Characterization of Reference Materials for Genetic Testing of CYP2D6 Alleles
2019; Elsevier BV; Volume: 21; Issue: 6 Linguagem: Inglês
10.1016/j.jmoldx.2019.06.007
ISSN1943-7811
AutoresAndrea Gaedigk, Amy Turner, Robin E. Everts, Stuart A. Scott, Praful Aggarwal, Ulrich Broeckel, Gwendolyn A. McMillin, Roberta Melis, Erin C. Boone, Victoria M. Pratt, Lisa V. Kalman,
Tópico(s)Chemical Reactions and Isotopes
ResumoPharmacogenetic testing increasingly is available from clinical and research laboratories. However, only a limited number of quality control and other reference materials currently are available for the complex rearrangements and rare variants that occur in the CYP2D6 gene. To address this need, the Division of Laboratory Systems, CDC-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Cell Repositories (Camden, NJ), has characterized 179 DNA samples derived from Coriell cell lines. Testing included the recharacterization of 137 genomic DNAs that were genotyped in previous Genetic Testing Reference Material Coordination Program studies and 42 additional samples that had not been characterized previously. DNA samples were distributed to volunteer testing laboratories for genotyping using a variety of commercially available and laboratory-developed tests. These publicly available samples will support the quality-assurance and quality-control programs of clinical laboratories performing CYP2D6 testing. Pharmacogenetic testing increasingly is available from clinical and research laboratories. However, only a limited number of quality control and other reference materials currently are available for the complex rearrangements and rare variants that occur in the CYP2D6 gene. To address this need, the Division of Laboratory Systems, CDC-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Cell Repositories (Camden, NJ), has characterized 179 DNA samples derived from Coriell cell lines. Testing included the recharacterization of 137 genomic DNAs that were genotyped in previous Genetic Testing Reference Material Coordination Program studies and 42 additional samples that had not been characterized previously. DNA samples were distributed to volunteer testing laboratories for genotyping using a variety of commercially available and laboratory-developed tests. These publicly available samples will support the quality-assurance and quality-control programs of clinical laboratories performing CYP2D6 testing. CYP2D6 is part of the cytochrome P450 gene family, a group of phase I metabolism enzymes that are responsible for the elimination or bioactivation of numerous drugs.1Zhou S.F. Polymorphism of human cytochrome P450 2D6 and its clinical significance: part I.Clin Pharmacokinet. 2009; 48: 689-723Crossref PubMed Scopus (580) Google Scholar, 2Zhou S.F. Polymorphism of human cytochrome P450 2D6 and its clinical significance: part II.Clin Pharmacokinet. 2009; 48: 761-804Crossref PubMed Scopus (221) Google Scholar CYP2D6 substrates belong to different drug classes, including antidepressants (eg, amitriptyline, doxepin, fluvoxamine, nortriptyline, fluoxetine, paroxetine), antipsychotics (eg, chlorpromazine, clozapine, haloperidol, risperidone), antiarrhythmics (eg, flecainide, propafenone), beta-blockers (eg, carvedilol, metoprolol), opioid analgesics (eg, codeine, tramadol), anticancer agents (eg, gefitinib, tamoxifen), and other drugs (eg, atomoxetine, dextromethorphan, ondansetron; Pharmacogenomics Knowledge Base, https://www.pharmgkb.org/vip/PA166170264, last accessed January 29, 2019). The highly polymorphic CYP2D6 gene is located on chromosome 22q13.2,3Gaedigk A. Complexities of CYP2D6 gene analysis and interpretation.Int Rev Psychiatry. 