Rapid and reliable genotyping procedure for detection of alleles with mutations, deletion, or/and duplication of the CYP2D6 gene Borros Arneth a, * ,1 , Mohammed Shams b,d, * ,1 , Christoph Hiemke b , Sebastian Härtter b,c a Department of Clinical Chemistry, University of Mainz, Germany b Department of Psychiatry, University of Mainz, Germany c Boehringer-Ingelheim Pharma, Department of Drug Metabolism and Pharmacokinetics, Germany d Department of Pharmaceutics and Clinical Pharmacy, Faculty of Pharmacy, Mansoura University, Egypt Received 20 December 2008; received in revised form 21 March 2009; accepted 8 April 2009 Available online 22 April 2009 Abstract Background: Polymorphisms of cytochrome P450 2D6 (CYP2D6) have a significant effect on the pharmacokinetics of most tricyclic antidepressants. More than 150 alleles lead to four distinct phenotypes of drug metabolism. The phenotypes are described as ultrarapid, extensive, intermediate, and poor metabolizers. Therapeutic plasma levels of CYP2D6 substrates may be difficult to achieve. Here we describe a rapid and reliable procedure for CYP2D6*4, *3, *6, and *9 genotyping. Design and methods: Serum concentrations of venlafaxine and its pharmacologically active metabolite, O-desmethylvenlafaxine, were measured in patients treated with the antidepressant venlafaxine, a substrate of CYP2D6. The ODV/V ratio was used as an indicator of the CYP2D6 phenotype, with a higher ratio reflecting more rapid metabolism. Real-time PCR with fluorometric melting point analysis of the PCR products (LightCycler ® ) is used to identify SNPs. Using quantitative PCR, gene deletion and gene duplication or multiplication are investigated by measurement of the fluorescence intensity quotient (q, N) of the CYP2D6 gene relative to that of the albumin gene as an internal standard. Results: Melting curves are verified using DNA samples of known genotypes and by sequencing the PCR products. The genotypes and phenotypes that were detected correspond to each other. Conclusion: A PCR procedure for the detection of CYP2D6 SNPs, deletions, and duplications is described and is rapid and reliable. © 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: CYP2D6; SNPs; Genetic polymorphisms; Real-time PCR; Light cycler Introduction Cytochrome P450 2D6 (CYP2D6) is involved in the oxidative metabolism of numerous commonly prescribed drugs, including many antiarrhythmics, β-blockers, neurolep- tics, selective serotonin reuptake inhibitors (SSRIs), and tricyclic antidepressants [1,2]. The CYP2D6 enzyme localizes mainly to the liver. Total deletion of the CYP2D6 gene or mutations that lead to an inactive or less-active enzyme has been described. Patients who are taking drugs that are CYP2D6 substrates and who have such mutations may have elevated drug levels. Conversely, gene duplication or multiplication is thought to lead to increased enzyme production, which results in an increased rate of drug metabolism [3], thereby leading to decreased drug levels. The importance of studying gene copy number variations (CNVs) is the subject of several recent publications [4–6], which suggests that the study of genetic variations will receive more attention in the near future. The characterization of CYP2D6 genotypes that are associated with specific phenotypes could lead to the practice of genotyping patients before initiating pharmacologic therapy with a CYP2D6 substrate. The genotype of an individual patient could inform the choice Available online at www.sciencedirect.com Clinical Biochemistry 42 (2009) 1282 – 1290 * Corresponding authors. B. Arneth is to be contacted at Department of Clinical Chemistry, University of Mainz, Langenbeckstr. 1, D-55131 Mainz, Germany. Fax: +49 6131 17 6627. M. Shams, Department of Psychiatry, University of Mainz, Langenbeckstr. 1, D-55131 Mainz, Germany. Fax: +49 6131 17 6789. E-mail address: arneth@zentrallabor.klinik.uni-mainz.de (B. Arneth). 1 Both authors contributed equally to this article. 0009-9120/$ - see front matter © 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2009.04.009