Association of CYP2C19*2 c.681G>A (rs4244285) Loss-of-function Allele with Cardiovascular Disease Risk in the Kosovo Population

Clopidogrel remains a cornerstone in dual antiplatelet therapy for reducing the risk of cardiovascular events in patients with coronary artery disease (CAD), as well as those undergoing percutaneous coronary interventions (PCI). It is particularly beneficial in preventing thrombotic events in patients at risk for or who have experienced acute coronary syndromes (ACS), including myocardial infarction (MI) with or without ST-elevation (STEMI or NSTEMI) or unstable angina [1]. Clinical trials (such as CAPRIE, PLATO, CLARITY-TIMI 28, COMMIT/CCS-2), have consistently demonstrated clopidogrel’s efficacy in various settings, including in secondary prevention and acute coronary syndromes. However, these clinical trials also emphasize the emergence of newer antiplatelet agents, such as prasugrel and ticagrelor that may demonstrate superior efficacy in certain clinical scenarios, challenging the traditional use of clopidogrel in some patient groups [2]. Clopidogrel is the only irreversible P2Y₁₂ (purinergic P2Y, G-protein coupled 12) inhibitor to have a class I indication in patients with stable CAD undergoing stent implantation and is recommended in those with a contraindication to ticagrelor or prasugrel or those taking an oral anticoagulant [3]. Despite its proven benefits, clopidogrel’s effectiveness as a prodrug is influenced by genetic and non-genetic factors, with genetic variations in the CYP2C19 gene being a key determinant of its therapeutic outcomes. CYP2C19 genetic variants, particularly loss-of-function (LOF) alleles like *2, *3, *4, and *5, have been shown to affect clopidogrel metabolism significantly. Individuals carrying two LOF-alleles exhibit reduced enzyme activity, leading to lower levels of the active metabolite and diminished antiplatelet effects. This reduction in efficacy has been associated with a 2-4 fold increased risk of ischemic events, stent thrombosis, and major cardiovascular and cerebrovascular events, particularly in high-risk patient populations [4–9]. As a result, genotype-guided clopidogrel therapy has gained attention, with clinical guidelines from the American College of Cardiology (ACC) and American Heart Association (AHA) recommending its consideration, especially for patients with CAD or those undergoing PCI [10–13]. Despite promising results from studies, such as from the POPular Genetics trial [12], conflicting findings from other studies (such as CURE, TRITON-TIMI 38, EAST-AFNET 4, GRAVITAS, ISAR-REACT 5) have raised concerns about the generalizability of genotype-guided therapy [14]. These inconsistencies in the evidence contribute to the challenges in achieving widespread adoption of routine CYP2C19 testing. The uncertainty surrounding the benefits of genotype-guided therapy has led to divergent recommendations from regulatory bodies, professional societies and pharmacogenetics consortia [14–16]. For example, The U.S. Food and Drug Administration (FDA) highlights reduced clopidogrel effectiveness in individuals with CYP2C19 LOF-alleles and suggests alternative therapies for poor metabolizers, whereas the European Medicines Agency (EMA) adopts a more cautious approach, advising against co-administration with CYP2C19 inhibitors but not mandating genetic testing [3]. Similarly, pharmacogenetic guidelines, such as those from the Clinical Pharmacogenetics Implementation Consortium (CPIC) and the Dutch Pharmacogenetics Working Group (DPWG), recommend CYP2C19 genotype-guided antiplatelet therapy, particularly in specific high-risk populations, but these recommendations differ in certain aspects and have not been universally integrated into clinical practice [3, 17, 18]. The ESC’s 2020 guidelines for ACS management propose genotype-guided therapy as an alternative to standard dual antiplatelet therapy with prasugrel or ticagrelor but refrain from endorsing routine testing for all PCI patients due to limited evidence. The AHA also calls for more research to establish the role of genotyping in improving clinical outcomes, particularly for ACS patients and secondary stroke prevention.

In addition to CYP2C19, other genetic polymorphisms such as those in CES1, PON1, ABCB1, and P2RY12 also influence clopidogrel’s pharmacokinetics and pharmacodynamics by affecting drug absorption, metabolism, and platelet receptor activity and further complicating the variability in clopidogrel response [19, 20]. Non-genetic factors, including age, comorbidities (e.g., chronic kidney disease and diabetes mellitus), and drug interactions (coadministration of medications that act as CYP2C19 substrates, inhibitors, or inducers) also play a significant role in modulating drug efficacy [21, 22]. Beyond cardiovascular outcomes, CYP2C19*2 variants have been associated with altered susceptibility to gastrointestinal disorders, such as peptic ulcers and gastroesophageal reflux disease, as well as certain neurological conditions, highlighting their multifaceted impact [21, 23, 24]. Inter-ethnic variability in the distribution of CYP2C19 genotypes adds another layer of complexity. Certain alleles, such as CYP2C19*2, are more prevalent in specific populations, with significant differences observed across ethnic groups [25, 26]. The lack of comprehensive data on the intra-ethnic distribution of CYP2C19 variants, coupled with challenges such as cost-effectiveness, logistical barriers, and limitations in the local healthcare infrastructure, restrict integration of PGx testing into routine clinical practice, particularly in resource-limited or under-researched settings like Kosovo, where the integration of genetic testing into routine clinical practice remains an ongoing problem.

