Ling-Yun Zhou1, Long Shen2, Xiao-Cong Zuo1 and Zhi-Chun Gu3,4*
1Clinical Pharmacy and Pharmacology Research Institute, Third Xiangya Hospital, Central South University, China
2Department of Cardiology, Renji Hospital, School of Medicine, Shanghai Jiaotong University, China
3Department of Pharmacy, Renji Hospital, School of Medicine, Shanghai Jiaotong University, China
4School of Pharmacy, Shanghai Jiaotong University, China
Received: 20 August 2018
Accepted: 15 September 2018
Version of Record Online: 27 September 2018
Zhou L-Y, Shen L, Zuo X-C, Gu Z-C (2018) PON-1 Q192R (rs662) Gene Polymorphism and The Risk of Major Adverse Cardiovascular Events in Clopidogrel-Treated Patients with Coronary Heart Disease: A Systematic Review and Meta-Analysis. J Cardiol Catheter 2018(1): 01-15.
Correspondence should be addressed to
Zhi-Chun Gu, China
Copyright © 2018 Zhi-Chun Gu et al. This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and work is properly cited.
Aims: To determine the association between the PON1 Q192R (rs662) gene polymorphism and the risk of Major Adverse Cardiovascular Events (MACE), especially the risk of Stent Thrombosis (ST), in clopidogrel-treated patients with Coronary Heart Disease (CHD).
Methods: We searched academic databases, book chapters and proceedings of international meetings for clinical trials investigating the relationship between the PON1 Q192R (rs662) gene polymorphism and the clinical outcome of CHD patients receiving clopidogrel as antiplatelet treatment. The pooled Odds Ratios (ORs) with 95% Confidence Interval (CI) were calculated using the STATA 12.0 software with random-effects model.
Results: We identified 18 clinical trials with a total number of 16801 CHD patients, seven using a case-control design (n=3098) and eleven a prospective cohort design (n=13703). Meta-analysis revealed that the PON1 Q192R (rs662) genetic polymorphism was not associated with an increased risk of MACE [Q allele vs. R allele: 1.10 (0.91-1.34), QQ vs. QR+RR: 1.08 (0.83-1.40), QQ+QR vs. RR: 1.19 (0.88-1.60), QQ vs. RR: 1.22 (0.83-1.79), QQ vs. QR: 1.05 (0.82-1.34), QQ+RR vs. QR: 0.98 (0.79-1.22)] in CHD patients treated with clopidogrel. However, when difference in ethnicity was taken into account, significant influence of PON1 Q192R (rs662) genetic polymorphism on the risk of ST was observed in Caucasus patients under PON1 Q192R (rs662) dominant [QQ vs. QR+RR: 1.69 (1.05-2.73), QQ vs. QR: 1.55 (1.06-2.28)], but not in Asians.
Conclusion: This meta-analysis suggests that the PON1 Q192R (rs662) polymorphism has no major impact on the risk of MACE. However, PON1 192 QQ genotype may be a determinant of high risk of ST in Caucasus CHD patients receiving clopidogrel, but not in Asians.
Keywords: Clopidogrel; Coronary Heart Disease; Gene Polymorphism; Major Adverse Cardiovascular Events; Stent Thrombosis; PON1
|MACE||:||Major Adverse Cardiovascular Events|
|CHD||:||Coronary Heart Disease|
|PCI||:||Percutaneous Coronary Intervention|
Clopidogrel is an irreversible inhibitor of the adenosine diphosphate P2Y12 receptor and can prevent recurrent ischemic events in CHD patients . Although novel inhibitors of the platelet P2Y12 receptor have more rapid, potent, and consistent inhibitory effect on platelets compared with clopidogrel, the reduction in ischemic complications with prasugrel or ticagrelor treatment is at the expense of excess major bleeding [2,3]. Furthermore, cost-effectiveness analyses reveal that, in the developing countries, such as China, patients without drug plans cannot afford the higher cost of novel agents and might less likely to be compliant . Thus, clopidogrel remains a better choice for those with increased bleeding risk or in developing countries. However, about 4~30% of the patients treated with clopidogrel display no or a low anti-platelet response [5,6], then inducing an increased risk of recurrent ischemic cardiovascular events .