2013; 25: 534-553Crossref PubMed Scopus (164) Google Scholar and, to date, more than 100 CYP2D6 star (*) allele haplotypes (not counting suballelic variants) currently are cataloged by the Pharmacogene Variation (PharmVar) Consortium (https://www.pharmvar.org/gene/CYP2D6, last accessed August 29, 2019). Genetic variation is an important factor explaining the wide range of enzymatic activity of drug-metabolizing enzymes that can be observed within and among populations.4Gaedigk A. Sangkuhl K. Whirl-Carrillo M. Klein T. Leeder J.S. Prediction of CYP2D6 phenotype from genotype across world populations.Genet Med. 2017; 19: 69-76Crossref PubMed Scopus (293) Google Scholar Based on the identified genotypes in a patient, a diplotype is assigned, which then is translated into a phenotype (ie, metabolizer status for drug-metabolizing enzymes and function status for drug transporters).5Caudle K.E. Dunnenberger H.M. Freimuth R.R. Peterson J.F. Burlison J.D. Whirl-Carrillo M. Scott S.A. Rehm H.L. Williams M.S. Klein T.E. Relling M.V. Hoffman J.M. Standardizing terms for clinical pharmacogenetic test results: consensus terms from the Clinical Pharmacogenetics Implementation Consortium (CPIC).Genet Med. 2017; 19: 215-223Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar For CYP2D6, there currently are four commonly inferred phenotype groups: poor metabolizers, intermediate metabolizers, normal metabolizers, and ultrarapid metabolizers. Normal and intermediate metabolizers generally are expected to tolerate and respond to standard doses of most medications metabolized by CYP2D6. Patients with no CYP2D6 activity (poor metabolizers) may experience adverse events because a drug either is not metabolized or is metabolized inefficiently, which causes levels above the therapeutic range for active drugs. Therapeutic failure also may be observed in patients with a poor metabolizer phenotype if the drug depends on CYP2D6 for bioactivation.6Weinshilboum R. Inheritance and drug response.N Engl J Med. 2003; 348: 529-537Crossref PubMed Scopus (917) Google Scholar Conversely, patients with diplotypes leading to higher than normal activity (ultrarapid metabolizers) may experience therapeutic failure because an active drug is metabolized too fast, causing drug levels that are below the therapeutic range. Alleles (or haplotypes) are named using the star (*) nomenclature referring to a particular allele or haplotype as *allele. An allele (or haplotype) defines variant(s) that are in cis and the combination of the two haplotypes is referred to as a diplotype. The majority of star (*) alleles are defined by single-nucleotide variants or insertions/deletions of one or a few nucleotides. However, the presence of copy number variations (deletions, duplications, and multiplications) and gene rearrangements with the CYP2D7 pseudogene, leading to CYP2D6–CYP2D7 and CYP2D7–CYP2D6 hybrid genes, pose additional challenges for genotype analysis.3Gaedigk A. Complexities of CYP2D6 gene analysis and interpretation.Int Rev Psychiatry. 2013; 25: 534-553Crossref PubMed Scopus (164) Google Scholar, 7Yang 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 (64) Google Scholar, 8Nofziger C. Paulmichl M. Accurately genotyping CYP2D6: not for the faint of heart.Pharmacogenomics. 2018; 19: 999-1002Crossref PubMed Scopus (24) Google Scholar Sequence variations and the structural variants can occur by themselves or in different combinations. Additional information regarding nomenclature can be found at https://www.pharmvar.org; last accessed August 29, 2019. PharmVar maintains nomenclature for most CYP450 genes, including CYP2D6, as well as other selected genes involved in drug metabolism.9Gaedigk A. Ingelman-Sundberg M. Miller N.A. Leeder J.S. Whirl-Carrillo M. Klein T.E. PharmVar Steering CommitteeThe Pharmacogene Variation (PharmVar) Consortium: incorporation of the Human Cytochrome P450 (CYP) Allele Nomenclature Database.Clin Pharmacol Ther. 2018; 103: 399-401Crossref PubMed Scopus (227) Google Scholar, 10Gaedigk A. Sangkuhl K. Whirl-Carrillo M. Twist G.P. Klein T.E. Miller N.A. PharmVar Steering CommitteeThe evolution of PharmVar.Clin Pharmacol Ther. 2019; 105: 29-32Crossref PubMed Scopus (75) Google Scholar For simplicity, the term allele will be used from here on forward. CYP2D6 genotyping assays are difficult to design and validate analytically because of the large number of star (*) alleles and variant types [there are 131 major star alleles (excluding suballeles) listed by PharmVar, https://www.pharmvar.org/gene/CYP2D6, accessed August 28, 2019; note that eight have been retired, making the actual count less than the highest numbered allele: *139]. Reference materials containing a wide variety of sequence and structural