1. Introduction
Cardiac-cerebral disease is common in clinical practice and is the leading cause of long-term disability and death around the world [Reference Tong, Yang and Ritchey1]. The global challenges of cardiac-cerebral diseases present an enormous health burden. Coronary heart disease and ischemic stroke are two common cardiac-cerebral diseases, which demonstrates a high-frequency of emergency department visits to manage acute and chronic symptoms [Reference Baptiste, Turkson-Ocran, Han, Dennison Himmelfarb and Commodore-Mensah2].
Antiplatelet therapy with aspirin and clopidogrel is frequently used for the secondary prevention of acute coronary syndrome, ischemic stroke, and other related ischemic cardiac-cerebral diseases to reduce recurrent ischemic events [Reference Liu, Hu, Wang, Guan, Chen and Liu3, Reference Hackam and Spence4]. Clopidogrel is an irreversible P2Y12 inhibitor, which is usually used for preventing ischemic complications related to cardiovascular diseases [Reference Elmariah, Doros and Benavente5]. However, many patients experience relapse or bleeding [Reference Lee, Lee and Lee6], which is associated with increased late mortality [Reference Eikelboom, Mehta, Anand, Xie, Fox and Yusuf7, Reference Armstrong, Fu and Chang8]. Clopidogrel resistance (CR), defined as a reduction in platelet aggregation rate by less than 10% from baseline after clopidogrel treatment [Reference Wang, Wang and Chen9], is considered to be critically associated with recurrent events after anti-platelet therapy.
Studies have reported that the potential mechanisms underlying the insufficient responses to clopidogrel involve several aspects, including epigenetic dysfunction (such as improper DNA methylation), clearance of active metabolites, variable absorption of precursor drugs, potential interactions between drugs, genetic polymorphisms of platelet receptors, adenosine diphosphate (ADP)-mediated variability of P2Y12 platelet receptor, and differences in signal transduction pathway of platelet [Reference Ohlmann, Eckly, Freund, Cazenave, Offermanns and Gachet10–Reference Yang, Yu and Xu12]. Additionally, clopidogrel is a prodrug that should be converted into active metabolites by hepatic metabolism through cytochrome (CYP) P450 isoenzymes, including CYP1A2, CYP2C9, CYP2C19, CYP3A, and CYP2B6 [Reference Cresci, Depta and Lenzini13, Reference Rotondo, Giari and Guerranti14]. Among these enzymes above, CYP2C19 plays the most important role in clopidogrel transformation [Reference Lee, Lee and Lee6]. The polymorphisms of CYP2C19 have been demonstrated to be implicated in CR [Reference Su, Li and Tang15, Reference Mega, Close and Wiviott16]. A recent study demonstrated that the CYP2C19∗2 or CYP2C19∗3 alleles were significantly related to attenuated platelet response to clopidogrel and increased CR risk among Chinese patients in the Beijing district. Nevertheless, CYP2C19 polymorphism considerably varies with both ethnic group and geographical location [Reference Zhong, Hou and Li17, Reference Shuldiner, O’Connell and Bliden18].
In this study, we intended to investigate the association of CYP2C19 polymorphisms with CR in patients with coronary heart disease and ischemic stroke among Han and Tibetan populations in Qinghai Province, China. The results may help to guide rational clinical drug use and reduce the incidence of cardiovascular adverse events.
2. Materials and Methods
2.1. Subjects
From June 2019 to January 2020, a total of 91 patients who were diagnosed with coronary heart disease or cerebral infarction in internal medicine of Qinghai Provincial People’s Hospital and had taken dual antiplatelet drugs were included in this study. Among them, 67 patients were ethnic Han and 24 were ethnic Tibetan. This study was approved by the Ethics Committee of Qinghai Provincial People’s Hospital. All participants had signed the informed consent.
2.2. Inclusion and Exclusion Criteria
The inclusion of patients with coronary heart disease was in line with the “coronary heart disease guidelines and expert consensus” in 2019, and patients with stroke or transient ischemic attacks met the “practical diagnosis and expert consensus of ischemic stroke in China” in 2020. The patients were permanent residents or long-time (10 years or more) residents in the Qinghai region. All patients received routine doses of aspirin (100 mg once daily) and clopidogrel (75 mg once daily) for 5–7 days.
