Interethnic Diversity of CYP2C9 Alleles on Warfarin Pharmacogenetics in Healthy Sindhi and Baluchi Population Samples: Correlation to Global Allelic Frequencies<br>

Interethnic Diversity of CYP2C9 Alleles on Warfarin Pharmacogenetics in Healthy Sindhi and Baluchi Population Samples: Correlation to Global Allelic Frequencies

Research Article

Open Access

Sana Abbas^1*, Uzma Sahar², Shahzad Bhatti^1,2, Ali Naqi¹, Muhammad Ali¹,
Noveen Nawaz¹ and Rabail Alam¹

¹Institute of Molecular Biology and Biotechnology, University of Lahore, Pakistan

²Department of Human Genetics, University of Health Sciences, Lahore, Pakistan

^*Corresponding authors:

Sana Abbas
Institute of Molecular Biology and Biotechnology
University of Lahore
E-mail: sanaabbas20@yahoo.com

Received September 18, 2012; Published November 05, 2012

Citation: Abbas S, Sahar U, Bhatti S, Naqi A, Ali M, et al. (2012) Interethnic Diversity of CYP2C9 Alleles on Warfarin Pharmacogenetics in Healthy Sindhi and Baluchi Population Samples: Correlation to Global Allelic Frequencies. 1:520. doi:10.4172/scientificreports.520

Copyright: Â© 2012 Abbas S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

CYP2C9 gene polymorphisms are extensively deliberated in several ethnic groups, however they are fewer identified in the Baluchi population. The aim of this work was to study the ethnic differences of the CYP2C9 allele distribution in a healthy Baluchi population in order to evaluate them with a healthy Sindhi population. A total of 100 Sindhi and 100 Baluchi volunteers were genotyped for the CYP2C9^*2 (Arg144Cys) and CYP2C9^*3 (Ile359Leu) allelic variants by PCR-RFLP assay. The frequencies of the CYP2C9^*1, ^*2 and ^*3 alleles in the Sindhi population were 0.877, 0.121, 0.097 and in Baluchi 0.817, 0.196 and 0.165, respectively. We constituted a significant dissimilarity in CYP2C9^*3 incidence between the Sindhi and Baluchi populations, which have therapeutic consequences (p*1/^*1, ^*1/^*2, ^*1/^*3, ^*2/^*2, ^*2/^*3, and ^*3/^*3 genotypes in Sindhi was 0.921, 0.152, 0.129, 0.000, 0.012 and 0.000 while in Baluchi, it was 0.731, 0.169, 0.170, 0.000, 0.021 and 0.000, respectively. A significant difference was found between the Sindhi and Baluchi populations regarding the ^*1/^*1, ^*1/^*3 and the ^*2/^*3 (p

Keywords

CYP2C9; Anticoagulants; Warfarin; Interethnic differences; Sindhi; Baluchi

Introduction

Cytochrome P450 CYP2C9 is mainly a significant enzyme in human drug metabolism [1]. Its genetic polymorphisms are wellknown and contribute to interindividual and interethnic variations in the metabolism of a number of drugs in humans [2]. CYP2C9 is key mediator that implicated in the metabolism of 20-30% of all drugs, including many clinically imperative pharmaceuticals such as, Non Steroidal Anti-Inflammatory Drugs (NSAIDS), losartan, tolbutamide, sulfonylurea drugs, warfarin and phenytoin, [3-10]. Although several allelic variants of CYP2C9 have been reported and identified, the most common variants are CYP2C9*2 (Cys144/Ile359) and CYP2C9*3, apart from the wild type allele CYP2C9*1 (Arg144/Ile359). CYP2C9*2 and CYP2C9*3 (Arg144/Leu359) allelic variants differ from the wild type allele by a single nucleotide substitution [11,12]. It was found that intrinsic clearance of warfarin in genotype CYP2C9*3 has 5% less than wild type and CYP2C9*2 has 12% less than wild type (CYP2C9*1) [12-14]. Currently, the total Baluchi and Sindhi population was estimated to be about 12 million and 30 million respectively, in Pakistan [15]. The Sindhis as a whole were the unique descendants of an ancient population known as Sammaat, [15] and Balochi have Persian, Kurdish origins, all of whom descending from the portions of the Iranian plateau. As the allelic variants of human cytochrome P450 CYP2C9 gene differ in frequency between different ethnic groups [7,16], we determined the major allelic variants of CYP2C9 gene in randomly selected healthy Baluchi and Sindhi population subjects, originated from subcontinent, and to compare them with results obtained in other ethnic populations.

