Evaluation of the VKORC 1 and CYP2C9 Gene Polymorphisms and Their Effects on the Emergency Complications in the Patients Using Warfarin.
Warfarin is a most commonly drug used for the prophylaxis and treatment of venous and arterial thromboembolism. It is now known that genetic factors also play a part in the effect mechanism of warfarin (1). So far, nearly 30 genes have been reported concerning the pharmacokinetics of warfarin. Cytochrome P450 2C9 (CYP2C9), vitamin K epoxide reductase complex subunit 1 gene (VKORC1) genes, and their polymorphisms are the ones that have the closest relationship with warfarin (2-4). VKORC1 is a gene and its polymorphism belonging to vitamin K epoxide reductase enzyme enabling vitamin K to be formed from reduced vitamin K epoxide. It has recently been reported that there are a lack of a resistance to warfarin (2), whereas the most common mutations that affect the dosage regulation of warfarin in VKORC 1 genotypes are 1173, 3730, and -1639 single nucleotide polymorphisms (5-8). Another important genetic factor is CYP2C9, a liver enzyme that plays a role in the oxidative metabolism of many other drugs as well as warfarin, and a series of gene polymorphisms were identified in the CYP2C9 gene area (3, 4). It has been demonstrated that these polymorphisms lead to difficulty in achieving a stable dosage in patients using warfarin, a decrease in the dosage requirement for warfarin, and a high risk of bleeding at an earlier period of drug use (8). The American Food and Drug Administration recommended that the starting dosage of warfarin should be regulated in accordance with the CYP2C9 and VKORC1 genotypes (5). This study aims to evaluate whether the patients who applied to the emergency unit with supratherapeutic international normalized ratio (INR) levels after using warfarin were different as regards CYP2C9 and VKORC1 genetic polymorphisms than the control group patients with therapeutic INR levels. I addition, this study attempts to reveal the effects of CYP2C9 and VKORC1 genetic polymorphisms on warfarin dosages, the frequency of application to the emergency unit, and the complications that develop due to use of warfarin.
Materials and Methods
The study was a prospective clinical study. After obtaining the approval of the local ethical board, we conducted the study at the Emergency Service of Kecioren Training and Research Hospital between December 15, 2015, and January 1, 2016. Seventy patients who applied to our emergency department for any complaints or for INR control were included the study. Forty patients who had been using warfarin for more than one month and who had INR levels of 3.5 or more were included consecutively in the case group. A control group consisting of 30 patients was included for comparison. The control group was composed of patients who had applied to the emergency service for INR control and who did not have any warfarin overdose (their INR levels were below 3.5). After the patients and their relatives were informed about the situation, the patients for whom confirmation was obtained were taken into the study.
The excluded criteria
Patients who were under the age of 18, who had been using warfarin for less than one month, who were using amiodarone, furosemide, heparin, quinolone, metronidazole, omeprazole, sulfonylureas, phenytoin, and tricyclic anti-depressants, and who had an infection were excluded from the study.
Analysis of blood samples
Three milliliters of blood were taken into tubes with sterile ethylenediamine tetraacetic acid. Isolation of genomic DNA was carried out from the blood samples, following an algorithm, and the distribution of CYP2C9 and VKORC1 alleles between the patients and their relationship with the other variables were examined.
CYP2C9 and VKORC1 alleles
In our study, we used the PGX-Thrombo Strip Assay (Vienna, Austria) test for laboratory and examined VKORC1-1639 G>A mutant and wild type, CYP2C9 1075 A>C mutant and wild type, and CYP2C9 430 C>T mutant and wild type alleles.
The presence of polymorphism in the patients is demonstrated in Table 1.
VKORC 1 genotypes
-1639 G>A, expressed as VKORC 1 AA, is made up of homozygote mutant genotypes,
-1639 G>A, expressed as VKORC 1 AG, is made up of heterozygote genotypes, and
-1639 G>A, expressed as VKORC 1 GG, is made up of homozygote normal (wild) genotypes.
CYP2C9 alleles formed through the mutations of 430 C>T and 1075 A>C are shown in Figure 1 as *1 (wild type), *2 and *3. Homozygotes and heterozygotes are shown as *1/*1, *1/*2, *1/* 3, *2 /* 2, and *2/*3.