This study aims to assess the frequency of the CY-P2C19*2 genotype in clopidogrel-treated patients with cardiovascular disease indications (CVDI) in Kosovo and evaluate its association with major adverse cardiovascular events (MACE), including cardiovascular death, nonfatal myocardial infarction, stroke, stent thrombosis, and revascularization.

Baseline characteristics of the 150 Kosovo patients treated with clopidogrel are summarized in Table 1. The mean age was 68 years, 78.7% of patients being older than 61 year and 40% being female. Among the patients, 43.3% presented with an ACS indication (STEMI/NSTEMI). All patients had at least one comorbidity including common conditions such as hypertension, diabetes, and dyslipidemia. Most patients had arterial hypertension (67.3%) and 35.3% of patients presented with more than one comorbidity.

Allele, genotype, and phenotype frequencies of CY-P2C19*2 polymorphism are detailed in Table 2. A total of 34.0% of patients were CYP2C19 intermediate or poor metabolizers (29.33% IMs and 4.67% PMs). The frequency of CYP2C19*2 LOF allele was 19.33%. The observed genotype distributions did not significantly deviate from Hardy–Weinberg equilibrium (χ²=0.532; p=0.465).

Table 3 provides a comparison of CYP2C19*2 allele and genotype frequencies observed in our cohort of patients with those reported for the healthy Kosovo population. According to the results, CYP2C19*2 LOF allele carriers have approximately 1.6 times higher probability for developing CVD compared to non-carriers (OR=1.6; 95% CI=1.08-2.37; p=0.018). The association between CYP2C19*2 allele and increased probability of developing CVD was further confirmed in dominant (NM vs. IM+PM) model of statistical analysis (OR=1.64; 95% CI=1.04-2.57; p=0.031) as well. Additionally, differences in the genotype/phenotype distribution were observed between the patient and historical healthy control groups (66.0%, 29.33% and 4.67% vs 76.07%, 21.76% and 2.14% for NM, IM and PM, respectively), although these differences did not reach statistical significance (codominant analysis: p=0.067 IM, p=0.112 PM; NM as reference). The present analysis does not allow a stratified assessment of association based on age and gender, since it is based on previously published data concerning CYP2C19*2 LOF allele in healthy Kosovo population. No statistically significant difference between stratified groups of patients (according to indication for clopidogrel treatment, coexistence of one or more risk factors) with respect to a CYP2C19*2 variant allele (Table 4; all p>0.05). The median observation time of follow up was not reached until this analysis was conducted.

The worldwide implementation of pharmacogenetics highlighted population-specific differences in allelic and genotype/phenotype frequencies of genes coding for drug-metabolizing enzymes. One significant challenge in translating treatment-associated polymorphisms into routine clinical use is the lack of knowledge regarding its frequency in the targeted population in comparison to the population frequency. In the context of clopidogrel treatment, understanding the prevalence of CYP2C19 LOF variants within a population is critical for assessing their clinical implications. Careful consideration is necessary when interpreting studies on the association between CY-P2C19 metabolizer phenotype and clopidogrel treatment outcomes. Evidence supporting this association primarily stems from studies involving ACS patients (with at least 50% undergoing PCI) and settings where clopidogrel was compared with an alternative P2Y₁₂ inhibitor [28]. Conversely, studies opposing this association often focus on lower risk, non-PCI patients or data that did not strongly justify the utility of PGx guided clopidogrel treatment for secondary stroke prevention or ACS patients [13, 16]. The presented study primarily aimed to determine the frequency of CYP2C19*2 LOF allele in clopidogrel treated patients with cardiovascular disease indications (CVDI) and other concurrent risk factors in Kosovo and to evaluate the prognostic value and association of CYP2C19 PM phenotype with the risk of MACE in this cohort during two-year term follow-up under routine clinical care. The prevalence of the CYP2C19*2 allele in this cohort of Kosovo patients (19%) and the historical genotype control group of healthy population (13%) was consistent with the range reported in other European populations [29, 30], with latter falling within the 9-18% range observed across the population from the Balkan region, including Turkey (12%), Greece (13%), Macedonia (14.4%), Serbia (11%), Republic of Srpska (16%) in Bosnia and Hercegovina) Croatia (15%) and Slovenia (16%). Intra-population comparisons, however revealed a significantly higher frequency of the CYP2C19*2 LOF allele in the patient cohort compared to the healthy Kosovo population reported by Krasniqi et al. (2017), suggesting a possible association between the CYP2C19*2 LOF allele and increased overall probability of CVD. This association appeared to be independent of the type and number of comorbidities as well as patients’ previous history on MI (STEMI or NSTEMI). Notably, the proportion of poor metabolizers (PMs) identified in our patient cohort was significantly higher than the reported in studies of other European populations (4.67% vs. 2.4%, respectively) [11, 29, 31].

