil

2Universidade Estadual de Campinas, Faculdade de Ciências Médicas - Departamento de Genética Médica, São Paulo, SP - Brazil

3Universidade Estadual de Campinas, Faculdade de Ciências Médicas - Departamento de Radiologia, São Paulo, SP - Brazil

4Universidade de São Paulo, Faculdade de Ciências Médicas - Laboratório de Lípides (LIM10), São Paulo, SP - Brazil

ABSTRACT

Background:

Evidences suggest that paraoxonase 1 (PON1) confers important antioxidant and anti-inflammatory properties when associated with high-density lipoprotein (HDL).

Objective:

To investigate the relationships between p.Q192R SNP ofPON1, biochemical parameters and carotid atherosclerosis in an asymptomatic, normolipidemic Brazilian population sample.

Methods:

We studied 584 volunteers (females n = 326, males n = 258; 19-75 years of age). Total genomic DNA was extracted and SNP was detected in the TaqMan® SNP OpenArray® genotyping platform (Applied Biosystems, Foster City, CA). Plasma lipoproteins and apolipoproteins were determined and PON1 activity was measured using paraoxon as a substrate. High-resolution β-mode ultrasonography was used to measure cIMT and the presence of carotid atherosclerotic plaques in a subgroup of individuals (n = 317).

Results:

The presence of p.192Q was associated with a significant increase in PON1 activity (RR = 12.30 (11.38); RQ = 46.96 (22.35); QQ = 85.35 (24.83) μmol/min; p < 0.0001), HDL-C (RR= 45 (37); RQ = 62 (39); QQ = 69 (29) mg/dL; p < 0.001) and apo A-I (RR = 140.76 ± 36.39; RQ = 147.62 ± 36.92; QQ = 147.49 ± 36.65 mg/dL; p = 0.019). Stepwise regression analysis revealed that heterozygous and p.192Q carriers influenced by 58% PON1 activity towards paraoxon. The univariate linear regression analysis demonstrated that p.Q192R SNP was not associated with mean cIMT; as a result, in the multiple regression analysis, no variables were selected with 5% significance. In logistic regression analysis, the studied parameters were not associated with the presence of carotid plaques.

Conclusion:

In low-risk individuals, the presence of the p.192Q variant ofPON1 is associated with a beneficial plasma lipid profile but not with carotid atherosclerosis.

Key words: Paraoxonase-1 / genetics; Polymorphism, genetic; Carotid Artery Diseases / physiopathology; Lipoproteins, HDL

Introduction

Human paraoxonase 1 (PON1 -OMIM 168820) is a calcium-dependent serum esterase composed of 354 amino acids, with a molecular weight of 43kDa, and coded in chromosome 7q21. It is expressed in a variety of tissues, but synthesized mainly in the liver. PON1 binds to high-density lipoprotein (HDL) through interaction of hydrophobic N-terminus with phospholipids and with apolipoprotein A-1 (apo A-1)1 leading to HDL important antioxidant and anti-inflammatory properties2.

Previous studies suggest that PON1 activity could influence HDL-C levels, probably because of the involvement of PON1 in the protection against HDL oxidation3. Furthermore, PON1 is present in small and dense HDL particles4, indicating that there is a relationship between PON1 activity and HDL size5.

The relationship between PON1 activity and coronary heart disease (CHD) risk in humans has been reported among various ethnic populations; thus, two meta-analyses confirm the association of a lower PON1 activity with increased CHD risk regardless of age and ethnicity6 , 7.

PON1 activity is under genetic and environmental regulation and varies widely among individuals and populations8. The SNP rs662, found in the coding region, leads to a substitution of glutamine (CAA) for arginine (CGA) at position 192 (p.Q192R)9.In addition to its effects on the enzymatic activity, several studies have been carried out to elucidate the relationship of this SNP with established cardiovascular disease (CVD) in different populations. The p.192R variant frequency was increased in CVD groups of Japanese, Caucasian and Asian-Indian populations10 - 12; however, the same associations were not found in Turkish and Finnish populations13 - 14.

In the Brazilian population, the p.Q192R SNP distribution varies among ethnic groups15; additionally, the impact of the p.192R variant on established CVD is conflicting16 - 18. Moreover, there are no studies in our population relating SNP to CVD risk in asymptomatic individuals; indeed, studies associating the polymorphism with subclinical carotid disease in healthy individuals are rare19.

In view of the results obtained in studies in different ethnic groups, and the scarcity of studies in low-risk populations, we investigated the relationships of p.Q192R SNP of PON1 with biochemical parameters and carotid atherosclerosis in an asymptomatic, normolipidemic Brazilian population.

