Association between PON1 rs662 gene polymorphism and serum paraoxonase1 level in coronary artery disease patients in Northern India

Coronary artery disease (CAD) is a leading cause of morbidity and mortality, with a shifting trend towards the younger population. Paraoxonase1 (PON1) is a glycoprotein enzyme associated with high-density lipoprotein (HDL) particles in the blood. It has the ability to protect against lipid oxidation, thereby reducing the risk of atherogenesis. PON1 rs662 gene polymorphism may affect serum PON1 levels as well as its activity and may have a significant role in the pathogenesis of CAD. The present study was conducted to identify the association of PON1 rs662 gene polymorphism with serum PON1 levels in CAD patients in the North Indian population. This case–control study included 71 angiography-proven CAD patients (with > 50% luminal stenosis in one or more coronary arteries) and 71 controls (with < 50% luminal obstruction in angiography). PON1 rs662 gene polymorphism was studied using PCR and RFLP under the standardized protocol. Serum PON1 levels were estimated by ELISA. The serum PON1 level was significantly lower in the CAD group than in the controls (7.79 ± 3.16 vs. 10.79 ± 3.19 ng/mL; p < 0.0001). Logistic regression analysis showed that homozygous GG genotype of PON1 rs662 SNP has ninefold increased risk of developing CAD in an Indian population (OR = 9.0, 95%CI 2.79–29.06, p = 0.0002). A significantly higher frequency of G allele was also observed in CAD patients than in controls (OR 2.64, 95%CI 1.61–4.33, p = 0.001). The reduced serum PON1 level is associated with CAD. PON1 rs662 gene polymorphism is significantly associated with CAD susceptibility in the North Indian population.


Background
Coronary artery disease (CAD) is a leading cause of morbidity and mortality, contributing to 15.5% of total deaths globally. At present, the phase of epidemiological transition is being observed in which there is an exponential increase in CAD mortality in developing countries as compared to declining drift in developed countries [1].
This may be linked to rapidly increasing industrialization, urbanization, sedentary lifestyle, and unhealthy food habits. According to the World Health Organization, the South Asian region has one of the highest cardiovascular mortality rates in the world [2]. Interactions between various genetic determinants and environmental factors have been implicated in the pathogenesis of premature atherosclerosis in the Indian population [3,4].
Human Paraoxonase (PON) gene family is located on the long arm of chromosome 7 (7q21.3-q22.1) and includes PON1, PON2, and PON3 [5]. Serum PON1, a 44 kDa Ca 2+ -dependent glycoprotein enzyme is one of the susceptibility genes which play a significant protective effect on the progression of atherosclerosis. PON1 is produced in the liver and associates with the surface of high-density lipoprotein (HDL) in blood [6]. The antioxidant activity of HDL cholesterol (HDL-C) is primarily due to the PON1 enzyme. It prevents oxidation of low-density lipoprotein cholesterol (LDL-C) and may also help in the metabolism of oxidized LDL-C (ox-LDL) [7][8][9].
PON1 gene has several genetic polymorphisms that can influence the levels and activity of PON1 in circulation [10]. The rs662 gene (or c.575A > G or p. Gln192Arg) polymorphism is located in exon 6 of the PON1 gene that exchanges an arginine (R) for glutamine (Q) at position 192 of the protein (CAA to CGA) [11]. The studies have shown conflicting results on the association between the Q192R gene variation and CAD as this association is not consistent among distinct population groups. In the present study, we examined the association between PON1 rs662 gene polymorphism and serum PON1 levels in CAD patients in Northern India.

Study population
The present study was conducted in a tertiary care center of Northern India after gaining approval from the institutional ethics review board. The study included randomly selected 142 participants (more than 18 year age) with written informed consent. The diagnosis of CAD was made by a cardiologist on the basis of medical history, clinical examination, and the relevant investigations including angiography (> 50% stenosis in one or more coronary arteries). Out of 142 study participants, 71 were CAD patients and 71 were age and gender-matched controls (with < 50% luminal obstruction on coronary angiography). The exclusion criteria included the recent history of acute coronary syndrome or cerebrovascular event (< 8 weeks), chronic liver and kidney insufficiency, chronic obstructive pulmonary disease, cancer, and repeated blood transfusions.

