Recommendations for Clinical CYP2D6 Genotyping Allele Selection: A Joint Consensus Recommendation of the Association for Molecular Pathology, College of American Pathologists, Dutch Pharmacogenetics Working Group of the Royal Dutch Pharmacists Association, and the European Society for Pharmacogenomics and Personalized Therapy

Abstract

The goals of the Association for Molecular Pathology Clinical Practice Committee's Pharmacogenomics (PGx) Working Group are to define the key attributes of pharmacogenetic alleles recommended for clinical testing, and to determine a minimal set of variants that should be included in clinical PGx genotyping assays. This document series provides recommendations on a minimal panel of variant alleles (Tier 1) and an extended panel of variant alleles (Tier 2) that will aid clinical laboratories in designing assays for PGx testing. When developing these recommendations, the Association for Molecular Pathology PGx Working Group considered the functional impact of the variant alleles, allele frequencies in multiethnic populations, the availability of reference materials, as well as other technical considerations with regard to PGx testing. The ultimate goal of this Working Group is to promote standardization of PGx gene/allele testing across clinical laboratories. This document is focused on clinical CYP2D6 PGx testing that may be applied to all cytochrome P450 2D6–metabolized medications. These recommendations are not meant to be interpreted as prescriptive but to provide a reference guide for clinical laboratories that may be either implementing PGx testing or reviewing and updating their existing platform. The goals of the Association for Molecular Pathology Clinical Practice Committee's Pharmacogenomics (PGx) Working Group are to define the key attributes of pharmacogenetic alleles recommended for clinical testing, and to determine a minimal set of variants that should be included in clinical PGx genotyping assays. This document series provides recommendations on a minimal panel of variant alleles (Tier 1) and an extended panel of variant alleles (Tier 2) that will aid clinical laboratories in designing assays for PGx testing. When developing these recommendations, the Association for Molecular Pathology PGx Working Group considered the functional impact of the variant alleles, allele frequencies in multiethnic populations, the availability of reference materials, as well as other technical considerations with regard to PGx testing. The ultimate goal of this Working Group is to promote standardization of PGx gene/allele testing across clinical laboratories. This document is focused on clinical CYP2D6 PGx testing that may be applied to all cytochrome P450 2D6–metabolized medications. These recommendations are not meant to be interpreted as prescriptive but to provide a reference guide for clinical laboratories that may be either implementing PGx testing or reviewing and updating their existing platform. To address variability in clinical pharmacogenomics (PGx) testing, and to facilitate standardization across laboratories, the Association for Molecular Pathology (AMP) PGx Working Group has developed a series of documents that recommend a minimal set of variant alleles for inclusion in clinical PGx assays. Previous documents have covered CYP2C19,1Pratt V.M. Del Tredici A.L. Hachad H. Ji Y. Kalman L.V. Scott S.A. Weck K.E. Recommendations for clinical CYP2C19 genotyping allele selection: a report of the Association for Molecular Pathology.J Mol Diagn. 2018; 20: 269-276Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar CYP2C9,2Pratt V.M. Cavallari L.H. Del Tredici A.L. Hachad H. Ji Y. Moyer A.M. Scott S.A. Whirl-Carrillo M. Weck K.E. Recommendations for clinical CYP2C9 genotyping allele selection: a joint recommendation of the Association for Molecular Pathology and College of American Pathologists.J Mol Diagn. 