Methods

Study population

This study comprised 584 individuals selected among 1536 normolipidemic and asymptomatic volunteers of both genders, aged 19 to 75 years, who had undergone free health checkups in primary health care centers between 2008 and 2012 in Campinas and Americana (São Paulo, Brazil), as previous described by Parra et al.20. At admission, the individuals underwent a complete clinical evaluation and answered a detailed questionnaire to provide data on family history of premature coronary artery disease (CAD), (defined as the occurrence of acute events and/or death in first-degree relatives), past and current health status, exercise and diet habits, alcohol and tobacco use, and medications. The exercise habits were evaluated through a questionnaire adapted from Baecke et al.21, consisting of 16 questions that include three indexes of habitual physical activities in the last 12 months, defined as (i) occupational or labor physical activity index; (ii) free time physical exercise index; and (iii) free time and locomotion activity index. Furthermore, diet habits were evaluated by a dietary frequency questionnaire (classified as daily, weekly and monthly/no consumption) adapted from Furlan-Viebig and Pastor-Valero22 of ten food classes as follows: (i) milk and dairy products; (ii) meat, fish and eggs; (iii) vegetables; (iv) fruits; (v) natural juices; (vi) breads, cereals and tubers; (vii) oils and fats; (viii) sweets, snacks and treats; (ix) non‑alcoholic beverages and (x) preparations and miscellaneous.

Exclusion criteria included regular use of medications, especially those which interfere with the lipid metabolism, such as statins, hormonal replacement therapy and contraceptives. The medications allowed in the protocol included: (1) vitamins, nutritional supplements and homeopathy; (2) antidepressants; (3) analgesics (sporadic use); (4) antiallergics and proton pump inhibitors; and (5) two or more of the mentioned medications combined. Additionally, we excluded volunteers with thyroid dysfunction; dyslipidemia; diabetes mellitus; metabolic syndrome; obesity; pregnancy; arterial hypertension; liver, lung or kidney diseases; alcohol abuse (> 14g/day); and smoking.

Venous blood samples were drawn after a 12h fasting period for serum and EDTA plasma separation by centrifugation (4°C, 1000 x g, 10 minutes), and stored at -80°C until analysis. In another visit, the volunteers underwent carotid ultrasonography.

The Medical Sciences Ethics Committee of the University of Campinas approved this study and all volunteers gave written informed consent.

Biochemical analysis

Fresh serum samples were analyzed for total cholesterol, HDL-C, triglycerides and glucose in Modular Analytics® EVO (Roche Diagnostics, Burgess Hill, West Sussex, UK) using Roche Diagnostics® reagents (Mannheim, Germany). Apolipoproteins A-1 and B were determined by nephelometry in BN II (Siemens Healthcare Diagnostics, Marburg, Germany) automated system using commercially available assays (Dade-Boehringer®, Deerfield, Illinois, USA). Low-density lipoprotein cholesterol (LDL-C) was calculated using the Friedwald formula23, while very-low density lipoprotein cholesterol (VLDL-C) was calculated using the formula triglycerides/521.

C-reactive protein (CRP) was measured by immunoturbidimetry in a high-sensitivity method (hsCRP), using the commercially available assay Tina-quant CRP (Latex) HS-Roche (Roche Diagnostics®, Mannheim, Germany).

HDL particle size was determined by the dynamic light scattering (DLS) technique after chemical precipitation of apo B-containing lipoproteins in plasma samples using polyethylene glycol (PEG) 8000 (Sigma-Aldrich, St. Louis, USA)24. The measurements were performed in triplicate in the Nanotrac Particle Size Analyzer (Microtrac, North Largo, Florida, USA) and a 100nm polymeric nanoparticle was used as the standard; a control sample obtained from the same individual plasma was used in all determinations.

Plasma activity of cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) was determined by radiometric assays using exogenous substrates25 , 26.Additionaly, PON1 activity was measured using paraoxon (diethyl-p-nitrophenylphosphate, Sigma, St. Louis, MO, USA) as substrate27.

SNP genotyping

Total genomic DNA was extracted from peripheral mononuclear blood cells, according to the standard phenol/chloroform method. Quality, integrity and concentration were determined and DNA samples were stored at −20 °C prior to use.

Genotype analysis for p.Q192R SNP of PON1 was performed using the OpenArray®Real-Time PCR Platform (Applied Biosystems, Foster City, CA), following the manufacturer’s standard protocol. The genotypes were determined using the TaqMan®Genotyper Software 1.0.1. (Applied Biosystems, Foster City, CA, USA). Individuals with the GG genotype were designated as RR (p.192R homozygous), while those with the AA genotype were named QQ (p.192Q homozygous) and those with the GA genotype (heterozygous) were designated as RQ.