Materials

Subjects

The study sample size was of 200 unrelated patients. The ages were between 40-85 years and were on anticoagulant warfarin therapy. The patients were determined as one whose warfarin maintenance dose was fixed and controlled with an INR range in between 1.5-3.0 during the last four clinical visits. The patientâ€™s information about their ages, sex, height, weight, Warfarin daily dose, INR value, primary reasons for anticoagulation and prescribed medications were obtained from clinical record of patients. The University of Lahore and hospitalâ€™s ethical review committee have approved this study. All patients, before participating in this study, have provided written informed consent.

Molecular genotyping

The DNA was extracted from the peripheral blood sample after digestion of blood leucocytes with the use of a QIA amp DNA blood midi kit. The protocol was carried out as it was mentioned in manufacturerâ€™s guide lines. The molecular genotyping of CYP2C9 allele was done using PCR-RFLP (Burian et al. 2002); the primers for exon 3 or exon 7 of CYP2C9 gene were used to amplify the DNA. The sequences of primers are given in table 1. For the PCR reaction, the final volume used was 20 Î¼l containing 2.0 mM MgCl₂ (from vivantis), 200 Î¼M dNTPs, 1XPCR buffer, 0.4 Î¼M CYP2C9 specific F and R primers, 1 U of Taq polymerase from vivantis, 90 ng genomic DNA (sample DNA), 0.1 Î¼M human growth hormone gene F and R primers for an internal positive control and sterilized distilled water is used to make up the final volume.

Table 1: Molecular genotyping of CYP2C9 allele.

The PCR amplification conditions for 45 cycles were; denaturation at 95Â°C for 45 seconds, 67Â°C for 10 seconds and 72Â°C for 15 seconds with an initial denaturation at 95Â°C for 10 minutes and final extension step at 72Â°C for 5 minutes (Seng et al. 2003). Afterwards, each PCR product of volume 12 Î¼l was digested with restriction enzyme from vivantis (Ava II for CYP2C9*2 and for CYP2C9*3 the Kpn I was used) at 37Â°C and incubated overnight to ensure complete digestion. The DNA segment was run on gel electrophoresis (2.5% agarose gel cast with ethidium bromide). Bands were detected via UV trans-illuminator and photographed using Chemi Doc XRS gel photo-documentation system of Bio rad. The 375 bp PCR product of CYP2C9*1 or CYP2C9*3 allele were cleaved by Ava II into two fragments of 296 bp and 79 bp. While the CP2C9*2 allele was resistant to cleavage. The allele CYP2C9*3 was cleaved in two fragments sized 85 bp and 20 bp by enzyme kpn I.

Statistical analysis of data

We applied Chi-square test (nonparametric test for discrete variables) to compare the differences between the two ethnic groups studied. The value of p

Results

The distribution of CYP2C9*1, *2, and *3 alleles as well the *1/*1, *1/*2, *1/*3, *2/*2, *2/*3, and *3/*3 genotypes in Sindhi and Baluchi populations is presented in table 2. All CYP2C9 allele and genotype frequencies were in Hardy-Weinberg equilibrium both in Sindhi and Baluchi subjects. Besides the wild-type allele, the CYP2C9*2 was the most common allele identified in Sindhi population, whereas in the Baluchi population, the CYP2C9*3 was most frequent. In addition, we found a significant increase in CYP2C9*3 prevalence in Baluchi population compared to Sindhi samples (p<0.001). Furthermore, the frequency of *1/*3 genotype observed was considerably higher in the Baluchi group than in Sindhi (0.170 versus 0.129, p<0.001). The *1/*1 genotype in the Sindhi population was more common than in Baluchi subjects (p<0.005).