2019; 21: 746-755Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar and genes important for warfarin PGx testing.3Pratt V.M. Cavallari L.H. Del Tredici A.L. Hachad H. Ji Y. Kalman L.V. Ly R.C. Moyer A.M. Scott S.A. Whirl-Carrillo M. Weck K.E. Recommendations for clinical warfarin genotyping allele selection: a report of the Association for Molecular Pathology and the College of American Pathologists.J Mol Diagn. 2020; 22: 847-859Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar The current document is focused on CYP2D6 and is intended to provide guidance for clinical laboratorians and assay manufacturers who develop, validate, and/or offer clinical CYP2D6 genotyping assays, and therefore to promote the standardization of PGx testing across clinical laboratories. This document should be implemented together with other relevant clinical guidelines, including those published by the Clinical Pharmacogenetics Implementation Consortium (CPIC),4Goetz M.P. Sangkuhl K. Guchelaar H.J. Schwab M. Province M. Whirl-Carrillo M. Symmans W.F. McLeod H.L. Ratain M.J. Zembutsu H. Gaedigk A. van Schaik R.H. Ingle J.N. Caudle K.E. Klein T.E. Clinical Pharmacogenetics Implementation Consortium (CPIC) guideline for CYP2D6 and tamoxifen therapy.Clin Pharmacol Ther. 2018; 103: 770-777Crossref PubMed Scopus (146) Google Scholar, 5Bell G.C. Caudle K.E. Whirl-Carrillo M. Gordon R.J. Hikino K. Prows C.A. Gaedigk A. Agundez J.A.G. Sadhasivam S. Klein T.E. Schwab M. Clinical Pharmacogenetics Implementation Consortium (CPIC) guideline for CYP2D6 genotype and use of ondansetron and tropisetron.Clin Pharmacol Ther. 2017; 102: 213-218Crossref PubMed Scopus (93) Google Scholar, 6Hicks J.K. Sangkuhl K. Swen J.J. Ellingrod V.L. Müller D.J. Shimoda K. Bishop J.R. Kharasch E.D. Skaar T.C. Gaedigk A. Dunnenberger H.M. Klein T.E. Caudle K.E. Stingl J.C. Clinical Pharmacogenetics Implementation Consortium (CPIC) guideline for CYP2D6 and CYP2C19 genotypes and dosing of tricyclic antidepressants: 2016 update.Clin Pharmacol Ther. 2017; 102: 37-44Crossref PubMed Scopus (249) Google Scholar, 7Hicks J.K. Bishop J.R. Sangkuhl K. Muller D.J. Ji Y. Leckband S.G. Leeder J.S. Graham R.L. Chiulli D.L. LLerena A. Skaar T.C. Scott S.A. Stingl J.C. Klein T.E. Caudle K.E. Gaedigk A. Clinical Pharmacogenetics Implementation Consortium (CPIC) guideline for CYP2D6 and CYP2C19 genotypes and dosing of selective serotonin reuptake inhibitors.Clin Pharmacol Ther. 2015; 98: 127-134Crossref PubMed Scopus (478) Google Scholar, 8Crews K.R. Gaedigk A. Dunnenberger H.M. Leeder J.S. Klein T.E. Caudle K.E. Haidar C.E. Shen D.D. Callaghan J.T. Sadhasivam S. Prows C.A. Kharasch E.D. Skaar T.C. Clinical Pharmacogenetics Implementation Consortium guidelines for cytochrome P450 2D6 genotype and codeine therapy: 2014 update.Clin Pharmacol Ther. 2014; 95: 376-382Crossref PubMed Scopus (434) Google Scholar, 9Brown J.T. Bishop J.R. Sangkuhl K. Nurmi E.L. Mueller D.J. Dinh J.C. Gaedigk A. Klein T.E. Caudle K.E. McCracken J.T. de Leon J. Leeder J.S. Clinical Pharmacogenetics Implementation Consortium guideline for cytochrome P450 (CYP)2D6 genotype and atomoxetine therapy.Clin Pharmacol Ther. 2019; 106: 94-102Crossref PubMed Scopus (60) Google Scholar Dutch Pharmacogenetics Working Group of the Royal Dutch Pharmacists Association (DPWG) Canadian PGx Network for Drug Safety,10Madadi P. Amstutz U. Rieder M.J. Ito S. Fung V. Hwang S. Turgeon J. Michaud V. Koren G. Carleton B. Hayden M.R. Ross C.J. MacLeod S. Rassekh R. Lauder G. Smith A. Brunham L. Shear N.H. Liu G. Kim R. Maher M. Flockhart D. Clinical practice guideline: CYP2D6 genotyping for safe and efficacious codeine therapy.J Popul Ther Clin Pharmacol. 