Carotid intima-media thickness (cIMT) measurements

All volunteers were invited to undergo carotid intima‑media thickness measurements and a subgroup of 317 individuals underwent the test. Carotid ultrasonography was performed by a single trained sonographer, blind to the study subjects. High-resolution B-mode ultrasonography was carried out using a 6-9 MHz linear array ultrasound imaging system (ATL HDI 1500 and 3500 Ultrasound System, Advanced Technology Laboratories Ultrasound, Bothell, EUA) and longitudinal measurements were made of segments of the common carotid arteries at the distal wall and at 1 cm from the bifurcation. Mean cIMT was calculated based on the average of five measurements of the right and left sides and expressed in millimeters (mm); the presence of carotid plaques was also investigated.

Statistical analyses

Statistical analyses were performed using the SPSS 16.0 (SPSS Inc., USA) and R software (R Development Core Team, Vienna, Austria). Normal distribution of the variables was determined by the Kolmogorov-Smirnov test. Normal data are expressed as mean ± standard deviation, and non-normal data are expressed as median (interquartile range).

Comparisons between genotypes were performed using ANOVA or Kruskal-Wallis, followed by the Bonferroni’s test for normal and non-normal data, respectively. Additionally, chi-square test was performed for categorical variables. When necessary, the comparisons were adjusted by ANCOVA for age, gender and body mass index (BMI).

Stepwise regression analyses were performed to verify the main determinants of PON1 activity, and included the variables age, gender, BMI, HDL particle size, CETP, PLTP and p.Q192R SNP of PON1 . The RR genotype was used as a reference group.

Univariate and multiple linear regression analyses were performed to evaluate the influence of HDL-C, apo A-1, HDL particle size, hsCRP, PON1 activity and p.Q192R SNP on mean cIMT; the analyses were controlled for age, gender and BMI. In addition, logistic regression analysis determined the odds ratio (OR) for the presence of carotid plaques according to HDL-C, apo A-1, HDL particle size, hsCRP, PON1 activity and p.Q192R SNP, also controlled for age, gender and BMI. The G* Power software was used in order to evaluate whether the sample had statistical power to detect associations, according to the following parameters: OR = 1.5; significance level α = 0.008 (adjusted for multiple comparisons); two-tailed; statistical power 1-β = 80%. In this way, the sample showed 99.9% and 54.98% of statistical power for multiple linear regression and logistic regression analysis, respectively.

P-values < 0.05 (corrected by Bonferroni for multiple tests) were considered statistically significant.

Results

The study population was comprised of 56% females and 44% males, and genotype frequency was 37%, 45% and 19% for RR, RQ and QQ groups, respectively. The p.Q192R SNP was in Hardy–Weinberg equilibrium. Furthermore, all volunteers were asymptomatic; non-smokers; had no past or current history of thyroid dysfunction, dyslipidemias, diabetes mellitus or other endocrine diseases; no pregnancy, obesity, metabolic syndrome, arterial hypertension; no liver, lung or kidney diseases.

Clinical and biochemical characteristics were described and compared between genotypes, as shown in table 1. QQ volunteers presented a significant increase in PON1 activity, total cholesterol, HDL-C and apo A-1 when compared to RQ or RR groups. No significant differences were observed regarding all the other biochemical parameters, mean cIMT, presence of plaques, family history of premature CAD, alcohol consumption, medication use and physical activity indexes. Additionally, the frequencies (daily, weekly and monthly/no consumption) of the ten studied food classes were not significantly different between the genotypes, as follows: milk and dairy products (p = 0.716); meat, fish and eggs (p = 0.269); vegetables (p = 0.766); fruits (p = 0.198); natural juices (0.412); breads, cereals and tubers (0.136); oils and fats (p = 0.435); sweets, snacks and treats (p = 0.545); non-alcoholic beverages (p = 0.454) and preparations and miscellaneous (p = 0.874).

Table 1  Descriptive and comparative analyses of clinical, anthropometrlcal and biochemical parameters among PON1 p.Q192R genotypes 

