P2Y 12 receptor gene polymorphism and the risk of resistance to clopidogrel: A meta-analysis and review of the literature

A number of investigators have evaluated the association between T744C, G52T and C34T polymorphisms in the P2Y 12 receptor gene and clopidogrel resistance (CR), but the results of their research are controversial. To quantify the evidence addressing this issue, we performed a meta-analysis of all available studies to evaluate the above association between the 3 different P2Y 12 genotypes and CR in patients suffering from cardiovascular diseases. This study included articles up to October 2015. We performed a systematic search of PubMed, Embase, Web of Science, Cochrane database, China National Knowledge Infrastructure (CNKI) and WanFang database. Articles meeting the inclusion criteria were included and accumulated by meta-analysis including 5769 participants from 15 individual studies. For G52T polymorphism, a significant relationship between the P2Y 12 receptor gene and CR was found under the dominant genetic model (p < 0.05). There was a clear positive correlation between the C34T polymorphism and CR under the dominant, recessive, additive genetic models, respectively (p < 0.05). The evidence from the present meta-analysis indicates that P2Y 12 receptor gene C34T and G52T polymorphism might be a risk factor for the poor response to the platelet in patients on clopidogrel therapy, whereas a lack of association was found for T744C polymorphism examined by various genetic models.

Increased platelet activation and thrombus formation are involved in the development and progression of cardiovascular diseases (CVDs). 1 Current oral anti-platelet agent thienopyridine clopidogrel inhibits adenosine diphosphate (ADP) binding to platelet ADP receptor P2Y 12 on the platelet surface, and thus inhibits platelet aggregation. P2Y 12 has been shown to trigger platelet activation when stimulated in this receptor. Clopidogrel is effective in decreasing platelet activation and the subsequent risk of atherosclerosis-related CVDs including myocardial infraction, coronary heart disease and ischemic stroke. 2 However, in recent years, the concept of clopidogrel resistance (CR) or poor responsiveness, is increasingly evoked in the cardiac literature. 3 The mechanisms of CR have not been fully characterized but are likely to be multifactorial, involving possible genetic polymorphisms, drug interaction and variable absorption or metabolism. 4 Five polymorphisms (T744C, C34T G52T, ins801A and C139T) of the P2Y 12 gene have been identified by Fontana et al. 5 It has been proposed that genetic polymorphisms of the platelet surface receptor affect the responsiveness to clopidogrel. Some have suggested that polymorphisms of T744C and C34T G52T contribute to CR. [6][7][8] However, this proposal is controversial, because other studies have shown that the correlations between the mutational statuses and poor response to clopidogrel therapy were not statistically significant. 7,9 Thus, in the present study, a meta-analysis including 5769 patients with CVDs was performed to clarify whether or not clopidogrel response to the platelet may be affected by P2Y 12 receptor gene polymorphism in patients with various types of CVDs. This study may help to identify the correct therapeutic approach for each individual patient with CVDs in order to maximize the therapeutic effect.

Search strategies
Published studies on the association between P2Y 12 receptor gene polymorphism and CR were retrieved by searching the following English and Chinese bibliographic databases: PubMed, Embase, Web of Science, Cochrane database, China National Knowledge Infrastructure (CNKI) and WanFang database. The search strategy was based on the following keywords: "P2Y 12 " or "T744C" or "C34T" or "G52T" and "polymorphism" or "mutation" or "genotype" or "allele" and "clopidogrel" and "resistance" or "response". The literature search was updated to October 2015.

Study selection
Studies included in the current meta-analysis had to be consistent with these criteria: a) the association of P2Y 12 gene polymorphism and CR was included; b) provides the genotype frequency in the target population; c) was a case-control study; d) all patients had received clopidogrel therapy. Studies were excluded if they were: (1) duplicates of previous publications; (2) based on incomplete data; (3) meta-analyses, letters, reviews, or editorial articles; (4) publications with non-English abstracts. If more than 1 study by the same author using the same case series was published, only the study with the largest sample size or the most recent publication was included.

Data extraction
Two reviewers independently extracted the information, including the name of first author, year of publication, country, disease name, diagnostic standard of CR and genotypes and the frequency in patients with and without CR. In cases of conflicting evaluations, disagreements were resolved through discussions among all of the 5 authors.

Statistical analysis
The strength of the association between P2Y 12 gene polymorphism and CR was represented by odds ratios (OR) and 95% confidence intervals (CI). The OR and 95% CI were calculated according to 3 genetic models of inheritance: dominant (heterozygotes + homozygotes vs wild-type homozygotes), recessive (wild-type homozygotes + heterozygotes vs homozygotes), and additive (heterozygotes + homozygotes vs wild-type homozygotes + + heterozygotes). Heterogeneity between the results of different studies was examined using a χ 2 test. A 2-side value of p < 0.05 was considered statistically significant as previously described. 10 A fixed effects model was used when p < 0.05, and a random effects was used when p > 0.05. All analyses were performed by RevMan, v. 5.2 for Windows (Cochrane Collaboration, Oxford, UK) using our previously described method. 11

Study selection and characteristics
As shown in Fig. 1, 297 relevant studies were identified using the key words by a computerized search of PubMed, Embase, Web of Science, Cochrane database, CNKI and WanFang database. According to the selection criteria as described in methods, 14 studies were included for meta-analysis. Of the studies, there were 9 comparisons for T744C polymorphism, 7 and 6 comparisons for G52T and C34T, respectively. 6,8,9,[12][13][14][15][16][17][18][19][20][21][22] The studies were conducted in France, the United States, China, Croatia, Egypt, and the Czech Republic. The characteristics of the studies included in this meta-analysis are presented in Table 1.

