Distribution of CYP2C9 and VKORC1 Gene Polymorphisms in Healthy Macedonian Male Population

Abstract Background: Distribution of CYP2C9 and VKORC1 gene polymorphisms may vary significantly among different ethnic groups, and eventually influence the variation in drug metabolism or even failure. Objective: The aim of this study was to evaluate the prevalence of CYP2C9 and VKORC1 alleles in the healthy population of Republic of Macedonia compared to the global geographic data reported from different ethnic populations. Also, to genotype CYP2C9 and VKORC1 genes and eventually to divide individuals in poor, extensive, or intermediate metabolizer. Material and Methods: Blood samples were collected after signing written consent, DNA was isolated from peripheral blood, and CYP2C9 and VKORC1 genes were typed (n=124). Genotyping was performed by commercially available kits (GeneID GmbH, Strassberg, Germany, AID Diagnostica), based on the method of polymerase chain reaction with a subsequent hybridization. The population genetics analysis package, PyPop ver. 0.6.0, was used for analysis of the data. Results: The frequency of alleles varies from 0.931 for CYP2C9*3 to 0.109 for CYP2C9*2 indicating common “wild type” allele in those genes. The frequency ranges spanned ~50% for each allele of VKORC1 gene, indicating no common “wild type” allele in this gene. Test of neutrality showed significant negative value for VKORC1 polymorphism that indicates balancing selection operating on the alleles at that locus. All polymorphisms of CYP2C9*2, CYP2C9*3 and VKORC1 showed a good fit with Hardy-Weinberg expectations. Conclusion: The results of polymorphic alleles of CYP2C9 and VKORC1 genes in Macedonian population can be used for the variation in drug metabolism studies as well for adapting dosage regimes for oral anticoagulant therapies


Introduction
The cytochrom P450 (CYP) family of heme monooxygenases comprise the most important group of Phase I enzymes. These enzymes oxidize a wide range of endogenous, as well as exogenous compounds. The cytochrome P450 gene family contains 60-100 different genes, of which only a small group is involved in drug and chemical transformations. The most important P450 isoenzyme is CYP3A4 (50% of the P450 metabolism) followed by CYP2D6 (20%), CYP2C9 and CYP2C19 (together 15%). The remaining is carried out by CYP2E1, CYP2A6 and CYP1A2 [1].
The genes for CYP2D6, CYP2C9, CYP2C19 and CYP2A6 are functionally polymorphic. Therefore, approximately 40% of human P450 dependent drug metabolism is carried out by polymorphic enzymes (for a list of all currently known cytochrome P450 gene alleles go to http://www.cypalleles.ki.se/ ) [2].
There are 3 CYP2C9 alleles -CYP2C9*1 allele is wild-type, but CYP2C9*2 and CYP2C9*3 are mutated alleles. In both mutated alleles, a point mutation leads to a substitution of an amino acid in the protein. In CYP2C9*2 an arginine at position 144 is converted to cystein (R144C), in CYP2C9*3 an isoleucine at position 359 is converted to leucine (I359) Cytochrom P450 CYP2C9 catalyse the metabolism of important drugs such as phenytoin, tolbutamide, losartan, warfarin, and several NSAILs [3]. Interestingly, significant variation in distribution of polymorphic variants of the CYP450 genes is documented among different populations worldwide . The vitamin K epoxide reductase (VKOR) is a membrane protein in the endoplasmic reticulum, which catalyses the transformation of vitamin K 2,3epoxide into vitamin K hydroxyquinone, which is essential for the synthesis of factors II, VII, IX and XI of the coagulation system, as well as proteins C and S. The enzyme vitamin K epoxide reductase is pharmacodynamic target of oral anticoagulants and is encoded by the VKORC1 gene.  [26]. The country seceded peacefully from Yugoslavia after an independence referendum, held in September 1991. This is the first study of the diversity of polymorphisms of CYP2C9*2, CYP2C9*3 and VKORC1 in Macedonian populations. The aim of this study was to determine the frequencies of CYP2C9*2, CYP2C9*3 and VKORC1 alleles and genotypes and to compare them with populations for which similar data are available.

Population
Unrelated healthy male volunteers of Macedonian origin (n=124), between the age of 18-55 years with a mean age of 36.43 ( 3.47) years, which are subjects of bioequivalnece studies, were included in the study. All individuals are of Macedonian origin, nationality, and residents of the geographical areas of different regions of the Republic of Macedonia. From each individual was obtained signed informed consent prior to his or her enrolment. The Ethical Committee of the Faculty of Medicine in Skopje has approved the study. After admission to the study, blood samples were collected from each subject (5 ml) into an EDTA tube, and the genomic DNA was isolated from peripheral blood leukocytes by the phenol-chloroform extraction method [27]. Samples were stored in Anthropology field of the Macedonian Human DNA Bank (hDNAMKD) [28].
Typing methods CYP2C9*2 (rs1799853), CYP2C9*3 (rs1057910), and VKORC1 C1173T (rs 9934438) polymorphysims genotyping was performed by commercially available kits from GeneID GmbH, Strassberg, Germany. It is based on the method of polymerase chain reaction (PCR) with a subsequent hybridization. A multiplex PCR is first carried out with DNA isolated from whole blood. In this reaction, two fragments of the CYP2C9 gene and one fragment of the VKORC1 gene are amplified with specific biotin labelled primers. The amplified gene fragments are then characterized in a hybridization reaction with sequence-specific oligonucleotide probes (SSOP), which are immobilized on nitrocellulose (reverse hybridization). Each nitrocellulose strip has gene probes for the wild-type and mutated sequences of the tested gene fragments, as well as control zones.