Parameters RR (n = 215) RQ (n = 260) QQ (n = 109) p
Gender (Female/Male) 112/103 153/107 61/48 -
Age (years) 45 (23) 44 (27) 46 (17) -
BMI (kg/m2) 23.6 ± 2.8 23.4 ± 2.9 24.5 ± 2.9 -
Waist circumference (cm) 78 (15) 76 (13) 74 (10) 0.413
SBP (mmHg) 120 (15) 120 (20) 120 (20) 0.334
DBP (mmHg) 80 (10) 80 (14) 80 (8) 0.260
Total cholesterol (mg/dL) 170 ± 32 174 ± 29 176 ± 30 0.044
HDL-C (mg/dL) 45 (37) 62 (39) 69 (29) < 0.001
HDL size (nm) 7.85 (0.91) 8.00 (1.02) 8.06 (0.88) 0.467
Triglycerides (mg/dL) 77 (43) 74 (45) 64 (33) 0.054
LDL-C (mg/dL) 100 ± 26 101 ± 23 101 ± 22 0.550
VLDL-C (mg/dL) 15 (8) 15 (9) 13 (7) 0.069
Apo A-I (mg/dL) 140.76 ± 36.39 147.62 ± 36.92 147.49 ± 36.65 0.019
Apo B (mg/dL) 76.37 ± 19.64 76.80 ± 17.11 75.54 ± 18.72 0.472
CETP(%) 13.55 ± 5.45 13.38 ± 5.92 13.20 ± 5.24 0.616
PLTP (nmolesFC/mL/h) 6321 (3074) 6086 (2253) 6088 (2393) 0.414
PON1 (Mmol/min) 12.30 (5.90) 46.95 (22.35) 85.35 (21.18) < 0.001*
Glucose (mg/dL) 84 (12) 83 (10) 84 (9) 0.499
hsCRP (mg/L) 0.80 (1.00) 0.80 (1.13) 0.90 (1.65) 0.231
Mean cIMT (mm) 0.60 (0.20) 0.60 (0.30) 0.58 (0.15) 0.207
Carotid Plaques (Yes/No, %) 19/81 18/82 11/89 0.712
Family history of premature CAD (Yes/No, %) 7/30 6/39 6/15 0.659
Alcohol consumption (Moderate/No consumption, %) 7/31 9/36 3/14 0.503
Medications (Yes/No, %) 5/31 8/37 3/16 0.903
Occupational or labor physical activity index 2.75 (1.00) 2.75 (0.87) 2.87 (0.62) 0.493
Free time physical exercise index 2.00 (1.00) 2.00 (1.25) 2.00 (1.50) 0.913
Free time and locomotion activity index 2.50 (1.00) 2.50 (1.00) 2.50 (1.00) 0.998

(n): number of individuals or as shown below; BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; VLDL-C: very low-density lipoprotein cholesterol; Apo: apoliprotein; hsCRP: high-sensitivity C reactive protein; CETP: cholesteryl ester transfer protein; PLTP: phospholipid transfer protein; FC: free cholesterol; PON1: paraoxonase 1; CAD: coronary artery disease; cIMT: carotid intima-media thickness: RR (119), RQ (132),QQ (66). Normal and non-normal data are presented as mean ± standard deviation and median (interquartile range), respectively P values - ANOVA or Kruskal-Wallis, adjusted with ANCOVA for age, gender and BMI; significant differences (p ≤ 0.05) by Bonferroni's were indicated by

*RR≠RQ;

RR≠QQ;

QQ≠RQ.

Plasma PON1 activity was influenced only by QQ (R2 = 39%) and RQ (R2 = 19%) genotypes, as demonstrated in table 2; thus, other non-studied factors have an influence of 42%.

Table 2  Stepwise regression analysis for PON1 activity in the studied population 

Parameters R2 Cumulative R2 F p
QQ 0.3876 0.3876 193.7 < 0.0001
RQ 0.1918 0.5794 210.1 < 0.0001
Non-studied factors 0.4206      

R2: coefficient of determination. Variables analyzed following the stepwise criteria are age, gender (female x male), body mass index, total cholesterol, HDL-C, HDL particle size, CETP, PLTP and p.Q192R SNP. The RR genotype was used as reference group.

Univariate linear regression analysis demonstrated that the studied parameters, including p.Q192R SNP, were not associated with mean cIMT, as demonstrated in table 3. As a result, in the multiple regression analysis no variables were selected with 5% significance. In addition, for the logistic regression analysis the studied parameters were not associated with the presence of plaques, as demonstrated in table 4.

Table 3  Univariate and multiple linear regression analysis for cIMT In the studied population 

  Univariate linear regression (n = 310)
Parameters Estimate SE p R2
p.Q192R (RR x QQ) 0.55 8.86 0.951 0.001
p.Q192R (QQ x RQ) -17.26 10.57 0.104 0.005
HDL-C 0.04 0.21 0.845 < 0.0001
Apo A-I 0.08 0.12 0.482 < 0.0001
HDL particle size 0.92 6.96 0.895 < 0.0001
hsCRP 0.52 0.87 0.554 < 0.0001
PON1 activity -0.08 0.14 0.548 < 0.0001
Multiple linear regression analysis (n = 310)        
Independent variable Dependent Variable Estimate p R2
Mean cIMT - - - -

The variables were transformed into ranks due the absence of Normal distribution and the analyses were controlled for age, gender and BMI. SE: standard error; R2: coefficient of determination; HDL-C: high-density lipoprotein cholesterol; apo: apolipoprotein; hsCRP: high sensitivity C reactive protein; PON1: paraoxonase 1.