The statistical analysis of the study was carried out with the IBM Statistical Package for the Social Sciences (IBM SPSS Statistics, Armonk, NY, USA) version 20.0 package program. In order to evaluate the compatibility of the distribution of alleles and haplotypes with the normal distribution, the Shapiro-Wilk test was employed. For the data that were compatible with the normal distribution, Student t-test was used. For the data that were not compatible, the Mann-Whitney U test was utilized. Descriptive statistics were shown in the form of mean [+ or -] standard deviation or median (minimum to maximum) for interrupted and continuous numeric variables. Categorical variables were shown in the form of case number and percentage (%). Pearson's X2 test was used for the evaluation of categorical variables. Logistic regression analysis was conducted to compare each polymorphism and the average warfarine dosage of the patients.
Seventy patients who aged 18-80 were taken into our study. The demographic features of the patients are shown in Table 2.
Vitamin K epoxide reductase complex subunit 1 heterozygote type AG haplotype was the most commonly seen allele identified in 17 patients in the patient group (42.5%) and in 12 patients in the control group (40%), whereas CYP2C9 *1/*1 was the most commonly seen allele identified in 21 patients in the patient group (52.5%) and CYP2C9 *1/*1 was the most commonly seen allele found in 17 patients in the control group (56.7%). No statistical difference was detected between the groups (p>0.05) (Table 3).
In terms of INR levels of the patient group, no statistical difference was found between VKORC 1 and CYP2C9 haplotypes (p=0.305 and p=0.088, respectively), whereas a significant difference was found on weekly warfarin dosages of VKORC 1 homozygote normal GG and CYP2C9 *1/*1 homozygote normal (wild) carriers (p=0.02 and p=0.034, respectively) (Table 4).
When the number of visits by the patients to the emergency service was evaluated, it was shown that patients from the case group had more frequest admission to emergency service. (p=0.001) (Table 2). In terms of the frequency of visits to the emergency service, no statistical difference was found either for CYP2C9 or for VKORC 1 (Table 5).
Gastrointestinal bleeding was the most frequently observed form of bleeding . Complications due to bleeding were present in patient group, whereas it was observed in 17 patients in the case group (42.5%). No statistical difference was established between CYP2C9 and VKORC 1 haplotypes in terms of complications due to bleeding (p>0.05 and p=0.576, respectively) (Table 6).
Finally, when we evaluated to association between the average warfarin dosage of the patients and the gene polymorphism by logistic regression analysis, we did not find any correlation (p=0.2).
In our study, in which we evaluated the effect of CYP2C9, VKORC1 genetic polymorphisms, warfarin dosages and complications, no difference was observed between the patient and control groups in terms of haplotypes in warfarin-using patients. Moreover, it was seen that carriers of VKORC 1 homozygote normal GG and CYP2C9 *1/*1 homozygote normal (wild) had a higher dosage requirement for warfarin compared with the other genotypes. No relationship was found between INR levels and VKORC 1 and CYP2C9 haplotypes. VKORC 1 and CYP2C9 had no effect on bleeding and frequent visits to the emergency services.
There are two points that require particular attention in patients using warfarin during the process of treatment: (1) specifying an effective and safe stable dosage in the first months of the treatment, during which bleeding is particularly frequent, and (2) regulating this continual dosage in accordance with any change in the diet, weight, any new disease or additional drugs. Recent studies have revealed that genetic factors should also evaluate, as well as these factors (9). So, far, nearly 30 genes have been reported in association with warfarin pharmacogenetics. CYP2C9 and VKORC1 genes are the genes with the highest association with warfarin (2-4).
The prevalence of CYP2C9 and VKORC1 alleles varies with ethnic groups. In our study, we found that CYP2C9 *1/*1 haplotype was at a rate of 54.3%, and we identified it more commonly than we identified the other five haplotypes. While CYP2C9 *5 and *6 alleles were more prevalent in African society, the prevalence of CYP2C9 *2 and CYP2C9 *3 was found to be lower among Chinese and Malaysian people (10). There is considerable research in the literature demonstrating that genetic polymorphisms of CYP2C9 *2 and CYP2C9 *3 reduce the dosage requirement of warfarin (11-13). In Sanderson's study, CYP2C9 *2 genetic polymorphism reduced the dosage requirement by 17%, while CYP2C9 *3 genetic polymorphism reduced the dosage requirement by 37% (11). Similarly in the study by Lindh et al. (12) CYP2C9 *1/*1 carriers had a reduced dosage requirement compared with the other carriers (12). Also in our study, when CYP2C9 *1/*1 (homozygote normal) carriers were compared with the other carriers, no significant difference was observed as regards warfarin dosage requirement, as has been demonstrated in the literature.