The patients with one or more of the following conditions were excluded: allergic or intolerant; contraindications of antiplatelets therapy; rupture and defect of gastrointestinal mucous membrane; inflammation of endocardium or heart valve due to microbial invasion of the body; serious decline in the ability of glomeruli to expel toxins and waste; continuous growth of cancer cells; decreased ejection fraction; combined with pulmonary congestion and inadequate peripheral perfusion with contemplated surgical operation; severe liver disease and/or abnormal coagulation function; and incomplete clinical case data.
2.3. Sample Collection, DNA Extraction, and Whole Exome Sequencing
Blood was collected on an empty stomach the next morning after admission for patients who met the inclusion and exclusion criteria, and routine items such as blood routine, biochemistry, thyroid function, and saccharification were completed. Basic clinical data, such as previous history, medication history, personal history, and others, were collected.
A total of 4 ml fasting venous blood was taken and placed in an anticoagulant tube containing ethylenediamine tetraacetic acid (EDTA), and the specimen was stored at −80°C. DNA was extracted using a TianGen DNA extraction kit (TianGen Ltd, Beijing, China). The DNA concentration was determined by fluorescence quantification. Genomic DNA (l μg) was sheared by sonication, and the fragments with an average size of 150–250 bp were selected by magnetic beads. Then, the fragments were ligated to adapters and amplified by pre-PCR. PCR products were then hybridized and washed with Agilent SureSelect or BGI Hybridization and Wash kits (Agilent, CA, USA), followed by PCR amplification. The reaction conductions were 18 cycles of 98°C for 10 s, 65 C for 30 s, and 72°C for 30 s, followed by a final incubation at 72°C for 5 min, and then hold at 4°C. The primers were CYP2C19∗2: forward 5′-ATT ACAACCAGAGCTTGGCAT-3′, reverse 5′-GTTGATGTCCATCGATTCTTG-3′; CYP2C19∗3: forward 5′-CTGCAATGTGATCTGCTCCAT-3′, reverse 5′-TTCAGGGCTTGGTCAATATAG-3′; and CYP2C19∗17: forward 5′-GATGAATGTGGTATATATTCA-3′, reverse 5′-GAGAACAGGACACCTGTTGGT-3′. The library concentration was measured using the Qubit kit (Invitrogen, USA). Sequencing was performed by the combinatorial probe-anchor synthesis method [Reference Fehlmann, Reinheimer and Geng19]. CYP2C19 genetic polymorphisms of CYP2C19∗2 (rs4244285), CYP2C19∗3 (rs4986893), and CYP2C19∗17 (rs12248560) were recorded.
2.4. Statistical Analysis
Statistical analysis was performed using SPSS 22.0 (IBM, Armonk, New York, USA). Quantitative data were assessed for normality by the Shapiro–Wilk test [Reference Hanusz, Tarasinska and Zielinski20]. If the data were normally distributed, they were expressed as the mean ± standard deviation, and the differences were compared by independent sample t-test. On the contrary, the data were expressed as median (interquartile range) and the differences were analyzed by Mann–Whitney Test [Reference Hart21]. Qualitative data were represented in the form of N (%) and the difference between groups was analyzed by chi-square test [Reference Tallarida and Murray22]. P < 0.05 was considered significant.
3. Results
3.1. Baseline Information
Finally, 91 patients (67 Han people and 24 Tibetan) meeting the inclusion and exclusion criteria were enrolled in this study. Patient demographics, and clinical and laboratory findings are shown in Table 1. Except for prothrombin time, urea nitrogen, and smoking history (P < 0.05), there was no significant difference in the other detection indexes between ethnic Han and Tibetan.
TABLE 1: Baseline information for participants.
#mean ± sd, P value, Han population vs. Tibetan population.
For all patients, according to the definition of CR, there were 52 cases with CR and 39 cases with non-CR. The differences between CR and non-CR groups were significant for age, glycosylated hemoglobin (HBALC), activated partial thromboplastin time (APTT), gender, aspirin resistance, diabetes, and coronary heart disease classification (P < 0.05), and the other indicators were not significantly different between the two groups (Table 2).
TABLE 2: Comparison of differences in baseline information between CR and non-CR for all participants.
For the Han population, the differences in age, HBALC, APTT, gender, aspirin resistance, and diabetes were statistically significant between CR and non-CR groups (P < 0.05), and the differences in the other indicators were not significant (Table 3). For the Tibetan population, the two groups showed no significant difference in all indicators (Table 4).