Table 4  Logistic regression analysis for the presence of carotid atherosclerotic plaques in the studied population 

  Univariate logistic regression (n= 308)
Parameters Estimate SE p OR 95%CI
p.Q192R (RR x QQ) 0.270 0.471 0.567 1.310 0.520; 3.303
p.Q192R (QQ x RQ) 0.266 0.464 0.566 1.305 0.526; 3.241
HDL-C -0.002 0.009 0.777 0.997 0.980; 1.015
Apo A-I 0.003 0.004 0.486 1.003 0.994; 1.012
HDL particle size -0.370 0.299 0.216 0.691 0.384; 1.241
hsCRP -0.032 0.040 0.418 0.968 0.895; 1.047
PON1 activity -0.005 0.005 0.334 0.994 0.983; 1.006
Multiple logistic analyses (n = 308)         95%CI
Independent variable Dependent variable Estimate p OR CI95%
Carotid plaques - - - - -

The variables were transformed into ranks due the absence of Normal distribution and the analyses were controlled for age, gender and BMI. SE: standard error; OR: odds ratio; CI: confidence interval; HDL-C: high-density lipoprotein cholesterol; apo: apolipoprotein; hsCRP: high sensitivity C reactive protein; PON1: paraoxonase 1.

Discussion

Studies conducted in different ethnic groups have reported diverse distribution of the p.192Q and p.192R variants. The p.192Q variant has been described as being more frequent in Caucasians28, and p.192R, in Japanese, Chinese and Hispanic populations29 - 31. In addition, the frequencies have been previously explored in a Brazilian population sample, in which significant differences between genotypes regarding European descendants and Afro-Brazilians were demonstrated15. In our study, the p.192R variant represents 59% of the total sample, and p.192Q, 41%, similar to the frequencies observed in the Afro-Brazilian population15.

Several SNPs in different regions of PON1 have been identified, including the amino acid 192, which is one of the most frequently studied in order to identify the influence on enzyme activity and cardiovascular disease32. In the present study, we demonstrated that the p.192Q variant significantly modulates PON1 activity and the levels of apo A-1, cholesterol and HDL-C.

Genetic variations have a major impact on PON1 catalytic activity; indeed, amino acid 192 is an important active-site of the enzyme and constitutes part of HDL surface anchoring33. Studies regarding the impact of p.Q192R on the hydrolytic activity demonstrated that the p.192R variant shows higher activity towards paraoxon and phenylacetate, compared to p.192Q8. Despite that, in this study, p.192Q was associated with increased activity towards paraoxon and the stepwise regression analysis suggests that 58% of PON1 activity was determined by this variant.

There has been evidence associating higher PON1 activity with increased HDL-C levels3. Indeed, Blatter Garin et al.34 observed positive correlations between PON1 activity, HDL-C and apo A-1 levels in a population with confirmed coronary artery disease as well as in control individuals. However, the p.Q192R SNP of PON1 was not associated with apo A-1 and HDL-C levels in any of the groups. On the other hand, our study demonstrated that the p.192Q variant is a significant predictor of increased apo A-1 and HDL-C levels in normolipidemic and asymptomatic individuals.

Low plasma PON1 activity has been positively associated with increased CVD risk regardless of age and ethnicity, as reported by numerous studies, including meta-analyses6 , 7. In this context, multiple clinical reports focused on the role of PON1 polymorphisms that directly affect PON1 activity on CVD35 - 37. In a meta-analysis including 88 studies, Wang et al.35 demonstrated that subjects with the p.192R variant presented a higher CVD risk than those with the p.192Q variant ofPON1 ; however, other studies still show controversies regarding the role of these alleles35. Indeed, there was one strong association correlating the p.192R variant with ischemic stroke, but the risk was confined to Caucasians36; furthermore, Birjmohun et al.37 demonstrated that p.Q192R SNP of PON1 did not predict the CVD risk in individuals from the EPIC-Norfolk cohort study37.

The impact of p.Q192R SNP was also explored in Brazilian population samples. Rios et al.16 studied both p.Q192R and p.M55L SNPs of PON1 in 712 patients, of whom 437 were Caucasians and 275 Afro-Brazilians with established CAD (obstructive lesions higher than 50%). The p.Q192R SNP was not associated with CAD; however, only in Caucasian men did the RR genotype lead to a decrease in HDL-C levels. Oliveira et al.17 also determined the influence of p.Q192R and p.M55L SNPs on 352 patients with angiographically defined CAD and 380 matched controls for age and gender at a high risk for CAD. Similarly, no significant associations were found between the p.Q192R SNP and CAD. In addition, Voetsh et al.18 evaluated the association of p.Q192R and p.M55L SNPs with the risk of arterial ischemic stroke in 118 patients (with a first nonfatal stroke occurring before 45 years of age) and 118 age- and sex-matched controls. The RR genotype was more frequent in patients with ischemic stroke and was independently associated with the disease risk. In this context, the Brazilian population presents controversial relationships regarding the p.Q192R SNP and cardiovascular disease.

