CYP2D6 genotyping strategy based on gene copy number determination by TaqMan real-rime PCR

Abstract

The genetic polymorphism of the cytochrome P450 monooxygenase, CYP2D6, comprises at least 43 alleles giving rise to distinct drug metabolism phenotypes termed ultrarapid, extensive, intermediate, and poor metabolizers. As a consequence, drug side effects or lack of drug effect may occur if standard doses are applied. Genetic prediction of drug oxidation phenotype as a basis for dose selection requires analysis of single nucleotide polymorphisms and of alleles with duplicated or deleted genes. Here we developed a novel method to determine the CYP2D6 gene dose per genome. A TaqMan real-time PCR assay to specifically amplify genomic CYP2D6 was established by using a specific set of amplification primers and probe, located in exon 9, which effectively prevent amplification of CYP2D7 and CYP2D8 pseudogenes. Quantitative CYP2D6 amplification data were normalized to albumin as an internal reference gene which was coamplified simultaneously in a single-tube biplex assay. The assay was validated with a selection of previously genotyped DNA samples containing none, one, two, or three CYP2D6 gene copies. The results were highly reproducible and closely matched the number of genes with no overlap between the groups. Analysis of DNA samples comprising all major alleles and genotypes revealed high sensitivity and specificity of the assay, as demonstrated by agreement of the determined gene dose with the presence of CYP2D6(*)2 x 2 (gene duplication) and CYP2D6(*) 5 (gene deletion) alleles. The predictability of the new strategy was systematically evaluated. The semiautomatic TaqMan assay allows high sample throughput and will be useful for pharmacogenetic studies and in the clinical setting.

Tamoxifen Resistance and CYP2D6 Copy Numbers in Breast Cancer Patients

“…Schaeffele et al in Germany used Realtime PCR for finding CYP2D6 copy numbers. Results were acceptable and similar to results of long-distance PCR and southern blot methods without any overlap on non metabolizer, poor metabolizer, intermediate metabolizer and extensive metabolizer alleles (Schaeffeler et al,2003 ). In 2005 in France, Bodin et al used CYP2D6 Real-time PCR for 43 samples that the long term PCR and Southern blot methods were done for them previously.…”

“…We in our study selected an area of exon 9 of CYP2D6 for PCR. Cause of choosing this exon was differences between this exon and pseudo gens of this gene (CYP2D7 and CYP2D8) to prevent making pseudo copies (Schaeffeler et al, 2003). We chose albumin (ALB) as internal reference gene.…”

“…CNV means one Kb or higher pieces of DNA with various numbers of copies in contrasting with a reference genome that is a part of genomic variation with single nucleotide polymorphism can be a factor in phenotype variation related to drug response (Schaeffeler et al, 2003;Yu et al, 2009). Copies of CYP2D6 can vary from 0-13 repeats of gene that possibly cause phenotypic variation in metabolism of drugs substrate of this enzyme.…”

“…All CNVs will not act in similar fashion. In CYP2D6, CNV can cause decreased enzyme activation (gene deletion) or increased enzyme metabolism (Ingelman-Sundberg et al, 1991;Johansson et al, 1993;Schaeffeler et al, 2003;Ingelman-Sundberg et al, 2007;Hosono et al, 2009). The aim of this study was to detect copy numbers variation of the CYP2D6 gene and its possible correlation with Tamoxifen resistance among breast cancer patients.…”

The clinical effectiveness and cost-effectiveness of testing for cytochrome P450 polymorphisms in patients with schizophrenia treated with antipsychotics: a systematic review and economic evaluation

“…This was particularly the case for CYP2D6, although five of the six US studies also investigated polymorphisms of 90 Heller 2006, 91 Hersberger 2000, 92 James 2004, 93 Lee 2007, 94 Melis 2006, 95c Muller 2003, 96 Neville 2002, 97 Nielsen 2007, 98 Roberts 2000, 99 Roche 2004, 100 Schaeffeler 2003, 101 Soderback 2005, 102 Stamer 2007, 103 Stuven 1996, 104 118 Muthiah 2004, 119 Oyama 1995, 120 Rohrbacher 2006, 121 Weise 2004, 122 Weise 2006, 123 Wen 2004, 124 Wu 2002 125 a The other CYP polymorphisms are CYP1A1 (n = 4), CYP1B1 (n = 1), CYP2B6 (n = 1), CYP2C8 (n = 3), CYP2E1 (n = 1), Studies varied in size from 40 subjects being genotyped in the smallest study 112 to 428 in the largest, 118 although the number of samples being compared by the reference method varied from just six samples tested by AS-PCR in Oyama et al 120 to 1400 samples in Lee et al 94 …”