The mechanisms of this phenomenon are not fully elucidated. One source of the variability may be the metabolism of clopidogrel, which is an inactive prodrug that requires enzymatic bioactivation into its active thiol metabolite before interacting with the P2Y12 receptor on blood platelets. Clopidogrel bioactivation is a two-step process in which the Cytochrome P450 (CYP450) system plays a major role . As a result, mutations in the genes for these CYP450 enzymes may affect clopidogrel responsiveness. Among these genes, CYP2C19 is of great concern. Previous studies have revealed that mutation of CYP2C19 gene was associated with the different platelet response to clopidogrel in CHD patients [9,10]. However, recent researches elucidated that less than 10% of the observed variability of clopidogrel responsiveness in cardiovascular patients could be explained by CYP2C19 genotype polymorphism .
Paraoxonase-1 (PON1) was reported to be strongly involved in clopidogrel bioactivation . Moreover, a common missense variant in PON1 Q192R (rs662) was a major determinant of clopidogrel efficacy. Several studies have been performed to evaluate the association between PON1 Q192R (rs662) gene polymorphism and the risk of MACE in CHD patients treated with clopidogrel [13-17]. The results from Asians demonstrated that PON1 192Q allele is an independent predictor of worse cardiovascular outcome in patients receiving drug-eluting stents . However, a rebuttal response by Dansette and Camps has stated that the antithrombotic activity of clopidogrel is mainly due to one of its cis-thiolmetabolites whose formation is major dependent on CYP enzymes and only to a minor extent on esterases such as PON1 [16,17]. In addition, above associations were also challenged by a meta-analysis of 11 trials, published up to July 2012, which suggested that PON1 Q192R polymorphism has no major impact on the risk of MACE in Western population .
However, it is worth noting that a high number of clinical studies, especially Asian population, have finished in order to test the determinate effect of the PON1 Q192R in clopidogrel-treated patients recently [15,19-24]. Thus, it is necessary to conduct an updated systematic review and meta-analysis of the eligible studies from different ethnic populations to determine the association between PON1 Q192R (rs662) gene polymorphism and risk of MACE, especially the risk of ST, in patients with CHD who use clopidogrel.
Ethical approval and patient consent were not necessary as the present study was a meta-analysis. We carried out a comprehensive search with no language limitations. The search was based on electronic academic databases (Medline, Embase, Cochrane Library Database, Wanfang database, CNKI database and VIP database). We also searched for book chapters, gray literature and unpublished work, such as conference papers, working papers, and reports published by research institutes or government organizations. The search was last updated on 5 July 2018. Key terms used for the systematic search were (‘PON1’ or ‘PON-1’ or ‘paraoxonase 1’ or ‘paraoxonase-1’) and ‘clopidogrel’. Articles were selected on the basis of the abstract before examining the full text. In addition, the reference lists of selected articles were hand-searched to identify additional relevant reports. Additionally, we wrote to authors asking for details of other published and unpublished studies. The reviewers were not blinded to the journal, authors or institution of the publications, as this has been shown to be unnecessary.
The following criteria were used to determine eligibility for including studies: (1) the study must be a clinical study; (2) the study must address the relationship between the PON1 Q192R (rs662) gene polymorphism and the clinical outcome of patients receiving clopidogrel as antiplatelet treatment; (3) all patients must have conformed to the diagnostic criteria of CHD; (4) the study must provide sufficient information about the frequencies of the PON1 Q192R (rs662) gene polymorphism. Article that did not meet the inclusion criteria were excluded. When authors published several studies using the same subjects, either the most recent or the largest sample size publication was included .
To assess the specificity and characteristics of the identified studies, we abstracted information from each report on the surname of first author, year of publication, country of origin, ethnicity, the total sample size, the study design, undergoing Percutaneous Coronary Intervention (PCI) or not, the type of clinical endpoint, and study population characteristics (age, sex, vascular risk factors, patients with acute events or stable ischemic disease, type of stent, and 192Q allele frequency).
Major clinical outcome was defined in terms of MACE, including the occurrence of death, non-fatal Myocardial Infarction (MI), ST, or stroke, and was expressed as the number of patients concerned in each genotype group. Secondary clinical outcome was ST, which was defined from the definition of The Academic Research Consortium . These numbers were extracted from the articles to prepare 2 × 2 tables.
