Introduction

Epidemiological studies have indicated that CAD is one of the leading causes of death in developed and developing countries [1]. The global death rate shows that one out of every five patients (more than 20% of global deaths) die due to CAD [2, 3]. In Iran, as one of the third-world countries in which patients are at risk of exposure to hazardous factors, more than half of them die from CAD [4, 5].

The occurrence of atherosclerosis in the cardiovascular system is one of the leading causes of CAD [6, 7]. Among different risk factors, high-density lipoprotein (HDL-cholesterol) is known as one of the essential parameters [8] which increases the risk of developing cardiovascular disease by 2–3% per 1% reduction in HDL [9]. Likewise, the mechanisms underlying the protective effects of HDL have been reported in many studies [10, 11]. The known anti-atherogenic properties of HDL are due to its role as a lipid transporter in the reverse cholesterol transportation pathway. LDL oxidation plays an essential role in atherosclerosis, and HDL can protect it against oxidation by several mechanisms. HDL also reduces the biological activity of oxidızed LDL. These antioxidants and anti-atherogenic properties depend on various proteins associated with HDL such as PON1, platelet-activating factor (PAF) for acetyl hydrazine, and lecithin [12, 13]. PON1 (EC 3.1.8.1, aryldialkylphosphatase) was first identified due to its ability for the hydrolysis of organophosphates. It is a calcium-dependent ester with 355 amino acids and a molecular weight of 43 kDa [14]. It has been shown in many studies that PON1 has two common polymorphisms (L55M, and Q192R) both of which are single-nucleotide polymorphisms (SNPs) [15, 16].

It has been reported that L55M polymorphism could be caused by a change in the CTG codon resulting in the substitution of methionine by leucine (L/M55) at the position 55. L55M polymorphism is associated with stroke [17], coronary artery disease [16], and Parkinson's disease [18]. Although L55M polymorphism does not affect the binding of the enzyme to the substrate, it could change the enzyme level. Research has revealed that individuals with MM genotype have the lowest expression level of the mRNA and serum concentration of PON1 [15].

Q192R polymorphism is caused by a change in the CAA codon. Hence, arginine replaces glutamine at position 192 (Q/R192). Q192R polymorphism seems to alter the PON1 enzyme activity, depending on the substrate. Some substrates like paraoxon are hydrolyzed by R isoforms, while Q isoforms rapidly hydrolyze substrates such as diazoxone. Several recent studies have indicated that R alleles decrease the ability to protect LDL against oxidation compared to the Q allele [19].

The purpose of the present study is to investigate the effect of L55M and Q192R polymorphism on the risk of atherosclerosis and PON1 activity in Western Iran. Furthermore, we examined the potential impact of L55M and Q192R polymorphism and PON1 activity on the level of oxLDL.

Methods

Study design and population

A total of 299 study subjects (154 cases with atherosclerosis and 145 controls) were examined. All participants in this study referred to Cardiology and Angiography Department of Shahid Madani Hospital, Khorramabad, Iran, between December 2016 and May 2017. The disease was confirmed in all subjects by the standard diagnostic angiography.

For all subjects, a permission form containing various clinical information, including age, sex, weight, smoking, family history, hypertension, diabetes mellitus, drug abuse, and alcohol consumption was recorded. The inclusion criterion was the confirmation of coronary atherosclerosis by Gold Angiography. Subjects with congenital heart disease, malignancy, chronic kidney disease, pulmonary obstruction, and the use of steroid hormones as treatments were excluded. Individuals without coronary atherosclerosis were considered healthy controls. They were matched for sex and age. Written informed consent was obtained from all subjects. The study was reviewed and approved by the Lorestan University of Medical Science's Ethics Committee (code: LUMS.REC.1395.123). The study was administered following the Declaration of Helsinki and its following revisions [20].

Biochemical measurements

Two types of blood samples were collected, one without anticoagulants (for biochemical assays) and another with Ethylenediaminetetraacetic acid (EDTA) (for genetic polymorphism evaluations), each one in a volume of 5 ml. The lipid profile was assessed using Auto Analyzer (BT-1000, USA). The blood samples were stored at 4 °C and transferred to the laboratory. Subsequently, the WBC was isolated at the same time and was saved at minus 70 °C.