The VKORC 1 allele most frequently seen in our study was heterozygote type AG haplotype, but in VKORC 1 genotypes, the most common mutation that affected the dosage regulation were 1173, 3730, and -1639 single nucleotide polymorphisms (5-7). Among Japanese people, while the ratio of VKORC1 -1639 G/G homozygote normal was extremely high (79.4%), the ratio of -1639 A/A homozygote mutant was negligibly low (0.8%) (6). In a study conducted by Gan et al. (14) revealed that VKORC 1 genetic polymorphism was quite common among Chinese and Malaysian people, but it had a low ratio among Indian people. These polymorphisms, which were identified in the VKORC 1 gene, could change the dosage requirement for warfarin. Ozer et al. (13), stated that VKORC1 -1639 G > A polymorphisms had the most important role. Similarly, in a study by Gan et al. demonstrated that patients with the GG genotype in the gene area of VKORC1 -1639 G > A had the highest dosage requirement for warfarin, patients with the AG genotype had a moderate dosage requirement for warfarin, and patients with the AA genotype had the lowest dosage requirement for warfarin (14).
Also in our study, a statistically significant difference was detected between haplotypes as regards warfarin dosage requirement both in the case group, in which the effect of overdose was seen at the time of the application, and in the control group, in which the ideal dosage was supposedly reached and no effect of overdose was seen. It was determined that while a higher dosage of warfarin was needed for VKORC 1 GG carriers, a lower dosage of warfarin was required for VKORC 1 AA carriers.
Warfarin is used in different indications and in increasing amounts today increases the likelihood of its side effects as well as its benefits (15). The most serious side effect resulting from an overdose of warfarin treatment is bleeding. When we evaluated the role of CYP2C9 and VKORC1 genetic polymorphisms in frequent applications to the emergency service and bleeding complications, we were unable to detect any effects of CYP2C9 and VKORC 1 haplotypes in terms of bleeding and frequent applications to the emergency service. We think that this may stem from the low number of the patients in our study.
Our study is a single-center study. The number of cases is low. Another limitation of our study is the fact that there were age differences between the patients in the patient and control groups. Apart from genetic factors, there are a number of factors that have an effect on the pharmacogenetics of warfarin. Among them, age is the leading factor. In our study, the difference in ages between the groups, as well as genetic polymorphism, might have had an effect on the weekly warfarin dosage that the patients used.
In this study, we could not clarify the effects of genetic polymorphisms on warfarin overdose and associated complications in emergency services. This may have to do with the low number of patients in our study. Further studies that include more patients and through which better results could be obtained with respect to cost and efficiency are required.
Ethics Committee Approval: Ethics committee approval was received for this study from the ethics committee of Kecioren Training and Research Hospital.
Informed Consent: Written informed consent was obtained from patients and patients' parents who participated in this study.
Peer-review: Externally peer-reviewed.
Correspondence to: Emine Emektar e-mail: firstname.lastname@example.org
Received: 17.01.2017 * Accepted: 22.02.2017
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: The authors declared that this study has received no financial support.