Two authors each independently implemented the search strategy, selected the studies and recorded the abstracted data. In cases of conflicting evaluations, disagreements were resolved through discussion and careful re-examination of the full text by the authors.
Methodological quality was evaluated separately by two authors using the Newcastle-Ottawa Scale (NOS) criteria . The NOS criteria is based on three aspects: (1) subject selection: 0-4; (2) comparability of subject: 0-2; (3) clinical outcome: 0-3. NOS scores range from 0 to 9 with scores ≥7 indicating good quality .
Meta-analysis was performed using the STATA statistical software (version 12.0, Stata Corporation, College Station, TX, USA). Descriptive data on the patients were recorded for each study as the mean and standard deviation or percentage. The OR for ischemic events associated with the PON1 Q192R (rs662) genotype was assessed in each study, along with its 95% CI. The pooled OR was computed by using the method of the inverse of the variance with random-effects model, and the hypothesis OR =1 was tested . Heterogeneity among studies was estimated with the Cochran’s Q-statistic and I2 tests. Predefined potential factors of heterogeneity, such as study design, sample size, ethnicity and undergoing PCI or not, were tested by comparing subgroups with random-effects model. Meanwhile, we did further interaction analysis for different subgroup. Meta-regression analysis was also performed to test the influence of stent type. In order to evaluate the influence of single study on the overall estimate, a sensitivity analysis was performed. The potential publication bias was explored for the OR by visual interpretation of the funnel plot, and the asymmetry of the funnel plot was checked with Begg’s test. Assessment of the quality of evidence was generated with the GRADEpro software.
The flow of references through the review is shown in figure 1. Initially, our search strategy identified 255 articles. We reviewed the titles and abstracts of all articles and excluded 193 articles. After systematically reviewing the remaining full texts, we excluded another 44 articles. Finally, eighteen clinical studies met our inclusion criteria for qualitative data analysis, with a total of 16801 CHD patients [12-15,19-24,29-35]. Characteristics of subjects are shown in table 1. Overall, four studies were conducted among Asians and fourteen studies among Caucasians. Genotyping was performed using the polymerase chain reaction-ligation detection reaction or TaqMan assay methods. The NOS scores of all included studies were ≥6, meaning that the quality of the included studies were considered to be moderate to high (Table S1).
Thirteen studies (n=14658) assessed the risk of MACE, two in a case-control design (n=956) and eleven in a prospective cohort design (n=13702). Nine studies (n=6520) assessed the risk of ST, five in case-control design (n=2142) and four in prospective cohort design (n=4378).
In terms of MACE, our results indicated that the PON1 Q192R (rs662) gene polymorphism was not correlated with an increased risk for MACE in CHD patients receiving clopidogrel as antiplatelet treatment (Q allele vs. R allele: 1.10 (0.91-1.34), QQ vs. QR+RR: 1.08 (0.83-1.40), QQ+QR vs. RR: 1.19 (0.88-1.60), QQ vs. RR: 1.22 (0.83-1.79), QQ vs. QR: 1.05 (0.82-1.34), QQ+RR vs. QR: 0.98 (0.79-1.22); respectively). Furthermore, subgroup analyses by study design, ethnicity, sample size and undergoing PCI or not demonstrated that the PON1 Q192R (rs662) polymorphism was not correlated with an increased risk of MACE in CHD patients (Table 2 and Figure 2).
Figure 2: Meta-analysis forest plots of the correlations between the PON1 Q192R (rs662) gene polymorphism and MACE: A: Q vs R; B: QQ vs QR+RR; C: QQ+QR vs RR; D: QQ vs RR; E: QQ vs QR; F: QQ+RR vs. QR. The horizontal solid lines mean the Confidence Intervals (CI) of OR in each population. And if the upper limit of OR exceed the top scale, arrow would replace the solid line. The size of solid squares corresponds to the weight of the study in the meta-analysis. The lengths of the hollow diamonds represent the CI of the pooled OR.
