Paraoxonase 2 Cys311Ser polymorphism and its association with the systolic blood pressure values in asymptomatic dyslipidemic individuals: a pilot study

Study design and subjects

The study was performed with asymptomatic dyslipidemic subjects (i.e. in individuals without history of clinically manifest atherosclerosis – coronary artery disease, heart failure, cerebrovascular ischemic disease, and peripheral vascular disease, with total cholesterol (TC) ≥5.0 mmol/L and/or TGs ≥1.5 mmol/L, n=264, 129 men and 135 women). They had been examined consecutively for the first time in the Lipid Centre of the 3^rd Department of Internal Medicine, University Hospital Olomouc, Czech Republic, during the period from January 2006 to March 2011. All subjects were tested for the signs of secondary hyperlipidemia: diabetes mellitus, hypothyroidism, renal or hepatic diseases, and nephrotic syndrome. Other exclusion criteria were as follows: history of clinically manifested atherosclerosis presented by coronary artery disease, cerebrovascular disease, and peripheral arterial disease, any hypolipidemic therapy in previous 8 weeks, hormone therapy, and clinical presence of acute infections. The subjects with E24 genotype of ApoE polymorphism were also excluded because of the opposite effects of E2 and E4 alleles on lipid levels (a total of seven individuals). All individuals filled out a questionnaire on their previous medical history, especially cardiovascular status, medication, and smoking habits. Body mass index (BMI) and systolic and diastolic blood pressures (SBP and DBP, respectively) were also determined. The study was approved by the Ethics Committee of Medical Faculty and University Hospital Olomouc and written informed consent was obtained from all participants. The authors have complied with the World Medical Association Declaration of Helsinki regarding ethical conduct of research involving human subjects.

Laboratory analysis

Venous blood samples were drawn in the morning after a 12-h fast. After centrifugation, the serum was used for other analyses. For the assessment of hemostatic markers, venous blood was collected in 3.8% sodium citrate tubes and plasma was obtained after centrifugation.

Routine serum biochemical parameters were analyzed on Modular SWA (Roche, Basel, Switzerland) on the same day of blood collection. Concentrations of other special analytes (i.e. insulin, C-peptide, adhesion molecules, and hemostatic markers) were measured in the sample aliquots stored at –20 °C, no longer than 6 months (see below).

TC, TG, and HDLc levels were determined enzymatically on Modular SWA system (Roche). LDLc levels were calculated using Friedewald formula (for TG <4.5 mmol/L). Other calculated parameters were as follows: non-HDL-cholesterol (non-HDLc=TC–HDLc), atherogenic index of plasma (AI=logTG/HDLc), and homeostasis model assessment (HOMA-R=glucose×insulin/22.5). Concentration of ApoB and ApoA1 were determined immunoturbidimetrically using Tina-Quant ApoB and ApoA-1 kits (Roche). Lipoprotein (a) [Lp(a)] was measured immunoturbidimetrically using Tina-Quant Lipoprotein(a) TQ kit (Roche). C-reactive protein (CRP) was assessed by an ultrasensitive immunoturbidimetric method using the kit Tina-Quant (Roche). Glucose was determined using GOD-PAP method (Roche). All tests were measured from fresh sera in the same day of blood collection.

Insulin was determined by the commercially available kit (Immunotech, Marseille, France) using specific antibodies by the IRMA method. C-peptide was determined using C-peptide Kit (Immunotech) by the IRMA method. Serum levels of the soluble adhesion molecules (soluble intercellular adhesion molecule 1 [sICAM-1] and soluble vascular cell adhesion molecule 1 [sVCAM-1]) were determined by immunoenzymatic assay using commercially available kits sICAM-1 and sVCAM-1 (both Immunotech). The sample aliquots were stored at –20 °C, no longer than 6 months.

The following hemostatic markers were examined from human plasma stored at –20 °C: fibrinogen (function coagulation method by Clauss; Technoclone, Vienna, Austria), vWF (immunoturbidimetric assay; Instrumentation Laboratory SpA, Milan, Italy), PAI-1, tPA (both ELISA; Technoclone), and soluble thrombomodulin (sTBM, ELISA Thrombomodulin; Diagnostica Stago, Asnieres sur Seine, France).

Genotyping

DNA was extracted from the peripheral leukocytes of all subjects using a standard commercial kit (QIAamp DNA Blood Mini kit, Qiagen, Hilden, Germany). After isolation, the extracts were stored at –20 °C, no longer than 3 months. PON2 Cys311Ser variants were identified using polymerase chain reaction (PCR) method followed by the restriction fragment length polymorphism analysis [22]. Briefly, after DNA extraction, a 262-bp product was amplified by PCR using the pair of primers with following sequences: 5′-ACA TGC ATG TAC GGT GGT CTT ATA-3′ and 5′-AGC AAT TCA TAG ATT AAT TGT TA-3′. PON2 Cys311Ser polymorphism was detected by digesting the PCR-amplified product with the DdeI restriction enzyme, followed by size fractionation in 3% agarose gel and visualization of bands with ethidium bromide. ApoA5 genotypes were determined by a melting curve analysis after a real-time PCR method adapted from the work of Frances et al. [23] using LightCycler, v.2.0 (Roche). Genotyping of ApoE alleles was performed using commercially available ApoE LightMix Kit (Roche) by melting curve analysis after a real time PCR. The metod was performed using the same instrument.