Table 2: Allele and genotype frequencies and the predicted phenotype of CYP2C9 in the healthy Sindhi and Baluchi population samples; data are compared with those reported for Indian and Caucasian populations.

Table 3: Genotype frequency in different ethnic groups worldwide.

Discussion

Genetic differences among different individuals and interethnic groups affect the warfarin drug metabolism and utilization [17]. A number of drugs are metabolized by the human cytochrome P450 system [7]. CYP2C9 is the most abundant of the CYP2C enzymes [18]. It is involved in the metabolism of more than 150 drugs [8,10]. The metabolism of S-warfarin of coumarins, acenocoumarol and phenprocoumon are significantly decreased by the presence of both the CYP2C9*2 and CYP2C9*3 alleles [19-23]. To reduce the toxic effects and faster anticoagulation of warfarin administration, a genotype adjusted dosage is required [24,25]. The results of this study were also compared with compiled data reported for other populations with particular observe to the Caucasian population and Indians (Tables 2 and 3) [16,26]. The prevalence of CYP2C9*2 and *3 alleles observed in our study of healthy Sindhi subjects were very close to that found in Baluchi patients [27] and other Caucasian populations [16-28,29-30], but proved to be higher than in Orientals [13,31,32-33]. CYP2C9*2 and *3 variants are more prevalent in Europeans and Americans of European descent (8-16% and 4-9%, respectively) [13,16,28,27-34,35] than in Asians (0% and 1-5%), or Americans of African descent (1-2% and 0.5-1%) [13,31,32-35]. Moreover, the allele frequencies of CYP2C9*2 and CYP2C9*3 tend to decrease from West to East. Although the *3/*3 genotype frequency in numerous studies was not detectable, in Sindhi population, it seems to be in a higher range among Caucasians [9,16,28,27-30]. Similarly, we did not find the homozygous mutants for the *3 polymorphism in Sindhi and Baluchi population samples considering the published literature (Table 3). The *2/*3 genotype is associated with clinically significant alterations in the pharmacokinetics of CYP2C9 substrates; therefore, our finding in healthy Sindhi and especially in Baluchi population is of special clinical importance [19,21,36]. We found significant differences in the genotype and allele frequencies of CYP2C9*2 and *3 variants in Baluchi population versus the Indian population (p<0.04) published in the literature, except the *2/*2 and *3/*3 genotypes, which were also not detected in Indians [26]. In addition, significant differences were found in *3 allele frequency and the *1/*1, *1/*3 genotype prevalence between the Baluchi group and other Caucasian population (p<0.04) [16]. It has been suggested that the CYP2C9*3 allele is responsible for the largest reduction in metabolic activity in vitro, as compared to *1 [36], thus the higher proportion of *3 allele (0.165) in Baluchi population compared to other ethnic groups was of special importance [13,16,28,26-37]. Taking these findings together, this is the first study to document the allele and genotype frequencies of two major variants of CYP2C9 gene in healthy Sindhi and Baluchi population samples. The distribution of CYP2C9 alleles in Baluchi population showed variation when compared with Hungarians, Indians and Caucasians. The analyses of allele and genotype frequencies both in Sindhi and Baluchi populations and their comparison with data obtained in other ethnic groups provide genetic evidence of the origin of different populations. Furthermore, it is suggested that CYP2C9 genotyping can be used routinely to obtain efficient drug therapy and to lower the risk of therapeutic failure, mainly of drugs with low therapeutic indices.

Acknowledgment

This work was supported by The University of Lahore, Pakistan. We are thankful to Dr. M.H. Qazi, Director, CRIMM, all doctors, nurses and subjects who participate in this study.

References