In terms of ST, as shown in table 3 and figure 3, no significant positive correlation was found between the PON1 Q192R (rs662) polymorphism and an increased risk of ST in CHD patients receiving clopidogrel as antiplatelet treatment (Q allele vs. R allele: 1.37 (0.94-1.99), QQ vs. QR+RR: 1.50 (0.97-2.33), QQ+QR vs. RR: 1.44 (CI 0.72-2.88), QQ vs. RR: 1.68 (0.71-3.95), QQ vs. QR: 1.43 (0.99-2.07), QQ+RR vs. QR: 1.25 (0.94-1.66); respectively). However, subgroup analysis by ethnicity demonstrated that the PON1 Q192R (rs662) polymorphism was associated with an increased risk of ST in CHD patients receiving clopidogrel among Caucasus patients with PON1 Q192R (rs662) dominant model, heterozygous model or over-dominant model, but not in Asians (QQ vs. QR+RR: 1.69 (1.05-2.73), QQ vs. QR: 1.55 (1.06-2.28), QQ+RR vs. QR: 1.29 (1.01-1.64); respectively).
Figure 3: Meta-analysis forest plots of the correlations between the PON1 Q192R (rs662) gene polymorphism and ST: A: Q vs R; B: QQ vs QR+RR; C: QQ+QR vs RR; D: QQ vs RR; E: QQ vs QR; F: QQ+RR vs. QR. The horizontal solid lines mean the Confidence Intervals (CI) of OR in each population. And if the upper limit of OR exceed the top scale, arrow would replace the solid line. The size of solid squares corresponds to the weight of the study in the meta-analysis. The lengths of the hollow diamonds represent the CI of the pooled OR.
Assessment of the quality of evidence was generated with the GRADE. Then a modified table was made to summarize the findings regarding the endpoints relating to MACE and ST (Table S3).
A sensitivity analysis suggested that no single study significantly influenced the pooled ORs of risk of MACE or ST (Figure S1 and Figure S2). However, most of the statistical heterogeneity was explained by two studies reported in one publication . When each of these studies was removed, I2 fell from 36.5%~62.9% to 11.4%~58.2% for the total group, whereas removal of any of the other studies only marginally affected the level of heterogeneity. When both of these two studies were removed, I2 fell from 36.5%~62.9% to 0%~25.8% for the total group, whereas removal of any of the other studies only marginally affected the level of heterogeneity. No publication bias was detected on funnel plot with Begg’s test (Figure 4 and Figure 5).
To our knowledge, this is the first meta-analysis to extensively investigate the association between the PON-1 Q192R (rs662) gene polymorphism and the risk of MACE, especially the risk of ST, both in clopidogrel-treated Caucasus patients and Asian patients with CHD.
Despite widespread use of clopidogrel, large inter-individual variability in clopidogrel response has been reported . To date, most researches have focused on genetic polymorphism of enzymes and transporters involved in clopidogrel metabolic activation [9-12]. Although many CYP450 enzymes have been implicated in the metabolic activation of clopidogrel, CYP2C19, as a major determinant of active thiol metabolite exposure, is generally considered to be the most important. However, recent researches elucidated that less than 10% of the observed variability of clopidogrel responsiveness in cardiovascular patients could be explained by CYP2C19 genotype polymorphism .
A new player in clopidogrel pharmacogenetics was recently reported by Bouman et al. , who identified PON1 as a potential major determinant of the efficiency of clopidogrel bioactivation and this drug’s clinical efficacy in European patients. In a series of biochemical, clinical and epidemiological studies, PON1 was shown to be the rate-limiting enzyme in clopidogrel metabolic activation . In a case-cohort study of patients who underwent PCI and received clopidogrel, PON1 Q192R (rs662) heterozygotes and 192 QQ homozygotes had reduced plasma PON1 activity, lower plasma concentrations of active thiol metabolite, lower platelet inhibition, and a 4- and 12-fold increased incidence of ST respectively compared with homozygous wild-type 192 RR patients .
Despite early excitement about the potential significance of this work, the effect of PON1 on clopidogrel efficacy found in the studies by Bouman et al.,  and Park et al.,  has recently been refuted. Subsequently, numerous clinical studies have failed to confirm these results and detailed biochemical studies have suggested that PON1 can metabolize 2-oxo-clopidogrel by hydrolytic opening of the thiolactone ring but this leads to the pharmacologically inactive endo-thiol isome . Hitherto, no clear correlation between PON1-Q192R (rs662) polymorphism and the risk of MACE or ST in CHD patients treated with clopidogrel as antiplatelet treatment has been demonstrated.