Sample collection
After overnight fasting, a blood sample was obtained from study participants by venipuncture, of which 2.5 mL each was collected in EDTA vacutainer and plain vacutainer (without anticoagulant). EDTA samples were stored at − 20 °C for the purpose of DNA extraction. The blood in the plain vacutainer was allowed to clot and then centrifuged to obtain serum for further analysis. These samples were stored at − 70 °C until batch analysis of enzyme assay.

Estimation of serum PON1
Estimation of serum PON1 level was done using a commercially available ELISA kit manufactured by Biovendor-Labratorni medicina A.S., following manufacturer's instructions. This was a two-step enzyme immunoassay for quantitative measurement of serum PON1. The absorbance was read at 450 nm. The measured optical density was directly proportional to the PON1 concentration.

Genotyping
The genomic DNA was extracted from nucleated blood cells using DNA Extraction Kit (Krishgen). This DNA was amplified by PCR using the following primers-forward, 5′-GCT GTG GGA CCT GAG CAC TT-3′; reverse, 5′-ATA CTT GCC ATC GGG TGA AATG-3′. The amplification was done in a 20 µL PCR reaction mixture containing 25-30 ng genomic DNA, 50 pmol of each primer, 10 µL Dream Taq green PCR Master-mix (Thermo-scientific) containing 3.2 µM Taq DNA polymerase, 3.2 µM 2X buffer, 32 µM MgCl2, and 3.2 µM of each dNTPs using a thermal cycler (Himedia Eco-96). The PCR conditions used were as follows: initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s; annealing at 60.7 °C for 30 s, then extension at 72 °C for 30 s, with a final extension step at 72 °C for 5 min. The amplified PCR product of 189 bp was incubated with MboI (New England Biolabs) restriction enzyme at 37 °C for 16 h, and digested products were then resolved on 1.1% agarose gel electrophoresis for the Q192R genotypes (AA-156 bp and 33 bp, AG-189 bp,156 bp, and 33 bp, GG -189 bp as depicted in Fig. 1).

Statistical analysis
The Statistical Package for the Social Sciences for Windows (SPSS), version 25.0 (Chicago, IL, USA), was used to analyze the data. The P value of < 0.05 was considered statistically significant. The Analysis of variance (ANOVA) test was used to determine statistically significant differences between the means of multiple independent groups. Any deviation from Hardy-Weinberg equilibrium was evaluated by the chi-square test. Odds Ratio (OR) was calculated with 95% confidence interval (CI) to estimate the association between genotypes of PON1 gene polymorphism and CAD.