Measurement of BP

The auscultatory method of the BP measurement with a properly calibrated and validated mercury sphygmomanometer was used. Persons were seated quietly for at least 5 min in a chair, with feet on the floor and arm supported at heart level. An appropriate-sized cuff was used to ensure accuracy. The first and fifth Korotkoff sounds were used to identify SBP and DBP. At least three sitting BP measurement with a 30-s interval were taken and the mean from the last two was calculated.

Statistical analysis

All values of quantitative parameters are expressed as means± standard deviation (SD), and parameters with skewed distribution were also expressed as medians. The Kolmogorov-Smirnov test was used to check for normal distribution. Variables with skewed distribution [MetS signs, CRP, TG, AI, Lp(a), tPA, PAI-1, sTBM, sICAM-1, insulin, HOMA-R, C-peptide] were log transformed in order to normalize their distribution before statistical analysis. Differences in variables between individual groups were analyzed with ANOVA, after adjustment for age and sex. The results have been corrected by the Bonferroni correction for multiple testing. Statistical analysis was performed by SPSS for Windows, version 12.0 (SPSS, Chicago, IL, USA). Probability values of p<0.05 were considered as statistically significant.

The genotype and allele frequencies for PON2, ApoA5, and ApoE gene polymorphisms were determined by a gene counting method and then evaluated by performing the Pearson χ² statistical analysis to identify whether followed polymorphisms were consistent with the Hardy-Weinberg equilibrium expectation. To create the common variants of genetic polymorphisms, a model with two variants of PON2 311 (CC+CS, and SS), two variants of T–1131C ApoA5 (TT, and CT+TT), and three variants of ApoE polymorphisms (E22+E23, E33, and E34+E44) was used.

PON2 variants

The PON2 expression in arterial cells apparently plays an important role in the body resistance to vascular disease [8]. Considering the effect of PON2 to vascular hemostasis, we hypothesized possible relationship of the endothelial/hemostatic marker levels with the PON2 Cys311Ser polymorphism even in asymptomatic individuals with dyslipidemia. In addition, the influence of the polymorphism on the level of these markers has not been evaluated in clinical studies so far. However, no significant differences between the PON2 subgroups were observed in the presented study, suggesting that the PON2 311C allele has probably no different effect on the expression of endothelial markers compared with S allele in asymptomatic dyslipidemic patients.

In contrast, the results showed significantly higher SBP values and CRP levels in the C allele carriers. Although the influence of PON2 Cys311Ser polymorphism on the risk of cardiovascular and other diseases is controversial in recent studies, some of them point to a link with the 311C variant. Cozzi et al. [8] described the presence of C allele as an independent possible cofactor in determining the risk of atherothrombotic events, together with hypertension and HDLc, in an Italian subpopulation. The frequency of C allele was significantly higher in the group of subjects with history of atherothrombotic events. CC homozygotes of the PON2 gene 311 polymorphism were more likely to have an increased risk of coronary artery disease in a Taiwanese population [24]. In the presence of MetS and diabetes, PON2 311 CC genotype was associated with an increased risk of significant coronary stenosis in Tunisian subjects, with no relationship of the PON2 Cys311Ser polymorphism to lipid profile. The authors hypothesize that the R allele of the PON1 192 and the C allele of the PON2 311 polymorphisms may promote oxidative stress, and this might increase cardiovascular risk in diabetic or the MetS patients [25]. Similarly, no association of PON2 Cys311Ser variants with lipid and lipoprotein parameters and C allele overrepresentation in subjects with diabetes and microvascular complications were observed in another study [12]. Thus, it appears that C allele might be a predisposing risk factor of adverse conditions in symptomatic patients, especially those with metabolic disturbances.

The study of Kokubo et al. [26] analyzed the association between 61 SNPs with BP variations and hypertension in a population-based study using randomly selected 1880 Japanese subjects. Analyses of covariance revealed that 17 polymorphisms in 16 genes were significantly associated with BP variations, including the PON2 gene. In contrast, the large genome-wide association study (GWAS) with 200,000 individuals of European descent identified 29 independent genetic variants associated with BP. Subsequent analysis confirmed the association of several independent genetic variant in individuals of non-European ancestry. No association was found between BP and the PON2 gene [27]. Previously, another GWAS performed with 34,433 subjects of European ancestry identified an association between SBP or DBP and common variants in eight regions, but not with the PON2 gene [28]. Based on these conclusions, our observation (i.e. association of the PON2 311C variant with elevated SBP) could be considered a random phenomenon. This discrepancy might be the result of different study design, including a small number of participants in particular. Nevertheless, two recent experimental studies revealed that renal PON2 is involved in the inhibition of reactive oxygen species and NADPH oxidase activity and contributes to the maintenance of normal BP [29, 30]. Therefore, the relationship of PON2 genotypes to the BP values should not be completely excluded. Similarly, because the expression of all three PON genes negatively correlates with a number of “inflammatory” diseases including atherosclerosis, it is highly probable that PON2 might play an important role in the process of inflammation. Thus, the relationship to CRP and other proinflammatory markers should be also evaluated.