Statistical methods
The population genetics analytics package, PyPop ver. 0.6.0 [29], was used for analysis of the gene data. Allele frequencies and expected Hardy Weinberg proportions (HWP) [30] for each single nucleotide polymorphisims (SNP) were determined. The exact test for genotype frequency deviation from HWP was calculated using the Arlequin implementation accessed via PyPop. Those SNPs that did not fit HWP were evaluated to determine wheather there was an excess of homozygotes or heterozygotes, or if any particular genotypes were significantly different fom expected frequencies by the Chi-square test. The Ewens-Watterson homozygozity test of neutrality with Slatkin's p-values [31] was used to indicate any deviations from the hypothesis of neutral selection for each locus. P-value less than 0.05 was taken as significant.

CYP2C9 and VKORC1 Alleles
Frequencies of polymorphic alleles, test of neutrality with Fnd statistic [Ewens-Watterson test of neutrality (EWN)], and Slatkin's Exact p-value (SEPV) with p of F statistics in macedonian population are shown on the Table 1. The frequency of alleles varies from 0.931 for CYP2C9*3 to 0.109 for CYP2C9*2, indicating common "wild-type" allele in these genes. The frequency ranges spanned ~50% for each allele of VKORC1 gene, indicating no common "wild type" allele in this gene (Table 1).

CYP2C9 and VKORC1 genotypes
Twenty-three individuals (18.6%) were heterozygous for CYP2C9*2, whereas 17 individuals (13.7%) were heterozygous for CYP2C9*3. There were two individuals (1.6%) which are homozygous for CYP2C9*2, but no individual was homozygous for CYP2C9*3. In addition, there were no individuals declared as heterozygotes (*2/*3). Thus, only 2 individuals can be predicted as subjects to have the lowest CYP2C9 enzymatic activity, with respect to *2 and *3; so they may be declared as PM (poor metabolizers). Overall, the CYP2C9*2 allele had a frequency of 10.1%, whereas the CYP2C9*3 allele had a frequency of 6.8%. The observed genotype frequencies for both polymorphisms for CYP2C9 were consistent with Hardy Weinberg equilibrium ( Table 2).
Observed versus expected genotypes for each SNP, Hardy-Weinberg proportion (HWP), and Guo and Thomson Hardy Weinberg Output (GTHWO) is given in the Table 3. All polymorphisms of CYP2C9*2, CYP2C9*3 and VKORC1 showed a good fit with HWP expectations (HWP p-value=0.824, 0.765 and 0.772, respectively). In two instances, χ 2 test cannnot be calculated because expected frequency was smaller than 5 (CYP2C9*2 *2/*2 and CYP2C9*3 *3/*3).  Table 3 shows the distribution of frequencies of the CYP2C9 and VCORC1 variant alleles in the healthy population of Republic of Macedonia compared to data reported from various ethnic groups and global geographic regions.
Allelic frequencies of rs9934438 (1173C>T) polymorphism in VKORC1 in different countries/ regions are presented in Table 4. The lowest frequency of the VKORC1 CC genotype was reported in Chinese populations (1%, 1.11% and 2.9% respectively ) [33,35,39]. The highest frequencies for VKORC1 CC genotype were reported in Netherlands, Italy and USA (37.2%, 36.8%, and 36.1% respectively). The frequency of VKORC1 CC genotype in Macedonians was similar to Caucasians (29.8%) ( Table 4). Homozygous genotypes for VKORC1 TT were dominant in the yellow populations (85%, 69% and 65% in China, Hong Kong and Egypt, respectively). Dominant heterozygous VKORC1 CT genotypes were reported in all Caucasian populations (Republic of Macedonia, Italy, USA, France and Netherlands) with frequencies between 46.9% and 50.7 (Table 4).

Discussion
In this study we present the distribution of the common alleles of CYP2C9 that has been found to vary across populations and different ethnic groups. The extent of variation is directly related to the genetic distance between populations and the allele being examined [40].
Results which are obtained from our study, showed that the allelic frequencies of CYP2C9*2 (10.1%) and CYP2C9*3 (6.8%) in tested population were similar to the general frequencies reported for the Caucasian population throughout Europe [41].
Compared to the frequency of CYP2C9*3 found in the Spanish ethnic group (16.2%) [4] a significant interethnic difference was noticed.
Several meta-analyses were published about the role of VKORC1 polymorphisms and different clinical conditions. To conduct a systemic review and metaanalysis to investigate the relationship between mean daily warfarin dose (MDWD) and VKORC1 single nucleotide polymorphisms (SNPs), a total of 19 studies were included in the meta-analysis. The frequencies of 1173TT and -1639 AA in Asian patients were higher than those in Caucasian and African populations. Sensitive analyses demonstrated that the impacts of gene polymorphism on warfarin dosage requirement were significantly different between Caucasian and Asian population, and the results of meta-analyses were stable and reliable [45]. Navigating the literature to determine how genotype will influence warfarin response in a particular patient is difficult, due to significant variation in patient ethnicity, outcomes investigated, study design, and methodological rigor. A paper was published where comprehensive search strategy was applied and 117 studies included [46]. Primary outcomes were stable dose, time to stable dose and bleeding events. Assessing study quality highlighted significant variability in methodological rigor. Notably, there was significant evidence of selective reporting, of outcomes and analysis approaches [46].

Conclusion
This study is the firsts on the distribution of the CYP2C9 and VKORC1 polymorphism in Macedonian population. This study shows that CYP2C9 and VKORC1 are polymorphic in the Macedonian population. Our data show that the distribution of CYP2C9 variant alleles frequencies are similar to the most of those reported for Caucasians of European descendant, but differ from those of North America Caucasians. Results from this study suggest that the genetically determined capacity of CYP2C9 has to be taken into account in process of designing, conducting and evaluation of pharmacokinetics for investigational drugs [44].