2013; 20: e369-e396PubMed Google Scholar and the American College of Medical Genetics and Genomics,11Lyon E. Gastier Foster J. Palomaki G.E. Pratt V.M. Reynolds K. Fernanda Sábato M. Scott S.A. Vitazka P. Laboratory testing of CYP2D6 alleles in relation to tamoxifen therapy.Genet Med. 2012; 14: 990-1000Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar which have mostly been focused on the interpretation of PGx test results and therapeutic recommendations on specific drug-gene pairs. The AMP PGx Working Group uses a two-tier strategy and selection criteria for recommending PGx variants for clinical testing.1Pratt V.M. Del Tredici A.L. Hachad H. Ji Y. Kalman L.V. Scott S.A. Weck K.E. Recommendations for clinical CYP2C19 genotyping allele selection: a report of the Association for Molecular Pathology.J Mol Diagn. 2018; 20: 269-276Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 2Pratt V.M. Cavallari L.H. Del Tredici A.L. Hachad H. Ji Y. Moyer A.M. Scott S.A. Whirl-Carrillo M. Weck K.E. Recommendations for clinical CYP2C9 genotyping allele selection: a joint recommendation of the Association for Molecular Pathology and College of American Pathologists.J Mol Diagn. 2019; 21: 746-755Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 3Pratt V.M. Cavallari L.H. Del Tredici A.L. Hachad H. Ji Y. Kalman L.V. Ly R.C. Moyer A.M. Scott S.A. Whirl-Carrillo M. Weck K.E. Recommendations for clinical warfarin genotyping allele selection: a report of the Association for Molecular Pathology and the College of American Pathologists.J Mol Diagn. 2020; 22: 847-859Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar Tier 1 PGx variant alleles are a minimal set recommended for clinical testing, while Tier 2 variant alleles do not meet all criteria for inclusion in Tier 1 but may be considered for clinical testing. As defined in previous guidelines,1Pratt V.M. Del Tredici A.L. Hachad H. Ji Y. Kalman L.V. Scott S.A. Weck K.E. Recommendations for clinical CYP2C19 genotyping allele selection: a report of the Association for Molecular Pathology.J Mol Diagn. 2018; 20: 269-276Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 2Pratt V.M. Cavallari L.H. Del Tredici A.L. Hachad H. Ji Y. Moyer A.M. Scott S.A. Whirl-Carrillo M. Weck K.E. Recommendations for clinical CYP2C9 genotyping allele selection: a joint recommendation of the Association for Molecular Pathology and College of American Pathologists.J Mol Diagn. 2019; 21: 746-755Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 3Pratt V.M. Cavallari L.H. Del Tredici A.L. Hachad H. Ji Y. Kalman L.V. Ly R.C. Moyer A.M. Scott S.A. Whirl-Carrillo M. Weck K.E. Recommendations for clinical warfarin genotyping allele selection: a report of the Association for Molecular Pathology and the College of American Pathologists.J Mol Diagn. 2020; 22: 847-859Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar Tier 1–recommended variant alleles meet the following criteria: i) have a well-characterized effect on the function of the protein and/or gene expression, ii) have an appreciable minor allele frequency in a population/ethnicity group, and iii) have publicly available reference materials (RMs). Tier 2–recommended variant alleles meet at least one but not all of the Tier 1 criteria, and may be moved to Tier 1 if RMs or additional information becomes available. The cytochrome P450 (CYP) 2D6 enzyme, encoded by CYP2D6, is a member of the 2D subfamily of CYP enzymes and is involved in the metabolism of many commonly prescribed medications, including some antidepressants, atypical and typical antipsychotics, β-blockers, opioids, antiemetics, atomoxetine, and tamoxifen.12Taylor C. Crosby I. Yip V. Maguire P. Pirmohamed M. Turner R.M. A review of the important role of CYP2D6 in pharmacogenomics.Genes (Basel). 