Figure 1. Restriction fragment products The Study on the Frequency of Xenobiotic-Metabolizing Enzyme Gene Polymorphisms CYP2C9 in a Turkish Population S. Vildan Akgül¹, Tuğçe Yeşil¹, Osman Ekinci², Berna Terzioğlu³, İ. Ömer Barlas4, Gulden Z. Omurtag¹, Semra Şardaş¹ ¹ Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Marmara University, Istanbul, Turkey ² Department of Anaesthesiology and Reanimation, Haydarpasa Numune Training and Research Hospital, Istanbul, Turkey ³ Dr. Siyami Ersek Thoracic and Cardiovascular Surgery Training and Research Hospital, Istanbul, Turkey 4 Department of Medicinal Biology and Genetics, Faculty of Medicine, Mersin University, Mersin, Turkey The functional CYP2C9*1 was the most frequent allele found in our study group (%88.33). 11.67% of the studied present population are predicted to be intermediate metabolizers, when classified according to Zainuddin et al . The study continues by covering also the analysis of CYP2C9*3 allele. Introduction The family of cytochrome CYP450 enzymes consists of more than 30 isoenzymes which are the major mediators of phase I metabolism of drugs and endogenous compounds. A mutation in a gene coding for a drug metabolizing enzyme can result in an enzyme with normal, low, or no activity. Genetic polymorphism of CYP450 enzymes can lead to adverse drug reactions or inadequate response to commonly prescribed doses of therapeutic agents. CYP2C9 represents the most abundant among human CYP2C isoforms and comprises approximately 20% of the total hepatic CYP450 content. It participates in the metabolism of various drugs, including nonsteroid antiinflammatory agents, S-warfarin, phenytoin and losartan. Polymorphisms in the CYP2C9 gene seriously affect the toxicity of drugs, especially warfarin, and may lead to severe and life-threatening bleeding episodes. Both CYP2C9*2 and *3 cause a reduction in S-warfarin clearance with10-fold variation observed from the genotype with the highest (CYP2C9*1/*1) to the one with the lowest (CYP2C9*3/*3) activity. (*1/*1>*1/*2>*1/*3>*2/*2>*2/ *3>*3/*3). The effect of the CYP2C9*3/*3 genotype is the most severe one with clearance of S-warfarin being 10% of the wild type genotype [1]. According to clinical studies, individuals with the CYP2C9*1/*2 and *1/*3 genotypes require 10–20% and 20–50% lower average maintenance doses of warfarin, respectively, compared to wild type individuals [2]. CYP2C9*2 and *3 polymorphisms occur in approximately 85% of PMs and the frequency reported for White populations varies from 2% to 6%. Figure 1. Restriction fragment products Figure 2. PCR products and Rest. Fragment products and their bp status on marker 88.33 11.67 Discussion Among multiple variant alleles of CYP2C9, the incidence of *2 is higher in European population than African and Asian populations as demonstrated in Table 1. However, the *2, the most prevalent mutant allele among Caucasians, has not been genotyped in East Asian populations. [5] Since these genetic variations are clinically unique, the findings of this study could be helpful for optimization of dosing of CYP2C9 substrates with a narrow therapeutic index and in identification of patients at risk of adverse drug reactions. Materials & Methods ALLELE GENOTYPE Population n *1 *2 *3 *1/*1 *1/*2 *2/*2 Turkish(*) 120 88.33 11.67 nsy 81.67 18.33 Chinese 165 97.3 3.3 93.33 Malays 202 95.7 1.9 2.4 91.43 3.81 Spanish 102 74.5 15.6 9.8 57.8 19.6 3.9 Ethiophian 150 4.3 2.3 86.7 8.7 Turkish [6] 499 79.4 10.6 1.0 61.72 18.04 1 ml blood sample was taken from 120 unrelated Turkish individuals who applied to Department of Anaesthesiology and Reanimation, Haydarpasa Numune Training and Research Hospital, Istanbul, Turkey. Ethical approval was obtained from Marmara University Ethical Commission. 66 male 54 female individuals whose ages were between 18-80 years old ( mean age 44) participated in the present study. DNA was isolated as using half of sample while other 0.5 ml was stored at -20 C for any further repitation. Roche’s DNA Isolation Kit for Mammalian Blood was used to obtain DNA from lymphocytes. DNAs were solved in 100 µl TE buffer and after isolation they were left overnight in water bath at 65 C. DNAs are stored at +4 C. RFLP-PCR was applied according to proceedure of Adithan et al.[3]. Ava II restriction enzyme was used. Table 1. Comparison of allele and genotype frequencies of CYP2C9 in different populations Results *in our study group in Turkey nsy: not studied yet References Scordo MG, Pengo V, Spina E, Dahl ML, Gusella M, Padrini R. Influence of CYP2C9 and CYP2C19 genetic polymorphisms. Pharmacol. Ther. 2002; 72: 702–710. Sanderson S, Emery J, Higgins J. CYP2C9 gene variants, drugdose, and bleeding risk in warfarin-treated patients: a HuGEnetsystematic review and meta-analysis. Genet. Med. 2005;797–104 Adithan C, Gerard N, Vasu S, R. Balakrishan R, Shasandrian CH, Krishnamoorty R, Allele and genotype frequency ofCYP2C9 in Tamilnadu population. Eur J Clin Pharmacol. 2003; 59: 707-709. Zainuddin Z., Teh L. K.,Suhaimi A. W. M., Ismail R. Malaysian Indians are genetically similar to Caucasians:CYP2C9 polymorphism. Journal of Clinical Pharmacy and Therapeutics; (2006) 31, 187–191 Bae JW, Kim HK, Kim JH, Yang SI, Kim MJ, Jang CG, Park YS, Lee SY. Alele and Genotyğe frequencies of CYP2C9 in a Korean population. British Journal of Clinical Pharmacology. 2005. 60:4; 414-422. Aynacioglu AS, Brockmoller J, Bauer S, Sachse C, Guzelbey P, Ongen Z et al. Frequency of cytochrome P450 CYP2C9 variants in a Turkish population and functional relevance for phenytoin. Br J Clin Pharmacol 1999; 48: 409–415. The frequency of the CYP2C9*2 was analysed by using RFLP-PCR. Although 153 unrelated Turkish individuals participated to our study 33 of the DNA samples failed to amplify using the current method. The frequencies of CYP2C9*1/*1 and CYP2C9**1/*2 added up to 240 and the genotyped allele were %88.333,and %11.6667respectively. No individual was found to be homozygous for CYP2C9*2/*2..