Balkan Journal of Medical Genetics

ASSOCIATION OF CYP2C19*2 C.681G>A (RS4244285) LOSS-OF-FUNCTION ALLELE WITH CARDIOVASCULAR DISEASE RISK IN THE KOSOVO POPULATION
Elshani N1*, Ukella K1, Staninova Stojovska M2, Naumovska Z2, Kurshumliu M3, Gorani D4, Kapedanovska Nestorovska A
*Corresponding Author: Corresponding Author: Nora Elshani, Glloku te Shelgjet “Veternik” 10000-Prishtinë, Kosova. Phone: +38344601032. E-mail: nora.elshani1@gmail.com; nora.elshani@rezonanca-rks.com.
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MATERIAL AND METHODS

Study population The study population for the current hybrid ob- servational study with retrospective data collection and prospective follow up was comprised of 150 adult pa- tients, 18 years and older, presenting at the cardiology clinic at the University Clinical Center of Kosovo in the period from December 2023 to May 2024. Major inclu- sion criteria were the use of clopidogrel therapy because of established cardiovascular diseases (CVD), coronary heart disease (CHD), coronary artery disease (CAD), and cerebrovascular disease, particularly patients with acute coronary syndromes (ACS), including myocardial infarction (MI) with or without ST-elevation (STEMI or NSTEMI) or unstable angina undergoing percutaneous coronary intervention (PCI) with or without stent. Patients were excluded from the study if they had: 1) a prior history of bleeding (e.g., peptic ulcer, intracranial hemorrhage, menstrual bleeding), 2) clinically significant abnormali- ties in platelet function or severe hepatic insufficiency, 3) drug addiction or alcohol use disorder, or 4) had donated blood within the last 2 months before starting clopidogrel therapy. Female patients on hormone replacement therapy or using an intrauterine contraceptive device were also ex- cluded. These exclusion criteria align with those in previ- ously conducted Phase 3 randomized, double-blind clinical studies of clopidogrel, ensuring a reliable, homogeneous study population while minimizing the risk of confound- ing variables and adverse effects. Demographic data (age and gender) and clinical data, including indications for clopidogrel therapy (e.g., acute myocardial infarction, stroke, cardiac catheterization for coronary artery disease, cardiac artery bypass graft surgery, carotid angiography, carotid stenting, carotid endarterectomy, and extremity arteriography), as well as risk factors and co-morbidities (such as heart disease, ischemic heart disease, heart fail- ure, hypertension, diabetes mellitus, stroke, and chronic ischemic heart disease), were manually abstracted from the medical health records (MHR). From each participant previously written informed consent was obtained. The follow-up period for Event-Free Survival (EFS) analysis was 24 months. The study complied with the principles of Helsinki Declaration and was approved from Com- mittee Ethics of Alma Mater Europaea Campus College “REZONANCA” (Protocol No: AD-762/23) and Chamber of Pharmacists of Kosova (No.105). Genotyping procedures Genomic DNA was extracted from 2 ml of peripheral blood using the NX-48S Genomic DNA Kit according to the manufacturer’s recommended protocol (Genolution Inc, Seoul, Republic of Korea) and stored at -20°C until further analysis. DNA purity and concentrations were veri- fied by UV absorption at 260/280 nm using the NanoDrop 2000TM spectrophotometer (Thermo Fisher Scientific). CYP2C19*2 c.681G>A (rs4244285) polymorphism was genotyped by allele discrimination PCR on a Stratagene Mx3005P (Agilent Technologies, Santa Clara, CA, USA) real-time PCR system using TaqMan® DME genotyping assay (C__25986767_70; Thermo Fisher Scientific). The genotypes were determined in a reaction mix containing 20 ng DNA in a total volume of 25 µL according to the manu- facturer’s recommended protocol. Positive and negative controls were included on each plate and reproducibility was checked by re-genotyping 15% of the cases. In accord- ance with the Clinical Pharmacogenetics Implementation Consortium (CPIC)–recommended genotype‐to‐pheno- type classifications, CYP2C19*2 metabolizer phenotypes were assigned and reported as follows: Poor Metabolizer (PM with 2 no function alleles; *2/*2 genotype), Inter- mediate Metabolizer (IM with 1 no function allele; *1/*2 genotype), normal metabolizer (NM; *1/*1 genotype) [16]. All genotyping procedures were performed at the Center for Biomolecular Pharmaceutical Analysis, Faculty of Pharmacy-University Ss. Cyril and Methodius, Skopje, Republic of North Macedonia. Statistical analysis The study population was analyzed descriptively, with demographic, clinical, and genetic data presented as counts and frequencies (percentages). Allele and genotype frequencies were assessed for Hardy-Weinberg equilib- rium using the Chi-squared (χ2) test. CYP2C19*2 data from patients were compared with a historical genotype control of the healthy Kosovo population as reported by Krasniqi et al. (2017) [27] utilizing Fisher’s exact test. Stratified analyses examined the association between pa- tients CYP2C19*2 status and risk factors. Odds ratios (OR) with 95% confidence intervals (CI) were calculated, with statistical significance set at p ≤ 0.05. All analyses were conducted using MedCalc Software v22.026 (Med Calc Software Ltd, Ostend, Belgium).

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