In addition to the reports involving the role of p.Q192R SNP of PON1on cardiovascular disease, previous studies conducted in disease states (mainly diabetes and/or hypercholesterolemia) showed inconsistent results regarding the associations between p.Q192R SNP and cIMT38 - 40, a surrogate marker of coronary atherosclerosis41.

Regarding the association of cIMT with p.Q192R in healthy individuals Dessi et al.19 observed an increase, although not significant, of cIMT in the p.192R variant among genotypes. Additionally, Karvonen et al.42 did not observe significant differences regarding cIMT in a large cohort of hypertensive and control individuals. Similarly, there is a lack of associations of p.Q192R with cIMT and carotid atherosclerotic plaques in our study, strongly suggesting that this SNP, in low-risk individuals, is not a major parameter associated with carotid atherosclerosis.

The results should be carefully interpreted since there are limitations that must be addressed, such as the relatively small sample size given the study design, especially its rigorous inclusion and exclusion criteria. In addition, the number of volunteers who underwent the carotid IMT exam was not enough to reach the minimum statistical power (80%) for the logistic regression analysis, since the predicted sample size to meet this condition was 480 individuals. Furthermore, the low compliance to a second visit for cIMT measurements limited our conclusions and the regression analysis could not provide evidences of a causal relationship betweenPON1 polymorphism and carotid plaques.

Conclusion

Despite the absence of associations of p.Q192R SNP with cIMT and carotid plaques, there is evidence in our study that the p.192Q variant exerts important beneficial influences on the plasma lipid profile. However, larger studies are needed to further establish the association of p.Q192R SNP with CVD in low cardiovascular risk individuals.

Sources of Funding

This study was funded by CNPq (processo 159980/2012-7) and FAPESP (processo 471380/2008-13).

Study Association

This study is not associated with any thesis or dissertation work.

Acknowledgments

This work was supported by the National Council for Scientific and Technological Development (CNPq), grant number 159980/2012-7, and by the State of São Paulo Research Foundation (Fapesp), grant number 471380/2008-13.

References

Sorenson RC, Bisgaier CL, Aviram M, Hsu C, Billecke S, La Du BN. Human serum paraoxonase/arylesterase's retained hydrophobic N-terminal leader sequence associates with HDLs by binding phospholipids: apolipoprotein A-I stabilizes activity. Arterioscler Thromb Vasc Biol. 1999;19(9):2214-25. [ Links ]

Aviram M, Kaplan M, Rosenblat M, Fuhrman B. Dietary antioxidants and paraoxonases against LDL oxidation and atherosclerosis development. Handb Exp Pharmacol. 2005;(170):263-300. [ Links ]

van Himbergen TM, Roest M, de Graaf J, Jansen EH, Hattori H, Kastelein JJ, et al. Indications that paraoxonase-1 contributes to plasma high density lipoprotein levels in familial hypercholesterolemia. J Lipid Res. 2005;46(3):445-51. [ Links ]

Kontush A, Chantepie S, Chapman MJ. Small, dense HDL particles exert potent protection of atherogenic LDL against oxidative stress. Arterioscler Thromb Vasc Biol. 2003;23(10):1881-8. [ Links ]

Razavi AE, Ani M, Pourfarzam M, Naderi GA. Associations between high density lipoprotein mean particle size and serum paraoxonase-1 activity. J Res Med Sci. 2012;17(11):1020-6. [ Links ]

Wang M, Lang X, Cui S, Zou L, Cao J, Wang S, et al. Quantitative assessment of the influence of paraoxonase 1 activity and coronary heart disease risk. DNA Cell Biol. 2012;31(6):975-82. [ Links ]

Zhao Y, Ma Y, Fang Y, Liu L, Wu S, Fu D, et al. Association between PON1 activity and coronary heart disease risk: a meta-analysis based on 43 studies. Mol Genet Metab. 2012;105(1):141-8. [ Links ]

Schrader C, Rimbach G. Determinants of paraoxonase 1 status: genes, drugs and nutrition. Curr Med Chem. 2011;18(36):5624-43. [ Links ]

Humbert R, Adler DA, Disteche CM, Hassett C, Omiecinski CJ, Furlong CE. The molecular basis of the human serum paraoxonase activity polymorphism. Nat Genet. 1993;3(1):73-6. [ Links ]

Imai Y, Morita H, Kurihara H, Sugiyama T, Kato N, Ebihara A, et al. Evidence for association between paraoxonase gene polymorphisms and atherosclerotic diseases. Atherosclerosis. 2000;149(2):435-42. [ Links ]