2020; 11: 1295Crossref Scopus (17) Google Scholar,13Brunton L.L. Hilal-Dandan R. Knollmann B.C. Goodman & Gilman's: The Pharmacological Basis of Therapeutics.ed 13. McGraw-Hill Education, New York2018Google Scholar About 21% of currently approved medications are metabolized by CYP2D6.14Saravanakumar A. Sadighi A. Ryu R. Akhlaghi F. Physicochemical properties, biotransformation, and transport pathways of established and newly approved medications: a systematic review of the top 200 most prescribed drugs vs. the FDA-approved drugs between 2005 and 2016.Clin Pharmacokinet. 2019; 58: 1281-1294Crossref PubMed Scopus (32) Google Scholar Interindividual variation in CYP2D6 activity varies substantially and can be attributed, at least in part, to numerous genetic variants in CYP2D6 in the general population. These variants cause increased-function, decreased-function, or nonfunctional CYP2D6 enzyme. Clinical CYP2D6 testing involves the determination of sequence and structural variants by targeted genotyping or sequencing, followed by empirical assignment of detected variants into star (∗) alleles or haplotypes. A haplotype is a combination of sequence variants from multiple loci on a single chromosome, and a diplotype is composed of two haplotypes from the maternal and paternal chromosomes. Although the terms genotype and diplotype are often used interchangeably in PGx literature, in this document, genotype refers to sequence variants identified by molecular platforms that will then be assembled to haplotypes (alleles) and diplotypes. Both genotype and copy number results are combined to assemble haplotypes and diplotypes. CYP2D6 diplotypes are typically stratified into four groups that represent a patient's predicted CYP2D6 metabolizer phenotype: ultrarapid metabolizer, normal metabolizer (NM), intermediate metabolizer, and poor metabolizer.15Caudle K.E. Sangkuhl K. Whirl-Carrillo M. Swen J.J. Haidar C.E. Klein T.E. Gammal R.S. Relling M.V. Scott S.A. Hertz D.L. Guchelaar H.J. Gaedigk A. Standardizing CYP2D6 genotype to phenotype translation: consensus recommendations from the Clinical Pharmacogenetics Implementation Consortium and Dutch Pharmacogenetics Working Group.Clin Transl Sci. 2020; 13: 116-124Crossref PubMed Scopus (140) Google Scholar Importantly, CYP2D6 metabolizer phenotype has significant consequences on drug safety and effectiveness, prompting the CYP2D6 gene to be broadly included in the US Food and Drug Administration's (FDA) Table of Pharmacogenetic Associations (https://www.fda.gov/medical-devices/precision-medicine/table-pharmacogenetic-associations, last accessed September 27, 2020), and Table of Pharmacogenomic Biomarkers in Drug Labeling (https://www.fda.gov/drugs/science-and-research-drugs/table-pharmacogenomic-biomarkers-drug-labeling, last accessed September 27, 2020). CYP2D6 has nine exons and is located on chromosome 22q13.2 next to two highly homologous pseudogenes, CYP2D7 and CYP2D8.16Yang Y. Botton M.R. Scott E.R. Scott S.A. Sequencing the CYP2D6 gene: from variant allele discovery to clinical pharmacogenetic testing.Pharmacogenomics. 2017; 18: 673-685Crossref PubMed Scopus (43) Google Scholar It is highly polymorphic, with over 130 star allele haplotypes defined by the Pharmacogene Variation Consortium (PharmVar, www.pharmvar.org, last accessed September 23, 2020).17Gaedigk A. Ingelman-Sundberg M. Miller N.A. Leeder J.S. Whirl-Carrillo M. Klein T.E. The Pharmacogene Variation (PharmVar) Consortium: incorporation of the human cytochrome P450 (CYP) allele nomenclature database.Clin Pharmacol Ther. 2018; 103: 399-401Crossref PubMed Scopus (163) Google Scholar Most of the CYP2D6 star alleles are defined by single-nucleotide variants or small insertion/deletions. CYP2D6 can also harbor copy-number variants (CNVs; deletions and duplications) and gene conversions with the nearby CYP2D7 pseudogene, leading to CYP2D6-2D7 and CYP2D7-2D6 hybrid alleles.16Yang Y. Botton M.R. Scott E.R. Scott S.A. Sequencing the CYP2D6 gene: from variant allele discovery to clinical pharmacogenetic testing.Pharmacogenomics. 