Small dense LDL (SdLDL) was measured using the method proposed by Hirano et al. As follows: lipoproteins (1.044 g/ml density) were precipitated via a reagent containing 40 U/ml heparin sodium salt and 30 mmol/l MgCl2 and then LDL-cholesterol was measured using LDL assay kits (Pishtaz Teb, Iran) [21]. OxLDL was measured using an ELISA kit (Mercodia, Sweden). Moreover, the measurement of PON1 enzyme activity was evaluated using the method developed by Paragh et al. In this method, the enzyme activity is measured using a paraoxon substrate. For this assay, 50 μl of serum was added to 1 cc of the ready-to-use solution, including Tris/HCl buffer (100 mmol/l pH 8), calcium chloride (2 mmol/l) and paraoxon (5 mmol/l). The speed of 4-nitrophenol production was measured at 412 nm and 25 °C using a spectrophotometer [22].

DNA extraction and PCR methodology

PON1 genotypes were determined by polymerase chain reaction (PCR) using a slightly altered version of the protocols proposed by Mackness et al. (Table 1). All PCR steps and the type of primer have been listed in Table 1. The PCR product of PON1Q192R polymorphisms was 99 bp, which was digested by 5U Alw l restriction enzyme (Thermo, Lithuania) in 5 h at 37 C. The products were separated by electrophoresis on a 3% agarose gel. The R allele contained a site for the Alw l restriction enzyme from which 66 and 33 bp products were made, but the Q allele did not [23]. (Fig. 1).

Table 1 Primer PCR product length and reaction conditions of selected SNPs
Full size table
Fig. 1
figure 1

PON1Q192R polymorphisms genotypes

Full size image

For the PON1L55M polymorphism, the PCR condition was the same as what was mentioned above except that 40 cycles were carried out. The PCR product (170 bp) was digested by Nla 111 (Thermo, Lithuania) and the digested products were separated via electrophoresis on a 2% agarose gel. The L allele did not contain the Nla 111 restriction site, but the M allele contained a restriction site that produced 126 bp and 44 bp [24]. (Fig. 2).

Fig. 2
figure 2

PON L55M polymorphisms genotypes

Full size image

Statistical analysis

The normality of the data was tested using the Kolmogorov–Smirnov test. The data of the numerical variable have been presented as mean ± SD. Accordingly, a t-test was used to compare continuous data, and the Chi-square test was used to recognize the significance of the difference between the proportions. Moreover, the age and gender between the two groups of case and control were matched. Logistic regression analysis was used for testing the independent association of various variables. Data were analyzed using SPSS software version 16 (SPSS Inc., Chicago, IL, USA). P-values of less than 0.05 were regarded as statistically significant.

Results

Subjects characteristics

The baseline characteristics of the 299 subjects have been indicated in Table 2. Our results showed a considerable distinction in age (P < 0.001), weight (P = 0.045) and diagnosis age (P < 0.001) between the cases and control groups but there was no significant difference in height (P = 0.112), BMI (P = 0.390), systolic blood pressure (P = 0.140) and diastolic blood pressure (P = 0.147) between the two groups. The biochemical characteristics of the participants with reference ranges have been presented in Table 3. A significant distinction was observed in lipid profile, Sd-LDL (P < 0.001), Ox-LDL (P < 0.001), serum glucose (P < 0.001), and PON1 activity (P < 0.001) between the cases and control groups. We also found a significant distinction between the family history of CAD, hypertension, cigarette smoking, and diabetes mellitus in the two groups (Table 2).

Table 2 Baseline and biochemical characteristics of the study population
Full size table
Table 3 The distribution of genotypes and allele frequencies of PON1L55M and PON1Q192R polymorphisms
Full size table

Genotype distribution and genotype frequencies

In the present study, a total of 299 participants (145 controls, 154 cases) were included. The results of the genotype of the PON1 gene and its relationship with atherosclerosis have been indicated in Table3. In the study groups, there were 128 (42.8%), 135 (45.7%) and 36 (12%) subjects with the LL genotype, the LM genotype, and the MM genotype respectively. Moreover, LM genotype distribution in the cases group was 1.71 times higher than healthy individuals (P = 0.031; OR 1.71; CI 1.051–2.791), and MM genotype distribution in the cases group was 3.114 times higher than healthy individuals (P = 0.005; OR 3.114; CI 1.412–6.780).