Zama T, Murata M, Matsubara Y, Kawano K, Aoki N, Yoshino H, et al. A 192Arg variant of the human paraoxonase (HUMPONA) gene polymorphism is associated with an increased risk for coronary artery disease in the Japanese. Arterioscler Thromb Vasc Biol. 1997;17(12):3565-9. [ Links ]

Pfohl M, Koch M, Enderle MD, Kuhn R, Fullhase J, Karsch KR, et al. Paraoxonase 192 Gln/Arg gene polymorphism, coronary artery disease, and myocardial infarction in type 2 diabetes. Diabetes. 1999;48(3):623-7. [ Links ]

Bayrak A, Bayrak T, Tokgozoglu SL, Volkan-Salanci B, Deniz A, Yavuz B, et al. Serum PON-1 activity but not Q192R polymorphism is related to the extent of atherosclerosis. J Atheroscler Thromb. 2012;19(4):376-84. [ Links ]

Antikainen M, Murtomaki S, Syvanne M, Pahlman R, Tahvanainen E, Jauhiainen M, et al. The Gln-Arg191 polymorphism of the human paraoxonase gene (HUMPONA) is not associated with the risk of coronary artery disease in Finns. J Clin Invest. 1996;98(4):883-5. [ Links ]

Allebrandt KV, Souza RL, Chautard-Freire-Maia EA. Variability of the paraoxonase gene (PON1) in Euro- and Afro-Brazilians. Toxicol Appl Pharmacol. 2002;180(3):151-6. [ Links ]

Rios DL, D'Onofrio LO, Cerqueira CC, Bonfim-Silva R, Carvalho HG, Santos-Filho A, et al. Paraoxonase 1 gene polymorphisms in angiographically assessed coronary artery disease: evidence for gender interaction among Brazilians. Clin Chem Lab Med. 2007;45(7):874-8. [ Links ]

Oliveira SA, Mansur AP, Ribeiro CC, Ramires JA, Annichino-Bizzacchi JM. PON1 M/L55 mutation protects high-risk patients against coronary artery disease. Int J Cardiol. 2004;94(1):73-7. [ Links ]

Voetsch B, Benke KS, Damasceno BP, Siqueira LH, Loscalzo J. Paraoxonase 192 Gln-->Arg polymorphism: an independent risk factor for nonfatal arterial ischemic stroke among young adults. Stroke. 2002;33(6):1459-64. [ Links ]

Dessi M, Gnasso A, Motti C, Pujia A, Irace C, Casciani S, et al. Influence of the human paraoxonase polymorphism (PON1 192) on the carotid-wall thickening in a healthy population. Coron Artery Dis. 1999;10(8):595-9. [ Links ]

Parra ES, Zago VH, Panzoldo NB, Alexandre F, Vendrame F, Virginio VW, et al. Development of a clinical laboratory data base of hyper and hypo alpha lipoproteins in Campinas-SP and neighboring region. J Bras Patol Med Lab. 2013;49(1):26-33. [ Links ]

Baecke JA, Burema J, Frijters JE. A short questionnaire for the measurement of habitual physical activity in epidemiological studies. Am J Clin Nutr. 1982;36(5):936-42. [ Links ]

Furlan-Viebig R, Pastor-Valero M. [Development of a food frequency questionnaire to study diet and non-communicable diseases in adult population]. Rev Saude Publica. 2004;38(4):581-4. [ Links ]

Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972;18(6):499-502. [ Links ]

Lima ES, Maranhao RC. Rapid, simple laser-light-scattering method for HDL particle sizing in whole plasma. Clin Chem. 2004;50(6):1086-8. [ Links ]

Lagrost L. Determination of the mass concentration and the activity of the plasma cholesteryl ester transfer protein (CETP). Methods Mol Biol. 1998;110:231-41. [ Links ]

Jauhiainen M, Ehnholm C. Determination of human plasma phospholipid transfer protein mass and activity. Methods. 2005;36(2):97-101. [ Links ]

Kleemola P, Freese R, Jauhiainen M, Pahlman R, Alfthan G, Mutanen M. Dietary determinants of serum paraoxonase activity in healthy humans. Atherosclerosis. 2002;160(2):425-32. [ Links ]

Chen J, Kumar M, Chan W, Berkowitz G, Wetmur JG. Increased influence of genetic variation on PON1 activity in neonates. Environ Health Perspect. 2003;111(11):1403-9. [ Links ]

Rojas-Garcia AE, Solis-Heredia MJ, Pina-Guzman B, Vega L, Lopez-Carrillo L, Quintanilla-Vega B. Genetic polymorphisms and activity of PON1 in a Mexican population. Toxicol Appl Pharmacol. 2005;205(3):282-9. [ Links ]

Wang X, Fan Z, Huang J, Su S, Yu Q, Zhao J, et al. Extensive association analysis between polymorphisms of PON gene cluster with coronary heart disease in Chinese Han population. Arterioscler Thromb Vasc Biol. 2003;23(2):328-34. [ Links ]