Results
In this study, we evaluated the association between PON1 rs662 (Q192R) gene polymorphism and serum PON1 levels in 142 participants. Of these, 71 patients were categorized as CAD group, and 71 as controls, on the basis of diagnostic coronary angiography. The mean age of CAD patients and controls was 56.5 ± 10.80 and 50.2 ± 9.50 years, respectively. The CAD group consisted of 75% males and 25% females. The case and the control groups were age and gender-matched. The demographic, clinical, and baseline biochemical characteristics of the study population are depicted in Table 1.
There was no significant difference between the frequencies of diabetes mellitus (DM) and hypertension between the groups. The CAD group had a significantly higher number of patients with smoking habits and a family history of CAD. In the lipid profile, serum total cholesterol, LDL-C, and triglycerides were not significantly different between the groups. However, HDL-C was significantly lower and the atherogenic index of plasma (AIP) was found to be significantly higher in the CAD patients. Further, serum levels of both total creatine kinase and its MB fraction were higher in the CAD group.
Serum PON1 levels in CAD patients and controls are shown in Table 2. The serum PON1 levels were significantly lower in the CAD group than in the control group (7.79 ± 3.16 vs. 10.79 ± 3.19 ng/mL, p < 0.0001).    The distribution of genotypes and alleles for the PON1 rs662 (Q192R) polymorphism is shown in Table 3. The frequencies of the AA, AG, and GG genotypes in the CAD patient and control group were 35.2%, 21%, and 35.2% vs. 50.7%, 43.7%, and 4%, respectively. Logistic regression analysis showed that the homozygous GG genotype of PON1 rs662 SNP has ninefold increased risk of developing CAD in the Indian population (odds ratio OR = 9.0, 95% CI 2.79-29.06, p = 0.0002). A significantly higher frequency of the G allele was also observed in CAD patients than in controls (OR 2.64, 95% CI 1.61-4.33, p = 0.001).
The inter-genotypic variation of the serum PON1 levels in the CAD and control group is shown in Table 4. Our results showed that the serum PON1 level in AA, AG, and GG genotypes of the CAD group was 9.8 ± 3.10, 8.04 ± 3.12, and 5.57 ± 1.26 ng/mL, respectively, whereas the respective values in different genotypes were 12.84 ± 2.71, 9.04 ± 1.92 and 5.91 ± 1.18 ng/ mL in the control group. The inter-genotypic difference in the serum PON1 values was statistically significant in both the CAD as well as the control group.
Our data also demonstrated that there was no significant difference between the PON1 level of the GG genotype of the CAD group when compared with that of the GG genotype of the control group (Table 5). Moreover, the PON1 level was significantly lower in the CAD group as compared to the controls in individuals with AA or AG genotypes, indicating the role of other factors in influencing the PON1 levels.
In our study, CAD patients were further subdivided into different subgroups on the basis of the degree of coronary vascular stenosis (Table 6), and the number of involved coronary vessels ( Table 7). The mean serum PON1 levels significantly decreased as the degree of coronary vascular stenosis increased from mild to severe in the CAD group. When compared within any two groups, the PON1 level was significantly different between the stenosis < 50% and stenosis > 90% groups, and also in the stenosis < 50-69% and stenosis > 90% groups. However, there was no difference between the stenosis < 50% and stenosis 50-69% groups or between the stenosis 50-69% and stenosis 70-90% groups. The PON1 levels did not differ significantly in CAD patients with the different number of involved coronary vessels (p = 0.32).