The results of our meta-analysis insights from Thirteen studies of 14658 patients do not support an increased risk of MACE of PON1 Q192R (rs662) polymorphism in patients receiving clopidogrel as antiplatelet treatment, independently of the study design (case-control or prospective cohort), ethnicity (Caucasians or Asians), sample size (n≥300 or n<300) and whether undergoing PCI or not. Those results suggest that the PON1 Q192R (rs662) gene polymorphism may not a determinant of MACE in both Caucasian and Asian CHD patients receiving clopidogrel. Our results are in line with the lack of correlation between PON1 activity and P2Y12 receptor inhibition (evaluated with the specific VASP assay) in 538 stable cardiovascular patients as well as with the lack of correlation between PON1 activity and clopidogrel active metabolite or other in vitro assays showing no impact of PON1 inhibition on the production of the clopidogrel active metabolite . The trend towards an association between the PON1 Q192R (rs662) gene polymorphism and MACE observed in our meta-analysis was mainly driven by one study, which accounted for most of the observed heterogeneity . A sensitivity analysis, in which we removed one study at a time, showed that a large part of the statistical heterogeneity was associated with the one study by Bouman et al.,  whereas heterogeneity was not noticeably affected when the study was removed.
The main reason for the discrepancy between the original work Bouman el al.,  and most subsequent studies may be that the paper of Bouman et al., used a flawed HPLC/MS method to measure the active thiol metabolite H4 of clopidogrel. However, their method nearly only detects the endo-thiol metabolite which is totally inactive on the P2Y12 receptor [16,17,39-50]. Previous studies, particularly by Dansette et al., [16,37,42-44] have elucidated the metabolic steps from clopidogrel to the active thiol acid in details. The metabolism of clopidogrel undergoes through two different pathways in the liver. Up to 85% of the administered parent drug might be transformed to carboxylic acid derivative of clopidogrel, which is inactive . For many years, because of the very low plasma concentration of the parent drug, the determination of this inactive metabolite was used for studying the pharmacokinetics of clopidogrel . However, the antithrombotic effect of clopidogrel depends mainly on its active thiol metabolite which was bio-transformed through a two-step hepatic pathway involving CYP450 isoenzymes including CYP2C19 and CYP3A4 . There were four diastereoisomers of the thiol metabolites in human body: H1, H2, H3 and H4. Furthermore, vitro studies confirmed that H4 isomer can be considered to be the only active isomer of thiol metabolites . However, only few HPLC-MS/MS assays have been published for determination of derivatized thiol metabolites in human plasma [54-57]. And most of the methods can’t distinguish between the four different isomers. According to the HPLC/MS method reported by Bouman et al., their method could only detect the endo-thiol, but not H4 isomer of clopidogrel. As all the mentioned above, though the paper of Bouman et al., was at the origin of a number of following clinical studies, we should re-interpret their conclusions. Differences in populations may be the second issue; the frequency of the Q192 allele is somewhat lower in their cohort study (63.9%) than in any of the other studies [21,23-26,31-35]. The observed clinical differences between PON1 gene variants may be due to clopidogrel-unrelated mechanisms, as PON1 plays a direct effect on atherogenesis by protecting low-density lipoprotein from oxidation . This may partly explain the trend towards an association of the PON1-Q192R variant with MACE, best seen in four studies in addition to Bouman’s studies (Figure 2) [15,20]. It could also be that PON1 has an effect on the steady state concentration of the intermediary thiolactone.