2017; 18: 673-685Crossref PubMed Scopus (43) Google Scholar,18Gaedigk A. Complexities of CYP2D6 gene analysis and interpretation.Int Rev Psychiatry. 2013; 25: 534-553Crossref PubMed Scopus (143) Google Scholar, 19Nofziger C. Paulmichl M. Accurately genotyping CYP2D6: not for the faint of heart.Pharmacogenomics. 2018; 19: 999-1002Crossref PubMed Scopus (19) Google Scholar, 20Nofziger C. Turner A.J. Sangkuhl K. Whirl-Carrillo M. Agúndez J.A.G. Black J.L. Dunnenberger H.M. Ruano G. Kennedy M.A. Phillips M.S. Hachad H. Klein T.E. Gaedigk A. PharmVar GeneFocus: CYP2D6.Clin Pharmacol Ther. 2020; 107: 154-170Crossref PubMed Scopus (60) Google Scholar Clinical laboratories offering CYP2D6 PGx testing mostly use a targeted genotyping approach. However, the large numbers of CYP2D6 alleles and structural variants make analysis of this gene challenging.16Yang Y. Botton M.R. Scott E.R. Scott S.A. Sequencing the CYP2D6 gene: from variant allele discovery to clinical pharmacogenetic testing.Pharmacogenomics. 2017; 18: 673-685Crossref PubMed Scopus (43) Google Scholar,19Nofziger C. Paulmichl M. Accurately genotyping CYP2D6: not for the faint of heart.Pharmacogenomics. 2018; 19: 999-1002Crossref PubMed Scopus (19) Google Scholar,20Nofziger C. Turner A.J. Sangkuhl K. Whirl-Carrillo M. Agúndez J.A.G. Black J.L. Dunnenberger H.M. Ruano G. Kennedy M.A. Phillips M.S. Hachad H. Klein T.E. Gaedigk A. PharmVar GeneFocus: CYP2D6.Clin Pharmacol Ther. 2020; 107: 154-170Crossref PubMed Scopus (60) Google Scholar A Genetic Testing Reference Material Program study21Pratt V.M. Everts R.E. Aggarwal P. Beyer B.N. Broeckel U. Epstein-Baak R. Hujsak P. Kornreich R. Liao J. Lorier R. Scott S.A. Smith C.H. Toji L.H. Turner A. Kalman L.V. Characterization of 137 genomic DNA reference materials for 28 pharmacogenetic genes: a GeT-RM collaborative project.J Mol Diagn. 2016; 18: 109-123Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar found little consistency in alleles included in CYP2D6 clinical tests across multiple laboratories. In addition, many assays were not designed for the detection of CNVs or other structural variants. A study in over 100,000 patient samples across all regions of the United States showed that structural variants, including CNVs, accounted for 7% of all CYP2D6 variants and could affect CYP2D6 metabolizer status.22Del Tredici A.L. Malhotra A. Dedek M. Espin F. Roach D. dan Zhu G. Voland J. Moreno T.A. Frequency of CYP2D6 alleles including structural variants in the United States.Front Pharmacol. 2018; 9: 305Crossref PubMed Scopus (50) Google Scholar Therefore, the variability in assay design, with or without the detection of CNVs and structural variants, can result in discrepancies in star allele calls and diplotype assignment, which directly affect phenotype translation, interpretation, and ultimately a patient's care.23Hoshitsuki K. Crews K.R. Yang W. Smith C.A. Hankins J.S. Turner A.J. Broeckel U. McMillin G.A. Relling M.V. Haidar C.E. Challenges in clinical implementation of CYP2D6 genotyping: choice of variants to test affects phenotype determination.Genet Med. 2020; 22: 232-233Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar,24Cavallari L.H. Van Driest S.L. Prows C.A. Bishop J.R. Limdi N.A. Pratt V.M. Ramsey L.B. Smith D.M. Tuteja S. Duong B.Q. Hicks J.K. Lee J.C. Obeng A.O. Beitelshees A.L. Bell G.C. Blake K. Crona D.J. Dressler L. Gregg R.A. Hines L.J. Scott S.A. Shelton R.C. Weitzel K.W. Johnson J.A. Peterson J.F. Empey P.E. Skaar T.C. Multi-site investigation of strategies for the clinical implementation of CYP2D6 genotyping to guide drug prescribing.Genet Med. 2019; 21: 2255-2263Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar