Rapid and Reliable Genotyping Procedure for Detection of Alleles with Mutation, Deletion or/and Duplication of the CYP2D6-Gene

Our study aimed to develop a rapid and reliable procedure for detection of CYP2D6 null alleles *3, *4, *6, *9 or alleles with reduced activity, gene deletion (*5) and gene duplication (*1x N) of the polymorphic CYP2D6 and relate the genotypes to venlafaxine pharmacokinetics. Twenty five depressed patients (14 men) with a mean age±SD (49.8±12.7) were treated with the antidepressant venlafaxine in dosages between 75 and 450mg/day and were preselected for genotyping analysis when abnormal metabolic ratios between the pharmacologically active metabolite O-demethylvenlafaxine (ODV) and venlafaxine (V), [ODV/V] were detected. These patients were genotyped with one normal individual [wild type (*1/*1)] as a control. EDTA blood was drawn and stored at -20°C until isolation of genomic DNA (Roche DNA Isolation Kit, Berlin, Germany). Real-time PCR reaction with subsequent fluorometric melting point analysis of the PCR product using the LightCycler (Roche Diagnostics), and Roche LightCycler-Software version 3.5 were used. Each mutation was investigated separately. One homozygous (*6/*6), and three heterozygous (two with*4/*5 and one with*4/*6) with a mean metabolic ratio±SD (0.25±0.0.04) and below the 10th percentile (0.26) could be classified as poor metabolizers (PMs). Five individuals were heterozygous (*4/*1) with a mean metabolic ratio±SD (1.1±0.8) and one patient carried [*4/gene duplication (*1xN)] with an intermediate metabolic ratio (4.8). The mean ODV/V ratio±SD in 100 depressed patients was 2.3±2.1. Six individuals had a gene duplication and showed high metabolic activities (10.3±2.7) well beyond the 90th percentile (5.08) of the metabolic ratios. Our new genotyping method allows rapid and reliable detection of PMs and UMs and that is of clinical importance for early adjustment of venlafaxine doses, and to assure therapeutic efficacy with a minimum risk of adverse effects.