Abstract
Cytochrome P450 2D6 (cytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6)), a highly polymorphic drug-metabolizing enzyme, is involved in the metabolism of one-quarter of the most commonly prescribed medications. Here we have applied multiple genotyping methods and Sanger sequencing to assign precise and reproducible CYP2D6 genotypes, including copy numbers, for 48 HapMap samples. Furthermore, by analyzing a set of 50 human liver microsomes using endoxifen formation from N-desmethyl-tamoxifen as the phenotype of interest, we observed a significant positive correlation between CYP2D6 genotype-assigned activity score and endoxifen formation rate (rs=0.68 by rank correlation test, P=5.3 × 10−8), which corroborated the genotype–phenotype prediction derived from our genotyping methodologies. In the future, these 48 publicly available HapMap samples characterized by multiple substantiated CYP2D6 genotyping platforms could serve as a reference resource for assay development, validation, quality control and proficiency testing for other CYP2D6 genotyping projects and for programs pursuing clinical pharmacogenomic testing implementation.
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References
Zhou SF . Polymorphism of human cytochrome P450 2D6 and its clinical significance: part II. Clin Pharmacokinet 2009; 48: 761–804.
Zhou SF . Polymorphism of human cytochrome P450 2D6 and its clinical significance: part I. Clin Pharmacokinet 2009; 48: 689–723.
Zanger UM, Raimundo S, Eichelbaum M . Cytochrome P450 2D6: overview and update on pharmacology, genetics, biochemistry. Naunyn Schmiedebergs Arch Pharmacol 2004; 369: 23–37.
Gaedigk A . Complexities of CYP2D6 gene analysis and interpretation. Int Rev Psychiatry 2013; 25: 534–553.
Kramer WE, Walker DL, O'Kane DJ, Mrazek DA, Fisher PK, Dukek BA et al. CYP2D6: novel genomic structures and alleles. Pharmacogenet Genomics 2009; 19: 813–822.
Broly F, Gaedigk A, Heim M, Eichelbaum M, Morike K, Meyer UA . Debrisoquine/sparteine hydroxylation genotype and phenotype: analysis of common mutations and alleles of CYP2D6 in a European population. DNA Cell Biol 1991; 10: 545–558.
Sachse C, Brockmoller J, Hildebrand M, Muller K, Roots I . Correctness of prediction of the CYP2D6 phenotype confirmed by genotyping 47 intermediate and poor metabolizers of debrisoquine. Pharmacogenetics 1998; 8: 181–185.
Bradford LD . CYP2D6 allele frequency in European Caucasians, Asians, Africans and their descendants. Pharmacogenomics 2002; 3: 229–243.
Pratt VM, Zehnbauer B, Wilson JA, Baak R, Babic N, Bettinotti M et al. Characterization of 107 genomic DNA reference materials for CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1: a GeT-RM and Association for Molecular Pathology collaborative project. J Mol Diagn 2010; 12: 835–846.
Hosono N, Kato M, Kiyotani K, Mushiroda T, Takata S, Sato H et al. CYP2D6 genotyping for functional-gene dosage analysis by allele copy number detection. Clin Chem 2009; 55: 1546–1554.
Qian JC, Xu XM, Hu GX, Dai DP, Xu RA, Hu LM et al. Genetic variations of human CYP2D6 in the Chinese Han population. Pharmacogenomics 2013; 14: 1731–1743.
International HapMap Consortium. A haplotype map of the human genome. Nature 2005; 437: 1299–1320.
Hosono N, Kubo M, Tsuchiya Y, Sato H, Kitamoto T, Saito S et al. Multiplex PCR-based real-time invader assay (mPCR-RETINA): a novel SNP-based method for detecting allelic asymmetries within copy number variation regions. Hum Mutat 2008; 29: 182–189.
Ramirez J, Liu W, Mirkov S, Desai AA, Chen P, Das S et al. Lack of association between common polymorphisms in UGT1A9 and gene expression and activity. Drug Metab Dispos 2007; 35: 2149–2153.
Fukuda T, Maune H, Ikenaga Y, Naohara M, Fukuda K, Azuma J . Novel structure of the CYP2D6 gene that confuses genotyping for the CYP2D6*5 allele. Drug Metab Pharmacokinet 2005; 20: 345–350.
Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 2007; 81: 559–575.
Antunes MV, Rosa DD, Viana Tdos S, Andreolla H, Fontanive TO, Linden R . Sensitive HPLC-PDA determination of tamoxifen and its metabolites N-desmethyltamoxifen, 4-hydroxytamoxifen and endoxifen in human plasma. J Pharm Biomed Anal 2013; 76: 13–20.
Box GEP, Cox DR . An analysis of transformations (with discussion). J R Stat Soc 1964; B: 211–252.
Venables WN, Ripley BD . Modern Applied Statistics with S 4th edn. Springer, 2002.
R Development Core Team. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing: Vienna, Austria, 2011.
Steen VM, Andreassen OA, Daly AK, Tefre T, Borresen AL, Idle JR et al. Detection of the poor metabolizer-associated CYP2D6(D) gene deletion allele by long-PCR technology. Pharmacogenetics 1995; 5: 215–223.
Abecasis GR, Auton A, Brooks LD, DePristo MA, Durbin RM, Handsaker RE et al. An integrated map of genetic variation from 1,092 human genomes. Nature 2012; 491: 56–65.
Gaedigk A, Bradford LD, Alander SW, Leeder JS . CYP2D6*36 gene arrangements within the cyp2d6 locus: association of CYP2D6*36 with poor metabolizer status. Drug Metab Dispos 2006; 34: 563–569.
Crews KR, Gaedigk A, Dunnenberger HM, Klein TE, Shen DD, Callaghan JT et al. Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines for codeine therapy in the context of cytochrome P450 2D6 (CYP2D6) genotype. Clin Pharmacol Ther 2012; 91: 321–326.
Heller T, Kirchheiner J, Armstrong VW, Luthe H, Tzvetkov M, Brockmoller J et al. AmpliChip CYP450 GeneChip: a new gene chip that allows rapid and accurate CYP2D6 genotyping. Ther Drug Monit 2006; 28: 673–677.
Kim J, Lee SY, Lee KA . Copy number variation and gene rearrangements in CYP2D6 genotyping using multiplex ligation-dependent probe amplification in Koreans. Pharmacogenomics 2012; 13: 963–973.
Sim SC, Daly AK, Gaedigk A . CYP2D6 update: revised nomenclature for CYP2D7/2D6 hybrid genes. Pharmacogenet Genomics 2012; 22: 692–694.
Gaedigk A, Fuhr U, Johnson C, Berard LA, Bradford D, Leeder JS . CYP2D7-2D6 hybrid tandems: identification of novel CYP2D6 duplication arrangements and implications for phenotype prediction. Pharmacogenomics 2010; 11: 43–53.
O'Donnell PH, Bush A, Spitz J, Danahey K, Saner D, Das S et al. The 1200 patients project: creating a new medical model system for clinical implementation of pharmacogenomics. Clin Pharmacol Ther 2012; 92: 446–449.
Acknowledgements
We acknowledge the help of Dr R Stephanie Huang and Bonnie LaCroix from the Pharmacogenomics of Anticancer Agents Cell Core and the kind remarks from Dr Kazuma Kiyotani. We also thank the Liver Tissue Procurement and Distribution System (NIH contract 3N01-DK-9-2310) and the Cooperative Human Tissue Network for providing the liver samples. This work is supported by NIH U01GM061393 (Pharmacogenomics of Anticancer Agents Research Group; to YN, MJR and NJC) and NIH K12 CA139160 and K23 GM100288-01A1 (to PHO). HF is supported by a NIH T32 GM007019 training grant in Clinical Pharmacology and Pharmacogenomics. MJR is a recipient of a Conquer Cancer Foundation of ASCO Translational Research Professorship, in memory of Merrill J Egorin, MD. Any opinions, findings and conclusions expressed in this material are those of the authors and do not necessarily reflect those of the American Society of Clinical Oncology or the Conquer Cancer Foundation.
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Fang, H., Liu, X., Ramírez, J. et al. Establishment of CYP2D6 reference samples by multiple validated genotyping platforms. Pharmacogenomics J 14, 564–572 (2014). https://doi.org/10.1038/tpj.2014.27
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DOI: https://doi.org/10.1038/tpj.2014.27