Frequencies of polymorphisms in genes affecting the pharmacokinetics of warfarin in the Zaporizhzhia region

Materials and methods. A total of 150 persons (62 male, 88 female) with a median age of 46 years (26; 58) undergoing preventive examination at the Medical Educational and Scientific Center “University Clinic” of Zaporizhzhia State Medical University (ZSMU). The CYP2C9, CYP4F2, VKORC1 genes polymorphisms in atrial fibrillation patients were determined in the Department of Molecular Genetic Studies of the ZSMU Medical Laboratory Center. Amplification of DNA fragments containing polymorphic regions was performed using multiplex real time polymerase chain reaction with Warfarin Pharmacogenetics kits (LLC NPO DNA Technology) in a CFX-96 thermocycler (BioRad) with a fluorescence detection scheme.

The global aspect that determines the safety of drug interventions, minimizing the occurrence of side effects, are the genetic characteristics of a patient, especially the polymorphism in genes involved in drug metabolism. Therefore, today a new area of personalized medicine is pharmacogenetics, which allows to optimize pharmacotherapy for individual patients [1,2]. In real clinical practice, pharmacogenetic testing is recommended for long-term use of drugs with a narrow therapeutic range, variable pharmacokinetics and a significant severity of side effects [2][3][4]. Such drugs include warfarin (WF) -an oral anticoagulant used to prevent thromboembolism in patients with atrial fibrillation (AF) [3,5,6]. Today, a whole range of genes that affect WF metabolism and cause different sensitivity to the drug is known. The largest contribution to WF dose variability make polymorphisms in the cytochrome P450 2C9 gene (CYP2C9), the vitamin K epoxide reductase complex subunit 1 gene (VKORC1) and the gene encoding an enzyme that is thought to inactivate vitamin K (CYP4F2) [7,8]. In this regard, the scientific litera ture describes algorithms for calculating the initial dose of WF based on the results of pharmacogenetic testing [9]. It is necessary to emphasize that genetic testing in practical medicine should take into account the population characteristics of the selected genetic variants prevalence, as well as their contribution to the dosage and the development of side effects in a particular population. [2]. In the Zaporizhzhia region, the prevalence of polymorphisms of the VKORC1, CYP2C9 and CYP4F2 genes was not studied.

Aim
To establish the frequencies of polymorphisms in VKORC1, CYP2C9 and CYP4F2 genes among residents of the Zaporizhzhia region.

Materials and methods
A total of 150 persons (62 male, 88 female) with a median age of 46 years (26; 58) undergoing preventive examination at the Medical Educational and Scientific Center "University Clinic" of Zaporizhzhia State Medical University (ZSMU).
All persons gave their informed consent to participate. CYP2C9, CYP4F2, VKORC1 genes polymorphisms were determined in the Department of Molecular Genetic Research of the Training Medical and Laboratory Center of ZSMU (director − Prof. A. V. Abramov). Peripheral blood samples were obtained from each participant and transferred to ethylenediamine tetraacetic acid (EDTA) containing tubes in order to prevent clotting. DNA samples were extracted from whole blood leukocytes using a set of reagents TEST-RAPID-GENETICS ("DNA-Technology"). Amplification of DNA fragments containing polymorphic regions was performed using a real-time polymerase chain reaction with Warfarin Pharmacogenetics kits ("DNA Technology") in a CFX-96 thermocycler (BioRad) with a fluorescent detection scheme. 20 μl of pre-centrifuged suitable mixture for amplification were added to the test tubes. A 20:1 mixture of PCR buffer with Taq-AT polymerase was prepared separately and centrifuged for 1-3 seconds. 10 μl of a mixture of PCR buffer with Taq-AT polymerase were added to the test tubes with the amplification mixture. 1 drop of mineral oil was added to each test tube. 5 μl of DNA was added to the appropriate tubes with aerosol barrier tips. The same manipulations were performed with the control sample. After centrifugation for 1-3 seconds, amplification was performed. PCR results were recorded automatically by software.
The principles of bioethics were observed in the study: the basic provisions of the European Council Convention on Human Rights and Biomedicine (dated 04.04.1997), GCP (1996), the World Medical Association's Helsinki Declaration
As shown in Tables 1, 2, 3, 4, no significant deviations from the Hardy-Weinberg equilibrium were observed.
There were no statistically significant differences in the distribution of genotype frequencies between males and females and between different age groups.

Discussion
We compared the obtained genotype frequencies with the all-Ukrainian population and other ethnic groups studied by a number of authors. The prevalence of the CYP2C9 gene polymorphisms is shown in Table 2.
As can be seen from the table, the prevalence of CYP2C9 C/T gene polymorphism was the same in Zaporizhzhia, all-Ukrainian population, Europe and South Asia. At the same time, the C allele was more common in America, East Asia, Oceania, and Africa compared with our study, and the T allele was more common in Central Asia. The A/C polymorphism did not show a significant difference in allele frequencies between different geographical regions, except Africa, where the C allele did not occur.
The prevalence of VKORC1 gene polymorphisms among different ethnic groups is shown in Table 3.
The G and A alleles of the VKORC1 gene prevalence varies in different geographical regions. Thus, the mutant A allele dominates in Japanese, is less common in Egyptians, Ukrainians (in our study and in the general population), Europeans and Latinos, is very rare in African Americans and is almost non-existent in Indians.
The prevalence of CYP4F2 gene polymorphism is shown in Table 4.
The frequency of polymorphic alleles of the CYP4F2 gene is comparable in our study, Europe, East and South Asia. The C allele is more common in America and Africa, and the T allele is more common in Central Asia and Oceania than in the Zaporizhzhia region.    graphical regions, which determines the feasibility of study on the genotypes distribution for each population. 2. CYP4F2 and VKORC1 genetic polymorphisms are more common in the Zaporizhzhia region, while the prevalence of CYP2C9*2 and CYP2C9*3 genetic polymorphisms is much lower.
Prospects for further research. Due to the widespread prevalence of genetic polymorphisms affecting WF pharmacokinetics, further research will focus on comparing the efficacy and safety of traditional and pharmacogenetic approaches to WF dosing.