Furthermore, there were 164 (54.8%), 108 (36.1%) and 27(9%) subjects with the QQ genotype, the QR genotype, and the RR genotype respectively. Distribution of QR genotype in the cases group was 0.952 times higher than the control group (P = 0.844; OR 0.952; CI 0.586–1.548), and RR genotype distribution in the cases group was 1.38 times higher than the healthy individuals (P = 0.44; OR 1.38; CI 0.606–3.166). Although we did not find any significant distinction between PON1Q192R polymorphism genotypes and alleles frequency in the cases and control groups, we found a significant relation between PON1L55M polymorphism genotypes and the alleles in the two groups (Table 3).

The controls with LL and LM genotype exhibited higher (P < 0.05) PON1 activity compared to the cases with LL and LM genotype respectively, but there was no significant difference between PON1 activity and MM genotype (P = 0.012) between the two groups. All subjects in the control group with PON1Q192R polymorphism showed higher PON1 activity compared to the cases group. However, this relationship was not significant between PON1 activity and RR genotype (P = 0.018) in comparison with the control group. The difference between the levels of enzyme activity in the different PON1Q192R polymorphism genotypes of the cases group is statistically significant. Nevertheless, it was not substantial in the control group. In this study, our results did not show any correlation between PON1L55M and PON1Q192R polymorphisms genotypes and enzyme activity in contrast with the control group. The results also indicated that the relationship between MM and LM genotypes interaction and PON1 activity in increasing the chances of developing atherosclerosis was significant.

In the present study, the serum levels of oxLDL in the cases and control groups were evaluated based on PON1L55M and PONQ192R polymorphism genotypes. In both polymorphisms, the mean serum level of oxLDL in the cases group was significantly different compared to the control group. However, there was no remarkable distinction between the ox-LDL level and PON1L55M and PONQ192R polymorphism genotypes in the two groups. The present research also revealed that the interactive effects of PON1L55M and PON1Q192R polymorphism among different genotypes of control and cases, and PON1 activity on oxLDL levels were statistically significant (Table 4).

Table 4 Comparison of PON activity and OxLDL level between different PON polymorphism genotype and alleles
Full size table

Using the multivariable logistic regression, the effects of PON1L55M and PONQ192R polymorphism genotypes on age, sex, history of blood pressure, smoking, and CAD were simultaneously examined. The results have been presented in Table 5. The results indicate significant variables such as MM (P = 0.011), tobacco (P < 0.001), history of CAD (P < 0.001), and age (P < 0.001). The chance of developing atherosclerosis in subjects with MM genotype is higher than those with LL genotype, which is statistically significant (P = 0.011; OR 3.39; CI 1.8–32.73). The risk of developing the disease in females is 70% higher than males but it is not statistically significant (P = 0.092; OR 1.7; CI 0.91–3.16). In terms of age, the risk of developing the disease is enhanced by 0.8% per 1-year-old increase, which is statistically significant (P < 0.001; OR 1.08, CI 1.05–1.11). With regard to the history of cardiovascular disease, the risk of cardiovascular disease is 3.36 times greater than those without an account, which is statistically significant (P < 0.001; OR 3.36, CI 2–6.33) (Table 5).

Table 5 Effect of PON1L55M, PON1Q192R genotypes, age, gender, history of hypertension, smoking, and family history of CAD on atherosclerosis using multivariate logistic regression
Full size table

Discussion

This research was planned to investigate the association between PON1L55M and PON1Q192R polymorphisms and the risk of developing atherosclerosis. In this Study, LM and MM genotypes of PON1L55M polymorphism had a meaningful relationship with the development of atherosclerosis, but we did not find any significant association between PON1Q192R polymorphism and atherosclerosis.

In our study population of Western Iran, the analysis of PON1L55M polymorphism showed that in the control group, 74 people had LL genotype, 60 people had LM genotype and 11 people had MM genotype. Moreover, in the cases group, 54 people with LL genotype, 75 people with LM genotype and 25 people with MM genotype were observed. Analysis of PON1Q192R polymorphism also showed 80 people with QQ genotype, 54 people with RQ genotype and 11 people with RR genotype in the control group and 84 people with QQ genotype, 54 people with RQ genotype and 16 people with RR genotype in the cases group. Hence, according to the analysis, people with LM and MM genotypes are more likely to get the disease while no significant results were found for PON1Q192R polymorphism.

Various studies, including this research, have found a meaningful relationship between the development of atherosclerosis and some determinants such as smoking [25, 26], obesity, and diabetes [27, 28]. Epidemiological studies have indicated that high levels of LDL cholesterol and triglyceride and low levels of HDL-cholesterol are among of the most influential risk factors for atherosclerosis [29, 30]. It was found out that PON1 is likely to prevent cardiovascular disease by inhibiting the oxidative damage of LDL through HDL [30, 31]. Due to the role of PON1 in the metabolism of lipids, various studies have shown evidence of a link between PON1Q192R and PON1 L55M polymorphism with CAD risk [17, 32, 33]. The activity of PON1 in the healthy subjects was higher than that of the cases, and it was statistically significant. However, no relationship was found between genotypes and enzyme activity.