TABLE 3: Comparison of differences in baseline information between CR and non-CR for Han populations.
TABLE 4: Comparison of differences in baseline information between CR and non-CR for Tibetan populations.
3.2. Comparative Analysis of CYP2C19 ∗2 and ∗3 Loci in Different Ethnic Groups
The genotypes of the CYP2C19∗2 locus in all participants included GG (67.0%), GA (26.4%), and AA (6.6%), respectively. The genotypes of the CYP2C19∗3 locus were GG (48.4%), GA (26.6%), and AA (23.0%), respectively. The genotypes of the CYP2C19∗17 locus were GG and GA, accounting for 65.1% and 34.9%, respectively. For the Han population, the genotypes of the CYP2C19∗2 locus were GG (67.2%), GA (23.9%), and AA (9.0%), respectively. The genotypes of CYP2C19∗3 locus were GG (50.7%), GA (25.4%), and AA (23.9%), respectively. The genotypes of the CYP2C19∗17 locus were GG and GA, accounting for 65.7% and 34.3%, respectively. For the Tibetan population, there were 66.7% GG and 33.3% GA for the genotypes of CYP2C19∗2, respectively. The genotypes of the CYP2C19∗3 locus were GG (41.7%), GA (37.5%), and AA (20.8%), respectively. For the genotypes of the CYP2C19∗17 locus, GG accounted for 54.2 and GA accounted for 45.8% (Table 1). There was no significant difference between ethnic Han and Tibetan.
3.3. Comparative Analysis of Different Locus between CR and Non-CR Groups
For all patients, the genotypes of CYP2C19∗2, CYP2C19∗3, and CYP2C19∗17 for CR and non-CR groups are shown in Table 2. The genotypes of CYP2C19∗2, CYP2C19∗3, and CYP2C19∗17 for the Han population in two groups are shown in Table 3 and for the Tibetan population in two groups are shown in Table 4. There was no significant difference between CR and non-CR groups for all genotypes in either Han or Tibetan populations.
3.4. Logistic Analysis of Risk Factors for CR
Variables with significant differences in baseline information were included for univariate and multivariate logistic regression analyses to explore the significant related factors of CR. As shown in Table 5, for all patients, age was a significant risk factor for CR, with an odds ratio (OR) (95% confidence interval (CI)) = 1.08 (1.02, 1.13), P = 0.005. The older the patients, the higher the risk of CR. APTT was a significant negative correlation factor for CR (OR (95% CI) = 0.81 (0.69, 0.95), P = 0.011), and the risk of CR decreased with the increase of APTT. Aspirin resistance was a significant positive correlation factor for CR (OR (95% CI) = 6.47 (2.02, 20.67), P = 0.002). Patients with aspirin resistance were at a significantly increased risk of developing CR. There was no significant association between coronary heart disease type and CR. For the Han populations, age, APTT, and aspirin resistance were significantly correlated with CR (P < 0.05) (Table 6).
TABLE 5: Univariate and multivariate logistics regression analyses for CR in all participants.
TABLE 6: Univariate and multivariate logistics regression analyses for CR in Han populations.
4. Discussion
Clopidogrel combined with aspirin is usually recommended for preventing ischemic events in patients with cardiovascular [Reference Thomson and Anderson23]. Despite the standard treatment, there are still a lot of adverse cardiovascular events, and CR is considered to be the main reason [Reference Joob and Wiwanitkit24]. In this study, we investigated the association between ∗2, ∗3, and ∗17 allelic variants of the CYP2C19 gene and CR in patients with coronary heart disease and ischemic stroke among Han and Tibetan populations. The results showed that three alleles were not statistically correlated with CR, while age, APTT, and aspirin resistance were significantly correlated with CR.
Presently, the mechanisms underlying CR have not been fully elucidated. The CYP2C19 genotype is the most important determinant of the pharmacodynamic and pharmacokinetic responses to clopidogrel [Reference Pereira, Rihal and So25]. It has been reported that CYP2C19∗2 and CYP2C19∗3, the main mutant alleles, are the most common genotypes in Asian populations [Reference Xie, Zou, Hu, Zhang, Ye and Chen26]. CYP2C19∗2 or CYP2C19∗3 allelic variants increase the risk of CR [Reference Zhuo, Xian and Long27]. CYP2C19∗17 allele is correlated with an increased risk of bleeding [Reference Sibbing, Koch and Gebhard28]. However, the present study showed that CYP2C19∗2, CYP2C19∗3, and CYP2C19∗17 alleles were not significantly different between Han or Tibetan populations as well as between CR and non-CR groups, which suggested that the three alleles were not statistically correlated with CR in this study.