This study is the first to report this correlation within the Balkan population. Comparable research conducted in Macedonia [32], Serbia [33] and Croatia [34] has demonstrated that CYP2C19*2 functions as an independent risk factor for adverse treatment outcomes, defined by the occurrence of MACE in patients receiving dual antiplatelet therapy with aspirin and clopidogrel. Notably, this is the first investigation that explores the relationship between the CYP2C19*2 allele and the risk of CVD among the Balkan population. The potential mechanism by which CYP2C19*2 may contribute to an increased risk of cardiovascular disease (CVD) extends beyond its pharmacogenetic role, encompassing disruptions in the metabolism of critical endogenous substrates, such as eicosanoids (arachidonic acid derivatives) and steroids. These substrates are integral to maintaining vascular homeostasis, modulating inflammatory processes, and regulating oxidative stress. Impaired CYP2C19 activity could alter the metabolism of these molecules, leading to endothelial dysfunction, compromised vascular function, and dysregulated blood pressure. Such metabolic disruptions may exacerbate conditions like hypertension and atherosclerosis, ultimately contributing to the pathophysiology of CVD [35, 36].

These preliminary findings highlight the importance of investigating intra-population genetic variations to identify specific genetic markers or health risks prevalent within a particular ethnic group. They supplement the growing body of evidence linking the CYP2C19*2 LOF allele with the occurrence and development of CAD, CVD and CHD. The results align with previously published studies suggesting that the CYP2C19 LOF allele may be involved in CVD susceptibility [37–41]. Rothenbacher et al. (2013) reported that stable CHD patients, carriers of the homozygous CYP2C19*2 LOF allele are at increased risk for subsequent CVD events during the long follow up, independent of other risk factors [37]. Similarly, Zhang et al. (2019) found that CYP2C19*2 not only increased the risk of CHD, but also worsened clinical outcomes in CHD patients during an extended follow-up period [38]. Cai et al. (2023) observed that among the Hakka population, carriers of the CYP2C19 LOF alleles, both heterozygous and homozygous, exhibited increased susceptibility to hypertension [39]. In Martínez-Quintana’s study (2017), patients with an acute coronary event and poor or normal CYP2C19 metabolizer phenotype were more likely to have insulin-dependent diabetes mellitus than those with rapid or ultrarapid metabolizer phenotypes [40]. More recently, Chen et al. (2024) reported that carriers of CYP2C19 LOF alleles are at increased risk for premature coronary artery disease, particularly when combined with other risk factors such as overweight, smoking, hypertension and diabetes mellitus [41].

When addressing the observed intra-population differences in CYP2C19*2 frequencies, it is important to acknowledge that relying on previously published data may introduce potential variability due to differences in study design, methodology, or population characteristics and recruitment between historical genotype controls and the study cohort might affect the validity of direct comparisons. However, we recognize this limitation arising from the lack of a contemporaneously recruited healthy control group, as well as the small sample size and absence of demographic data for the healthy population. Despite these limitations, the internal validity of our findings remains intact. Importantly, there were no differences in the sample collection, handling and storing, DNA extraction or genotyping procedures, and both studies used DNA extraction kits comparable in terms of sensitivity and specificity and employed the same PCR-based methods and genotyping assays. Additionally, both the patient cohort and the historical control group primarily consist of ethnic Albanians from all parts of Kosovo. Nevertheless, it is essential to consider that differences in exposure to environmental factors, diet, or healthcare access between the cohorts could act as confounding variables, potentially masking genetic associations. Overall, these results provide valuable insights into the allele, genotype, and phenotype frequencies of the CYP2C19 drug-metabolizing enzyme in Kosovar patients indicated for clopidogrel treatment. They add to the growing evidence advising the need for careful consideration of CYP2C19 genetic diversity as a population-specific factor when recommending PGx-guided clopidogrel therapy [25, 26], particularly in populations like Kosovo’s, where genetic determinants are not yet fully elucidated. The observed association between the CYP2C19*2 LOF allele and increased probability of developing CVD for clopidogrel treatment in our study hinders the ability to conclusively demonstrate a clinical benefit of CYP2C19*2 PGx guided treatment in the population of Kosovo. Variability in treatment outcome, if identified during the study follow up period, could potentially be attributed to additional patient-specific determinants (such as comorbidities, concomitant drug treatments or as-yet undiscovered population-specific genetic factors). Future studies incorporating a broader range of demographic and clinical variables are needed to better elucidate the role of CYP2C19*2 PGx guided therapy in optimizing clopidogrel treatment strategies for this population. Moreover, additional investigations should evaluate whether the carriage of the CYP2C19*2 allele itself is associated with an adverse outcome in patients not taking clopidogrel.

This study identifies a possible association between the CYP2C19*2 allele and an increased probability of CVD within the Kosovo population, offering novel insights into the intra-ethnic variability of this LOF allele and its clinical relevance for PGx-guided therapy within the region. The findings should be considered when optimizing the implementation of the clopidogrel PGx testing in the routine clinical practice at the national level. Further studies examining the relationship between the CYP2C19 PGx data, patient-specific follow up outcomes, and plasma concentration of clopidogrel’s active metabolite are necessary to validate these observations and assess the utility of PGx-guided clopidogrel treatment in Kosovo population.