Studies conducted by Özkök et al. and Kaman et al. indicated the association between PONQ192R and PONL55M polymorphism and the risk of developing cardiovascular disease. These studies revealed that individuals with MM genotype have lower enzyme activity and a higher risk of developing the disease [34, 35]. Moreover, a research carried out by Schmidt et al. on the Australian population showed a strong association between PONL55M polymorphism and atherosclerosis [36]. Odawara et al. indicated an association between PONQ192R polymorphism and cardiovascular disease in patients with diabetes mellitus. They also showed that individuals with RR genotype are exposed to a higher risk of developing atherosclerosis [37]. Furthermore, studies by Sanghera et al., Pati et al., and Agraval et al. confirm the association between PONQ192R polymorphism and the risk of cardiovascular disease in the Indian population. Both of these studies found that individuals with RR genotype and R allele have lower enzyme activity and a higher risk of the disease [16, 38, 39].

However, some studies have failed to find an association between the PON1 gene polymorphism and the increased risk of atherosclerosis. Several researches were conducted by Ferre et al. on the Spanish population but none of them found any correlation between PONQ192R and PONL55M polymorphisms and the incidence of the disease in individuals [40]. Furthermore, the studies carried out by Robertson and Lawlor studies did not show any relationship between PONQ192R and PONL55M polymorphisms and the risk of developing the illness in the British population [41, 42]. The studies conducted by Hong et al. on the community of Korea [43], Arca et al., on the Italian population [44], and Balcerzyk et al. on the Dutch population [45] also reported similar results observed in previous investigations. Distinct results from multiple studies could be due to the complex interactions between genotype-genotype, genotype-phenotype, and genotype-environment.

There are shreds of evidence that LDL peroxidation is one of the most critical risk factors for atherosclerosis. LDL oxidation seems to be the most crucial trigger for the development of atherosclerosis. Oxidative damage is a significant contributor to vascular wall damage and oxidative changes in LDL. Hence, it is an essential factor in the development of atherosclerosis [11, 39].

In the present study, the level of oxLDL was significantly higher in the cases group in contrast with the control group. Furthermore, we did not find a significant difference in the levels of oxLDL in PONQ192R and PONL55M polymorphism genotypes. Lakshmy et al. launched an investigation into oxLDL levels in cases with cardiovascular disease. The results showed that the oxLDL level was significantly higher in cases than in the control group, and the total antioxidant capacity in the cases was lower. In this research, the relationship between the PONL55M and PONQ192R polymorphism genotypes and the level of oxLDL was also investigated, but no significant correlation was found [46].

In addition to genetic testing, we investigated biochemical parameters. In this study, we measured factors such as blood sugar levels, lipid profiles, systolic and diastolic blood pressure, as well as sdLDL levels. As expected, the levels of these factors in the patient group were higher than the control, which was consistent with previous studies [47]. Moreover, other factors such as age, gender, weight, history of smoking, family history of the disease and blood pressure were also examined in this study. Our research also revealed that people with the MM genotype were more likely to develop the disease due to the simultaneous effects of these risk factors.

Limitations

The limitations that can be mentioned in this study include: At first, this study only collected samples from several western provinces in Iran. However, due to the genetic diversity and racial differences in different populations in Iran, this study could have broader examine the relationship between this SNP and various ethnic groups in Iran, which may have different outcomes in diverse communities. Moreover, this study did not investigate the effects of environmental pollution factors, which, given Iran's third world, is affected by numerous contaminating factors such as air pollution, which, by increasing the amount of oxidative stress, can affect the development of heart disease.

Conclusion

To sum up, it can be mentioned that LDL peroxidation is one of the most critical risk factors for atherosclerosis, and our results confirmed that. It has been concluded that LM and MM genotypes were associated with an increased risk of coronary artery disease in Iranian patients with atherosclerosis. We did not find any relationship between PON1Q192R polymorphism and atherosclerosis. There was no association between the type of genotypes, enzyme activity, and oxLDL level. On the other hand, to investigate the risk factors in the population, biochemical factors were examined, which showed a significant relationship.