The observed effect of PON2 Cys311Ser polymorphism on the levels of ApoA1 was surprising. In contrast to PON1 and PON3, PON2 is not associated with HDL, and no similar information was found in recent literature. Formerly, two studies showed that PON2 Ala148/Ser311 homozygotes exhibited significantly higher plasma TC, LDLc, and ApoB than subjects with the other two genotypes [31, 32]. Another study revealed that PON2 Gly148 homozygotes had the highest plasma concentrations of TC, HDLc, and ApoA1; Ala148 homozygotes the lowest; and heterozygotes had an intermediate phenotype [33]. In our work, the association of higher ApoA1 levels with the PON2 311C allele was apparent in analysis of the common PON2/ApoE variants as well. But as the study was conducted as a pilot, one with a limited number of participants, we can speculate about a random phenomenon in this case.

Since our study was performed with individuals without the symptoms of cardiovascular disease, we had no clinical correlates to assess the impact of the PON2 Cys311Ser variants. Nevertheless, higher levels of proinflammatory CRP and SBP in the C allele carriers suggest that this variant could represent the symptom of higher risk of vascular complications compared with SS genotype carriers.

PON2/ApoA5 common variants

As the majority of genetic polymorphisms exert their effects in accordance with other polymorphisms as haplogroups, we evaluated a relationship between the PON2/ApoA5 combined variants to the levels of all measured parameters. Numerous studies have confirmed the association of ApoA5 naturally occurring variants, including –1131C allele of the ApoA5 gene, with an increased TG that may itself represent elevated risk of endothelial dysfunction [19]. Formerly, we have observed that the presence of the ApoA5 –1131C variant was associated with higher TG and tPA levels in dyslipidemic patients [34]. In the present study, influence of the ApoA5 –1131C variant was also confirmed. The presence of the –1131C minor allele has been associated with higher levels of TG in comparison with T allele carriers, regardless of PON2 genotype (both in the 311C/–1131C and the 311S/–1131C common variant subgroups). In context of the above mentioned data, the carriers of the 311S/–1131T combination had also significantly lower CRP compared with the 311C/–1131T variant that probably represents the action of the PON2 311 polymorphism (see PON2 variants section).

PON2/ApoE common variants

The ApoE gene has been suggested as a factor influencing susceptibility to atherothrombotic diseases. The minor alleles E2 and E4 of ApoE common polymorphism were found to be associated with higher plasma TG in comparison with E3 common variant, which we demonstrated previously in the cohort of dyslipidemic patients [35]. This effect, particularly of the E4 variant, was evident in the presented study, with elevated TG concentration in the 311S/E4 subgroup. As no elevation of TG was observed in PON 311S carriers alone, we hypothesize the action only of ApoE polymorphism on TG levels. No effects of combined PON2/ApoE variants were found on the level of endothelial/hemostatic markers and parameters of IR, but with one exception. The 311S/E2 variant carriers had increased sVCAM-1 compared with 311C/E3 subjects. According to some authors, sVCAM-1 levels may be related to the extent of atherosclerotic lesions and a prediction of subsequent cardiovascular progression in subject with coronary artery disease [36, 37]. A significant increase in the adhesive molecule levels was detected in hypertensive patients with coexisting impaired glucose tolerance, hyperlipidemia, obesity, and diabetes [38, 39]. Elevated sVCAM-1 levels in the 311S/E2 subgroup are surprising, as asymptomatic patients were included in the study. However, the small number of participants in subgroups has to be taken into account and any general conclusions should not be deduced.

Probably the most interesting finding was the detection of decreased values of SBP in all S allele subgroups, compared with the similar C allele cohorts (i.e. 311S/E2 vs. 311C/E2, 311S/E3 vs. 311C/E3, 311S/E4 vs. 311C/E4). The lowest SBP values were observed in the 311SS/E33 homozygotes, i.e. in the patients carrying the most frequent variant, and the significant decline was observed only in this subgroup in comparison with the 311C/E3 variant. This observation could support the above-mentioned speculation that the C allele carriers might be at higher risk of elevated SBP. However, it is necessary to bear in mind the limited number of individuals in the followed subgroups.

Limitation of the study

The major limitation of the study is represented particularly by the small number of subjects in the PON2/ApoA5 and the PON2/ApoE minor variant subgroups, which could reduce the power of significance of the observed relationships. For this reason, our results require validation in larger cohorts of individuals, both in symptomatic and healthy control subjects.