Discussion
Asian Indians have more susceptibility to develop CAD and more so, a decade earlier than the Caucasian population [12]. Further, a recent study stated that the risk of developing cardiovascular disease among North Indians is higher as compared to other parts of India [13]. It needs to be emphasized that the conventional risk factors have somewhat not been able to justify completely the excess risk of CAD among Indians, raising the possibility of a genetic vulnerability. PON1 Q192R gene polymorphism has been implicated in increased CAD risk in many populations. However, there is a relative paucity of studies on the genotypic and allelic distribution of    the PON1 Q192R genotypes in CAD in India. Thus, we examined the implications of this single nucleotide polymorphism (SNP) and its association with PON1 concentration in CAD patients and controls. Oxidative damage plays an important role in chronic inflammatory diseases such as CAD. PON1 is atheroprotective due to its antioxidant and anti-inflammatory role in the body [14,15]. It prevents LDL oxidation, thereby reducing the risk of atherosclerosis. [16]. The diverse genetic variations in the PON1 gene may affect its functional role and, thereby, its ability to protect against CAD. Our findings showed that PON1 levels in CAD patients were significantly lower as compared to the controls. Our data also indicated that individuals with GG genotype had a ninefold amplified risk of developing CAD. Furthermore, the serum PON1 level in GG genotypes was significantly lower as compared to PON1 level in AA or AG genotypes in both CAD as well as the control group. This demonstrates that individuals with GG genotype have lower PON1 levels, thereby indicating a significant role of PON1 Q192R gene polymorphism in enhanced CAD risk. Many studies have documented the association between PON1 Q192R gene variants with increased susceptibility to CAD; however, the results have been inconsistent among different population groups. Table 8 lists down some of the relevant studies showing the relationship of this particular polymorphism in distinct ethnic groups.
A meta-analysis study by Saadat [24] reported that RR genotype is significantly associated with increased PON1 enzyme activity in healthy individuals; however, a remarkable heterogeneity between the included studies was also present. As evident from a multitude of studies, the frequency of the polymorphic alleles and haplotypes of PON1 differs among various ethnic groups [24,25]. The conflicting results in the association of the Q192R polymorphism with the increased risk of CAD may also be due to variations in sample size, genotyping methods, pathological states, and diverse environmental influences [26][27][28].
Chronic inflammation plays a pivotal role in the progression of CAD. Among the plasma lipoproteins, ox-LDL has an extensive role in inflammatory and atherogenic processes [29]. There are several antioxidant enzymes in our body, of which PON1, with its anti-atherogenic properties, has been widely studied. PON1 is a 354 amino acid calcium-dependent protein mainly synthesized in the liver, and present in blood in association with HDL. Reduced PON1 levels may lead to an increase in the circulating levels of ox-LDL, thereby increasing the susceptibility to CAD [30,31]. Ox-LDL significantly contributes to key processes in the pathophysiology of CAD such as the formation of foam cells [32], endothelial dysfunction [33], oxidative stress [34], and microvascular injury [35].
The serum levels of PON1 along with its enzymatic activity are important determinants of the degree of oxidative stress, thereby playing an important role in diseases characterized by chronic inflammation such as CAD. As with other proteins, the expression and catalytic activity of PON1 are greatly influenced by SNPs [36,37]. Among these SNPs, PON1 rs662 gene polymorphism has been implicated in modulating the functionality of the PON1 enzyme [38]. A recent study reported that Q alloenzyme has more ability to hydrolyze oxidized lipids, and also protects LDL particles against the oxidative processes more efficiently than R alloenzyme [39]. The R allele variant has been associated with a less active isoform of PON1 against lipoprotein oxidation, resulting in an enhanced risk of vascular disease [40]. A meta-analysis study found that the R allele gene variant is significantly associated with increased levels of ox-LDL, triglycerides, Association of paraoxonase-1 Q192R (rs662) single nucleotide variation with cardiovascular risk in coffee harvesters of Central Colombia [22] Central Colombia Q allele 7 The Gln-Arg192 polymorphism of human paraoxonase gene is not associated with coronary artery disease in Italian patients [23] Italy No association total cholesterol, and LDL-C, which may lead to a higher risk of CAD [22]. In our study, the aggregate allele frequency in the total study population was 61.3% for wild allele and 38.7% for polymorphic allele, which corroborated with global frequency. Our study demonstrated that within the GG genotype, PON1 levels did not differ significantly between CAD and control group, indicating that GG genotype is a major genetic determinant for decreased PON1 levels. However, the PON1 level was significantly lower in the CAD group as compared to the controls within AA or AG genotypes. Thus, it can be interpreted that although the serum PON1 level was affected by PON1 rs662 gene polymorphism and is significantly lower in individuals with GG genotype, however, there are other exogenous factors also including environmental modulators, which affect PON1 levels in CAD patients. It is well known that a multitude of environmental factors (such as diet, lifestyle, smoking, and alcohol consumption) have a significant effect on PON1 activity. Some pathological states also influence the enzyme levels as well as antioxidant activity of PON1 independently from the genotype, resulting in a state of oxidative stress, and accelerated development of atherosclerosis [26][27][28]41]. Nevertheless, a recent study stated that the genetic determinants of the PON1 phenotype have stronger influences than the contributions of the lifestyle risk factors [42]. Thus, it is suggested that evaluation of genotype, as well as serum PON1, might be potential markers of CAD [43].
Our study showed that the PON1 levels significantly decreased in severe (> 90%) luminal stenosis, irrespective of the number of involved coronary vessels. A recent study found a positive association between low PON1 activity and CAD severity, particularly in smokers and diabetes patients [44]. Thus, serum PON1 may have a prognostic value in determining the severity of CAD.

Conclusions
Our findings support the association between PON1 rs662 (Q192R) gene polymorphism and low PON1 levels in CAD patients. Individuals with GG genotype have low PON1 levels compared to AA and AG genotypes. Polymorphic G allele may be responsible for altering the functionality of the PON1 enzyme, leading to an increased risk of CAD. Moreover, as CAD is a multifactorial disease due to interaction between various environmental and genetic influences, there may be other modulators also, that alter the serum PON1 levels in these individuals. Moreover, PON1 may be of prognostic importance in severe coronary vascular stenosis.
The limitation of our study was the relatively small sample size, which represented only a fraction of CAD patients admitted to a tertiary care hospital in the given time period. However, our study reveals the importance of additional risk factors, including genetic polymorphisms, and valuable markers such as PON1 levels, which may be useful to predict CAD in the Indian population. Prospective studies with a larger population are warranted to have a better understanding of the role of PON1 in enhanced CAD susceptibility. PON1 as a potential therapeutic candidate is also a point of interest for further studies.