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Oguz Yardim , Emine Emektar , Yunsur Cevik , Aeref Kerem Corbacioglu , Ali Ekber Karabulut 
 Clinic of Emergency Medicine, Bayburt State Hospital, Bayburt, Turkey
 Clinic of Emergency Medicine, Kecioren Training and Research Hospital, Ankara, Turkey
Caption: Figure 1. CYP2C9 genotypes
Table 1. Types of polymorphism Wild type line Mutant line Genotype Normal Positive Negative Normal Heterozygote Positive Positive Heterozygote Homozygote Negative Positive Homozygote mutant Table 2. Clinical characteristics of study Case, n (%) Control, n (%) p Sex, n (%) Female 31 (77.5) 22 (73.3) 0.687 Male 9 (22.5) 8 (26.7) Age median (IQR 25-75) 76 (50-80) 68.5 (50-80) 0.012 Comorbidity, n (%) Diabetes mellitus 8 (20) 10 (33.3) 0.207 Hypertension 26 (65) 22 (73.3) 0.457 Coronary artery disease 16 (40) 14 (46.7) 0.577 Other 19 (47.5) 10 (33.3) 0.234 Indication for warfarin therapy, n (%) Atrial fibrillation 17 (42.5) 9 (30) 0.010 Heart valve replacement 9 (22.5) 17 (56.7) Thromboembolism 14 (35) 4 (13.3) Bleeding, n (%) 17(42.5) 1 (3.3) - Bleeding field, n (%) Gastrointestinal bleeding 5 (12.5) Hematuria 2 (5) - Epistaxis 2 (5) 1 (3.3) Skin 2 (5) Hemoptysis 1 (2.5) Other 2 (5) The number of overdose in the last year (%) 0 - 19 (63.3) 1 time 25 (62.5) 6 (20) 2 times 11 (27.5) 4 (13.3) 3 times and higher 4 (4) 1 (3.3) <0.05 IQR: interquartile range Table 3. Distribution of CYP2C9 and VKORC1 genotypes Frequency, Frequency, Genotype n (%) case n (%) control p VKORC1 AA 14 (35) 9 (30) AG 17 (42.5) 12 (40) GG 9 (22.5) 9 (30) 0.53 CYP2C9 *1 / *1 21 (52.5) 17 (56.7) Others 19 (47.5) 13 (53.7) 0.630 VKORC1: vitamin K epoxide reductase complex subunit 1 gene; CYP2C9: cytochrome P450 2C9; Others: CYP2C9 *1 / *2, *1 / *3, *2 / *2, *2 / *3, *3 / *3 haplotypes Table 4. The relationship with the weekly warfarin dose and INR of genotypes in case groups INR median Weekly warfarin dose median (min-max) (min-max) Genotypes Case p Case p VKORC1 AA 8.36 (3.83-15.63) 0.3 26.2 (17.5-35) 0.02 AG 7.76 (4.03-15) 30 (13.75-35) GG 4.82 (3.62-13.48) 35 (17.5-45) CYP2C9 *1/*1 8.77 (3.62-15) 0.08 35 (17.5-40) Others 2.32 (3.83-15.63) 27,5 (17.5-40) 0.03 VKORC1: vitamin K epoxide reductase complex subunit 1 gene; CYP2C9: cytochrome P450 2C9; INR: international normalized ratio; Others: CYP2C9 *1/*2, *1/*3, *2/*2, *2/*3, *3/*3 haplotypes Table 5. The relationship between recurrent admission due to warfarin overdose of VKORC1 and CYP2C9 genotypes Recurrent VKORC1 CYP2C9 admission AA AG GG p *1/*1 None 6 (8.6%) 6 (8.6%) 7 (10%) 12 (17.1%) 1 time 8 (11.4%) 14 (20%) 9 (12.9%) 0.268 17 (24.3%) 2 and more 9 (12.9%) 9 (12.9%) 2 (2.9%) 19 (12.9%) Total 23 (32.9%) 29 (41.4%) 18 (25.7%) 38 (54.3%) Recurrent CYP2C9 admission Others p None 7 (10%) 1 time 14 (20%) 0.52 2 and more 11 (15.7%) Total 32 (45.7%) VKORC1: vitamin K epoxide reductase complex subunit 1 gene; CYP2C9: cytochrome P450 2C9; Others: CYP2C9 *1/*2, *1/*3, *2/*2, *2/*3, *3/*3 haplotypes Table 6. The relationship between bleeding complications of VKORC1 and CYP2C9 genotypes VKORC1 Bleeding AA AG GG p Yes 6 (8.6%) 9 (12.9%) 3 (4.3%) No 17 (24.3%) 20 (28.6%) 15 (21.4%) 0.57 Total 23 (32.9%) 29 (41.4%) 18 (25.7%) CYP2C9 Bleeding *1/*1 Others p Yes 10 (14.3%) 8 (11.4%) No 28 (40%) 24 (34.3%) >0.05 Total 38 (54.3%) 32 (45.7%) VKORC1: vitamin K epoxide reductase complex subunit 1 gene; CYP2C9: cytochrome P4502C9; Others: CYP2C9 *1/*2, *1/*3, *2/*2, *2/*3, *3/*3 haplotypes
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|Title Annotation:||Original Article|
|Author:||Yardim, Oguz; Emektar, Emine; Cevik, Yunsur; Kerem Corbacioglu, Aeref; Ekber Karabulut, Ali|
|Publication:||Eurasian Journal of Emergency Medicine|
|Date:||Jun 1, 2017|
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