Our result was in accordance with a recent study that investigated the association between CYP2C19∗2, CYP2C19∗3, and CYP2C19∗17 variants of the CYP2C19 gene and CR in patients with acute coronary syndromes in Morocco, and demonstrated that none of the three alleles showed a statistical correlation with CR. Different from the results of our study, that study identified a synergic effect among the three alleles on CR [Reference Hassani Idrissi, Hmimech, Khorb, Akoudad, Habbal and Nadifi29]. In fact, the correlation between polymorphisms of CYP2C19 and platelet responsiveness to clopidogrel has been widely recognized among patients with acute coronary syndrome and percutaneous coronary intervention, but the association with other indications, such as arterial fibrillation and stable angina, is negative [Reference Bhatt, Paré and Eikelboom30, Reference Johnson, Roden, Lesko, Ashley, Klein and Shuldiner31]. The inconsistent results may be due to the magnitude of the influence of CYP2C19 on the effectiveness of clopidogrel and may be consistent with the influence of this molecule on specific clinical indications [Reference Roden32, Reference Paré, Mehta and Yusuf33].
In this study, APTT, age, and aspirin resistance were significantly correlated with CR. The APTT is a widely available test used to screen for hypercoagulable states in bleeding disorders [Reference Abdullah34]. Shortened APTT is an independent risk factor for ischemic stroke [Reference Lin, Kuo and Kuo35], but its role in CR has not been reported to our knowledge. Age was a positively correlated factor of CR, which was inconsistent with previous studies. Prabhakaran et al. [Reference Prabhakaran, Wells, Lee, Flaherty and Lopes36] have reported that being older than 55 years contributed to a low response to clopidogrel loading. It has been reported that patients with aspirin resistance have increased platelet reactivity [Reference Eikelboom, Hirsh, Weitz, Johnston, Yi and Yusuf37]. High on-treatment platelet reactivity has become the most important factor inhibiting the antiplatelet effect of clopidogrel, resulting in the ineffectiveness of this agent [Reference Fiolaki, Katsanos and Kyritsis38]. Clopidogrel’s high on-treatment platelet reactivity could negatively influence the clinical course of a stroke and increase the risk of recurrent vascular events [Reference Wiśniewski and Filipska39]. Therefore, platelet function testing is necessary for stroke individuals, especially those predisposed to CR.
There were several limitations in the present study. First, there was a lack of sequence analysis that could provide more robust information on the investigated CYP2C19 polymorphisms. Second, the study only comprised Chinese patients, while multicentric investigation might have been more informative in terms of data robustness. Third, there was a lack of functional correlation between examined gene polymorphisms and enzyme activity in patients. At last, no control group represented by healthy individuals was included in the analysis. Furthermore, studies including larger sample sizes and control groups may help to better understand the phenomenon of heterogeneity in clopidogrel response.
5. Conclusion
In conclusion, the present study indicated that CYP2C19∗2, CYP2C19∗3, and CYP2C19∗17 alleles were not correlated with CR for both Han and Tibetan populations in Qinghai Province, while age, APTT, and aspirin resistance were independent risk factors of CR in this region. Our results may provide useful data for precision medicine based on individual gene sequencing results.
Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.
Ethical Approval
This study was approved by the Ethics Committee of Qinghai Provincial People’s Hospital.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Authors’ Contributions
RC and JW were responsible for conception and design of the research, YY, WZ, and XZ were responsible for acquisition of data, YY and WZ were responsible for analysis and interpretation of data, RC and JW were responsible for statistical analysis, RC and JW were responsible for obtaining funding, RC and JW were responsible for drafting the manuscript, and YL was responsible for revision of the manuscript for important intellectual content. All authors have read and approved the final manuscript.
Acknowledgments
This work was supported by the Natural Science Foundation of Qinghai Province Science and Technology Department (grant no. 2018-ZJ-904), Qinghai Provincial Health Commission Fund Project (grant no. 2020-wjzdx-36), and The Special Funds for Medical and Health Work in Longhua District (grant no. YL20180201).
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