According to the National Institutes of Health Genetic Testing Registry (https://www.ncbi.nlm.nih.gov/gtr/all/tests/?term=1565%5bgeneid%5d, last accessed October 29, 2020), CYP2D6 variants tested in US clinical laboratories range from a few targeted haplotype-defining variants to the analysis of selected exons or the entire coding region of the gene. Testing methods include targeted genotyping employing various laboratory-developed procedures or commercial platforms, single-nucleotide polymorphism–based microarrays, and full-gene Sanger or next-generation sequencing approaches, with or without deletion/duplication and structure variant analysis. For gene duplications, some laboratories offer additional testing, for example using long-range PCR12Taylor C. Crosby I. Yip V. Maguire P. Pirmohamed M. Turner R.M. A review of the important role of CYP2D6 in pharmacogenomics.Genes (Basel). 2020; 11: 1295Crossref Scopus (17) Google Scholar,25Qiao W. Yang Y. Sebra R. Mendiratta G. Gaedigk A. Desnick R.J. Scott S.A. Long-read single molecule real-time full gene sequencing of cytochrome P450-2D6.Hum Mutat. 2016; 37: 315-323Crossref PubMed Scopus (55) Google Scholar, 26Kramer W.E. Walker D.L. O'Kane D.J. Mrazek D.A. Fisher P.K. Dukek B.A. Bruflat J.K. Black J.L. CYP2D6: novel genomic structures and alleles.Pharmacogenet Genomics. 2009; 19: 813-822Crossref PubMed Scopus (45) Google Scholar, 27Gaedigk A. Jaime L.K.M. Bertino J.S. Bérard A. Pratt V.M. Bradford L.D.A. Leeder J.S. Identification of novel CYP2D7-2D6 hybrids: non-functional and functional variants.Front Pharmacol. 2010; 1: 121Crossref PubMed Scopus (49) Google Scholar to determine which allele is duplicated in order to provide a more accurate activity score assignment and phenotype prediction.15Caudle K.E. Sangkuhl K. Whirl-Carrillo M. Swen J.J. Haidar C.E. Klein T.E. Gammal R.S. Relling M.V. Scott S.A. Hertz D.L. Guchelaar H.J. Gaedigk A. Standardizing CYP2D6 genotype to phenotype translation: consensus recommendations from the Clinical Pharmacogenetics Implementation Consortium and Dutch Pharmacogenetics Working Group.Clin Transl Sci. 2020; 13: 116-124Crossref PubMed Scopus (140) Google Scholar,28Gaedigk A. Simon S.D. Pearce R.E. Bradford L.D. Kennedy M.J. Leeder J.S. The CYP2D6 activity score: translating genotype information into a qualitative measure of phenotype.Clin Pharmacol Ther. 2008; 83: 234-242Crossref PubMed Scopus (528) Google Scholar Regardless of whether a targeted genotyping or a full-gene sequencing approach is used, most laboratories specifically test for haplotype-defining variants and empirically assign diplotypes. Next-generation, short-read sequencing presents many challenges, including the accurate distinction of CYP2D6 sequence variants versus interfering pseudogenes, the characterization of structural variants, phasing of variants for the assignment of haplotype and diplotype, and the interpretation of novel or rare haplotypes. Recent studies have addressed some of these technical challenges by using full-gene, single-molecule, real-time sequencing with the Pacific Biosciences platform21Pratt V.M. Everts R.E. Aggarwal P. Beyer B.N. Broeckel U. Epstein-Baak R. Hujsak P. Kornreich R. Liao J. Lorier R. Scott S.A. Smith C.H. Toji L.H. Turner A. Kalman L.V. Characterization of 137 genomic DNA reference materials for 28 pharmacogenetic genes: a GeT-RM collaborative project.J Mol Diagn. 2016; 18: 109-123Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar,29Gaedigk A. Riffel A.K. Leeder J.S. CYP2D6 haplotype determination using long range allele-specific amplification: resolution of a complex genotype and a discordant genotype involving the CYP2D6∗59 allele.J Mol Diagn. 