Suehiro T, Nakamura T, Inoue M, Shiinoki T, Ikeda Y, Kumon Y, et al. A polymorphism upstream from the human paraoxonase (PON1) gene and its association with PON1 expression. Atherosclerosis. 2000;150(2):295-8. [ Links ]

Lawlor DA, Day IN, Gaunt TR, Hinks LJ, Briggs PJ, Kiessling M, et al. The association of the PON1 Q192R polymorphism with coronary heart disease: findings from the British Women's Heart and Health cohort study and a meta-analysis. BMC Genet. 2004;5:17. [ Links ]

Gaidukov L, Rosenblat M, Aviram M, Tawfik DS. The 192R/Q polymorphs of serum paraoxonase PON1 differ in HDL binding, lipolactonase stimulation, and cholesterol efflux. J Lipid Res. 2006;47(11):2492-502. [ Links ]

Blatter Garin MC, Moren X, James RW. Paraoxonase-1 and serum concentrations of HDL-cholesterol and apoA-I. J Lipid Res. 2006;47(3):515-20. [ Links ]

Wang M, Lang X, Zou L, Huang S, Xu Z. Four genetic polymorphisms of paraoxonase gene and risk of coronary heart disease: a meta-analysis based on 88 case-control studies. Atherosclerosis. 2011;214(2):377-85. [ Links ]

Dahabreh IJ, Kitsios GD, Kent DM, Trikalinos TA. Paraoxonase 1 polymorphisms and ischemic stroke risk: a systematic review and meta-analysis. Genet Med. 2010;12(10):606-15. [ Links ]

Birjmohun RS, Vergeer M, Stroes ES, Sandhu MS, Ricketts SL, Tanck MW, et al. Both paraoxonase-1 genotype and activity do not predict the risk of future coronary artery disease; the EPIC-Norfolk Prospective Population Study. PLoS One. 2009;4(8):e6809. [ Links ]

Gnasso A, Motti C, Irace C, Di Gennaro I, Pujia A, Leto E, et al. The Arg allele in position 192 of PON1 is associated with carotid atherosclerosis in subjects with elevated HDLs. Atherosclerosis. 2002;164(2):289-95. [ Links ]

Cao H, Girard-Globa A, Serusclat A, Bernard S, Bondon P, Picard S, et al. Lack of association between carotid intima-media thickness and paraoxonase gene polymorphism in non-insulin dependent diabetes mellitus. Atherosclerosis. 1998;138(2):361-6. [ Links ]

Leus FR, Wittekoek ME, Prins J, Kastelein JJ, Voorbij HA. Paraoxonase gene polymorphisms are associated with carotid arterial wall thickness in subjects with familial hypercholesterolemia. Atherosclerosis. 2000;149(2):371-7. [ Links ]

Hodis HN, Mack WJ, LaBree L, Selzer RH, Liu CR, Liu CH, et al. The role of carotid arterial intima-media thickness in predicting clinical coronary events. Ann Intern Med. 1998;128(4):262-9. [ Links ]

Karvonen J, Kauma H, Paivansalo M, Kesaniemi YA. Paraoxonase-1 gene Leu-Met55 and Gln-Arg192 polymorphisms are not associated with carotid artery atherosclerosis in a population-based cohort. Eur J Cardiovasc Prev Rehabil. 2004;11(6):511-2. [ Links ]

Received: October 16, 2014; Revised: January 12, 2015; Accepted: January 19, 2015

Mailing Address: Daniel Zanetti Scherrer, R. Tessália Vieira de Camargo, 126. Cidade Universitária “Zeferino Vaz”, Postal Code 13083-887. Campinas, SP - Brazil. E-mail: zanettis@hotmail.com

Author contributions

Conception and design of the research:Scherrer DZ, Quintão ECR, Faria EC. Acquisition of data:Scherrer DZ, Zago VHS, Vieira IC, Parra ES, Panzoldo NB, Alexandre F, Baracat J. Analysis and interpretation of the data: Scherrer DZ, Zago VHS, Vieira IC, Secolin R, Baracat J, Faria EC. Statistical analysis: Scherrer DZ, Zago VHS, Secolin R. Obtaining financing: Quintão ECR, Faria EC. Writing of the manuscript:Scherrer DZ, Zago VHS, Faria EC. Critical revision of the manuscript for intellectual content: Scherrer DZ, Zago VHS, Vieira IC, Parra ES, Panzoldo NB, Alexandre F, Secolin R, Quintão ECR, Faria EC.

Potential Conflict of Interest

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

Creative Commons License This is an Open Access article distributed under the terms of the Creative Commons Attribution NonCommercial License which permits unrestricted noncommercial use, distribution, and reproduction in any medium provided the original work is properly cited.