Subgroup analysis and sensitivity analysis
For T744C and C34T polymorphisms, high heterogeneity was observed (I 2 = 63%) according to the reported quantifying heterogeneity approach. 10 Subgroup analysis of the T744C genetic polymorphism was performed to determine the potential sources of the heterogeneity ( Table 2). We classified the studies based on the geographic region (European and American, Asian). In the results, whether geographic region was adjusted or not, this association did not change (p > 0.05). For the G52T polymorphism, we did not perform a subgroup analysis due to the limited number of included studies (only 2 studies included from European and American regions).
We removed one study by Galic et al. in the review due to T744C genotype distribution in the control groups of these studies deviating from the Hardy-Weinberg equilibrium (HWE) and found that the association (OR= 1.17, 95% CI: 0.86-1.60, p = 0.32) was not significantly altered after exclusion of the study, indicating that the result of the meta-analysis was stable. 6 In addition, potential publication bias or heterogeneity was detected using visual as-  13 Lev 14 Wang 18 Sun 17 Galic 6 Zoheir 8 Shi 16 Chen 12

Discussion
The strength of our study is the statistically most extensive studies to date addressing whether the risk of CR is associated with the frequencies of P2Y 12 receptor gene T744C, C34T and G52T genotypes in patients with CVDs. In the current review, 15 independent studies were included, comprised of 1829 patients with CR and 3940 patients without CR. For the first time, we used meta-analysis to summarize that there was probably a significant association between P2Y 12 receptor gene G52T/ C34T polymorphism and CR.
The P2Y 12 receptor, activated by ADP, plays a critical  role in platelet aggregation and is a target of antiplatelet therapeutic agents that has proven therapeutic value. 23 One of the first successful drugs is clopidogrel. Clopidogrel is a prodrug and its metabolite inhibits ADP receptor-mediated platelet aggregation. Clopidogrel is a milestone in the development of antiplatelet therapy and entails a reduction in the risk of atherothrombotic event, which is the leading cause of CVDs. 24,25 However, the concept of CR or poor responsiveness is increasing evoked in the cardiac literature in antiplatelet response. 3 Although numerous association studies reflecting the influence of P2Y 12 receptor gene polymorphisms on CR have been published, the results are controversial, possibly because of variations in heterogeneous population, sample sizes and other issues. A meta-analysis by systematically combining all the results of individual studies increases the power to detect an association. 26 In the current meta-analysis, we did not find a significant association between the T744C polymorphism and CR in various genetic models. This finding suggests that the T744C polymorphism may be not susceptible to CR in patients with CVDs. In contrast, we also found that patients with a higher frequency of homozygous and heterozygous (TT + GT) genotypes of G52T had a higher risk of CR than wild-type (GG) genotypes (p = 0.003). For C34T polymorphism, despite the statistical heterogeneity of the studies associating P2Y 12 receptor gene C34T polymorphism under the dominant genetic model (p = 0.002, Variability in the definitions of CR and different regions of population possibly contributed to the heterogeneity in the comparisons. The use of antiplatelet therapy, including differing doses and lengths of the treatment, may also result in the heterogeneity. Antiplatelet drugs could influence the association of the P2Y 12 gene polymorphisms with clopidogrel resistance as this might affect clopidogrel and ADP binding. 27 In addition, an association was also found for the C34T gene polymorphism determined under the dominant, additive, and genetic models (p < 0.05). Conversely, in a previous study excluded in this meta-analysis, it was shown that there is lack of association between the frequency of C34T polymorphism and the antiplatelet aggregation response of clopidogrel. 28 Our results clearly indicate augmented CR for C34T polymorphism.
Although considerable effects have been found in the current study, there are some inherent limitations. First, the sample size is still relatively small in the current metaanalysis. Although 5489 total participants from 14 individual studies were included in the analysis, < 1600 participants were analyzed in the G52T polymorphism group. Fewer case subjects were included in some studies. Of the included studies, 3 had less than 100 participants. 6,8,19 Second, 3 different definitions of CR were adopted by researchers since a standard definition of CR was not available. This may slightly weaken the comparability of the data. Third, the overall ORs were calculated according to the unadjusted estimates. It has been reported that drugdrug interaction or coexisting polymorphisms of P2Y 12 and other genes may influence CR. 22,29 This may exist in the included studies and could interfere with the metaanalysis. Fourth, different clopidogrel doses (75-600 mg) and various times after initial therapy (6 h-7 days) were used in the included studies. Those may be a confounder in the analysis of the platelet aggregation test. Such confounding factors could not be extracted from the included studies due to a lack of information, but they limit the potential to draw robust conclusions.
In summary, this is the first meta-analysis to provide evidence that P2Y 12 G52T/C34T polymorphism is related to a poor response of clopidogrel in patients, reflected by platelet function assay. In contrast, a lack of association between T744C polymorphism and CR was found. This finding warrants further studies of larger sample sizes and multiple countries for a more sensitivity analysis. Furthermore, this study raises concerns about future studies addressing individual alternative clopidogrel-based antiplatelet therapy for patients with P2Y 12 C34T and/or G52T polymorphism, and encourages a precision medicine approach to the treatment of CVDs.