Differences in clinical endpoints and ethnicity across studies might also be involved. Previous studies and meta-analysis evaluating the influence of PON1 Q192R (rs662) polymorphism on clinical outcomes have been confined mostly to Western populations [12-14,18,20,21,29-31]. In our meta-analysis, most of the studies addressing the association of the PON1 Q192R (rs662) variant with clinical events involved composite endpoints whose pathogenetic mechanisms are more intricate. Although, consistent with previous studies, we also did not observe a positive association between the PON1 Q192R (rs662) gene polymorphism and the development of ST in CHD patients undergoing antiplatelet treatment with clopidogrel, we conducted the subgroup analyses of eligible studies from different ethnic populations to determine the association between the PON1 Q192R (rs662) polymorphism and the risk of ST, in patients with CHD who use clopidogrel. Subgroup analysis by ethnicity demonstrated that a significant association between PON1-Q192R (rs662) gene polymorphism and the risk of ST was found among Caucasus patients with PON1 Q192R dominant model, heterozygous model or over-dominant model, but not in Asians. It revealed that there are ethnic differences in the effects of the PON1 Q192R (rs662) gene polymorphism on the risk of ST in clopidogrel-treated patients, but not all clinical outcomes. In previous study, Bouman et al., also found a strongest association with definite ST, while the association with all MACE was smaller. The low incidence of ST, small sample size and different prevalence of PON1 Q192R (rs662) allele may partly explain the different results between the different subgroups. Low incidence of ST and small size of clinical studies in Asian CHD patients may possibly leading to an underestimation of the clinical importance of the PON1 Q192R (rs662) genotype. It is worth noting that the distribution of the PON1 polymorphisms varies with ethnicity. The frequency of the 192R allele increases the further from Europe a population originates, the frequency in Caucasians of 15-30% increases to 70-90% in Far Eastern Oriental and Sub-Saharan African populations . In the southern USA African-Americans are 5 times more likely to be RR than Caucasians . These ethnic differences in SNP distribution can lead to large activity differences between populations . The results of our meta-analyses show that there are approximately 63.9%~70.9% 192 Q allele in Caucasians and 21.0%~37.2% 192 Q allele in Asians, consistent with previous report. Ethnic heterogeneity across the different studies, resulting in genetic differences that might have influenced the response to clopidogrel . Due to the very low frequency of 192 RR patients, there was no significant association between PON1 Q192R (rs662) genetic polymorphism and the risk of ST among Caucasians under recessive and homozygous models. To identify this result, further studies with large sample size may be needed to get the results with reliable statistical power.
Furthermore, CHD patients may co-administrated with different drugs, resulting in different drug-drug interactions. Drug-drug interactions, mainly via CYP3A4/5, are another important issue when analyzing the variability of clopidogrel responsiveness [61,62]. For example, platelet inhibition by clopidogrel is attenuated by co-administration of ketoconazole, a known CYP3A4/5 inhibitor, or atorvastatin, a competitive inhibitor, though it is debated for this latter drug [63,64]. Conversely, increased platelet inhibition is observed in hypo-responsive patients when CYP3A4/5 activity is induced by St. John’s Wort, an herbal remedy used for the treatment of depression . Significant pharmacokinetic interactions also exist with proton pump inhibitors, probably through a CYP2C19 inhibition [63,66,67]. Hence, an imbalance of co-medication with drugs affecting CYP450 activities across genotype groups might have influenced the findings.
The present meta-analysis has some limitations. First, the numbers of clinical studies and sample size of studies in CHD Asians still too small to get a reliable result. Second, studies enrolled in the meta-analysis were observational studies, this conclusion should be interpreted cautiously, and hence high-quality and adequately powered RCTs are warranted. Third, the inclusion criteria of cases and controls were not well defined in all included studies, which might have also influenced our results. Last, because of the nature of the meta-analysis that has been conducted among published studies and not on individual patients’ data, we were not able to obtain information on some of the relevant issues, such as different clinical end-points, comorbidities or concomitant therapies.
Our meta-analysis provides evidence that the PON1 192 QQ genotype may increase the risk of ST in Caucasian CHD patients receiving antiplatelet treatment with clopidogrel. However, due to the limitations mentioned above, high-quality and adequately powered RCTs are still needed to provide a more representatively statistical analysis.
This work was supported by the Research Funds of Shanghai health and family planning commission (20184Y0022), Program for Key Discipline of Clinical Pharmacy of Shanghai (2016-40044-002), Program for Key but Weak Discipline of Shanghai Municipal Commission of Health and Family Planning (2016ZB0304), and China Health Promotion Foundation (KNKT-ZX-1601).
Copyright © 2019 - All Rights Reserved - ocimumpublishers.com