2015; 17: 740-748Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar and allele-specific amplification combined with long-range PCR sequencing for the comprehensive characterization of full-length CYP2D6 haplotypes.21Pratt V.M. Everts R.E. Aggarwal P. Beyer B.N. Broeckel U. Epstein-Baak R. Hujsak P. Kornreich R. Liao J. Lorier R. Scott S.A. Smith C.H. Toji L.H. Turner A. Kalman L.V. 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Allelic decomposition and exact genotyping of highly polymorphic and structurally variant genes.Nat Commun. 2018; 9: 828Crossref PubMed Scopus (23) Google Scholar Clinical PGx guidelines are available from professional societies, including the CPIC, Dutch Pharmacogenetics Working Group of the Royal Dutch Pharmacists Association (DPWG), Canadian PGx Network for Drug Safety, and American College of Medical Genetics and Genomics. At the time of the publication of the present article, CPIC has published six CYP2D6-related guidelines with dosing recommendations on 14 medications.4Goetz M.P. Sangkuhl K. Guchelaar H.J. Schwab M. Province M. Whirl-Carrillo M. Symmans W.F. McLeod H.L. Ratain M.J. Zembutsu H. Gaedigk A. van Schaik R.H. Ingle J.N. Caudle K.E. Klein T.E. Clinical Pharmacogenetics Implementation Consortium (CPIC) guideline for CYP2D6 and tamoxifen therapy.Clin Pharmacol Ther. 2018; 103: 770-777Crossref PubMed Scopus (146) Google Scholar, 5Bell G.C. Caudle K.E. Whirl-Carrillo M. Gordon R.J. Hikino K. Prows C.A. Gaedigk A. Agundez J.A.G. Sadhasivam S. Klein T.E. Schwab M. Clinical Pharmacogenetics Implementation Consortium (CPIC) guideline for CYP2D6 genotype and use of ondansetron and tropisetron.Clin Pharmacol Ther. 2017; 102: 213-218Crossref PubMed Scopus (93) Google Scholar, 6Hicks J.K. Sangkuhl K. Swen J.J. Ellingrod V.L. Müller D.J. Shimoda K. Bishop J.R. Kharasch E.D. Skaar T.C. Gaedigk A. Dunnenberger H.M. Klein T.E. Caudle K.E. Stingl J.C. Clinical Pharmacogenetics Implementation Consortium (CPIC) guideline for CYP2D6 and CYP2C19 genotypes and dosing of tricyclic antidepressants: 2016 update.Clin Pharmacol Ther. 2017; 102: 37-44Crossref PubMed Scopus (249) Google Scholar, 7Hicks J.K. Bishop J.R. Sangkuhl K. Muller D.J. Ji Y. Leckband S.G. Leeder J.S. Graham R.L. Chiulli D.L. LLerena A. Skaar T.C. Scott S.A. Stingl J.C. Klein T.E. Caudle K.E. Gaedigk A. 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Clinical Pharmacogenetics Implementation Consortium guideline for cytochrome P450 (CYP)2D6 genotype and atomoxetine therapy.Clin Pharmacol Ther. 2019; 106: 94-102Crossref PubMed Scopus (60) Google Scholar Although some clinical PGx guidelines have included summaries of known CYP2D6 alleles, frequencies in various populations, and their functional and/or clinical relevance, they do not explicitly recommend specific variant alleles for inclusion in CYP2D6 genotyping in clinical laboratories. Specific considerations for diagnostic laboratories, such as allele selection, testing platforms, and the availability of RMs, have not been the focus of the other clinical practice guidelines. However, consistency of genotyping tests among clinical laboratories will facilitate the use of these important clinical PGx practice guidelines and the clinical implementation of PGx testing in general. The AMP PGx Working Group comprises PGx experts from the CPIC, College of American Pathologists (CAP), DPWG and the PGx clinical testing and research communities. CYP2D6 alleles, including the ∗5 gene deletion, alleles with gene duplications, and alleles having one or more gene copies consisting of a combination of portions of CYP2D6 and CYP2D7 (commonly referred to as hybrid genes or hybrid alleles), were reviewed and stratified into tiers on the basis of four criteria: i) functional characterization status of the allele, that is, whether it is known to affect the function of the gene or encoded protein; ii) presence at an appreciable minor allele frequency (MAF) in a population