variants, including copy number variants and hybrid genes, are invaluable for developing and validating assays and test platforms that can accurately detect all of the variants that the assay or platform is designed to detect and ensure that sequence variations found on other alleles are not affecting assay or platform results. Laboratories developing test panels and testing patients for CYP2D6 need a large and diverse set of reference materials, ideally representing all defined alleles.11Chen B. O'Connell C.D. Boone D.J. Amos J.A. Beck J.C. Chan M.M. et al.Developing a sustainable process to provide quality control materials for genetic testing.Genet Med. 2005; 7: 534-549Crossref PubMed Scopus (44) Google Scholar These materials also are important for quality control and proficiency testing. Although many clinical laboratories offer CYP2D6 testing, reference materials are not publicly available for many of the complex structural or low-frequency variants. The CDC Genetic Testing Reference Material Program (GeT-RM) has conducted two previous pharmacogenetic (PGx) studies,12Pratt V.M. Zehnbauer B. Wilson J.A. Baak R. Babic N. Bettinotti M. Buller A. Butz K. Campbell M. Civalier C. El-Badry A. Farkas D.H. Lyon E. Mandal S. McKinney J. Muralidharan K. Noll L. Sander T. Shabbeer J. Smith C. Telatar M. Toji L. Vairavan A. Vance C. Weck K.E. Wu A.H. Yeo K.T. Zeller M. Kalman L. 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: 835-846Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 13Pratt 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 (83) Google Scholar referred to here as PGX1 and PGX2, with the goal of creating publicly available and well-characterized genomic DNA reference materials for pharmacogenes, including CYP2D6. Together, these studies identified cell line–based genomic DNA samples containing many, but not all, of the star (*) alleles that are included in most clinical CYP2D6 genotyping tests. For a significant number of the genomic DNA samples, results were ambiguous or genotype could not be determined with certainty (eg, gene copy number remained unknown or structural variants were only partially characterized). In addition, no reference materials were identified for many of the rare star (*) alleles, including many of the structural variants. This was because of the low population frequency of these variants as well as the design of the test platforms used to characterize the samples. To address the need for a more comprehensive set of reference materials for CYP2D6 testing, the GeT-RM program, in collaboration with the genetic testing community, have further characterized selected samples previously characterized by Get-RM for CYP2D6 as well as additional samples that harbor rare and/or complex alleles or allele combinations. DNA from 179 cell lines was selected from the National Institute of General Medical Sciences and the National Human Genome Research Institute Repositories at the Coriell Cell Repositories (Camden, NJ) for this study. Of these, 135 were characterized in the most recent GeT-RM study (PGX2),13Pratt 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 (83) Google Scholar and two (NA17222 and NA17287) were characterized in PGX1.12Pratt V.M. Zehnbauer B. Wilson J.A. Baak R. Babic N. Bettinotti M. Buller A. Butz K. Campbell M. Civalier C. El-Badry A. Farkas D.H. Lyon E. Mandal S. McKinney J. Muralidharan K. Noll L. Sander T. Shabbeer J. Smith C. Telatar M. Toji L. Vairavan A. Vance C. Weck K.E. Wu A.H. Yeo K.T. Zeller M. Kalman L. 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: 835-846Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar Forty-two additional DNA samples carrying rare variants that were not represented in the PGX2 reference material panel13Pratt 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 (83) Google Scholar were selected based on data supplied by the authors of this article or identified by searching the NCBI 1000 Genomes Project (https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes, last accessed August 13, 2018). The five laboratories that participated in this follow-up study, Agena Bioscience, ARUP Laboratories, Children's Mercy Kansas City, Medical College of Wisconsin/RPRD (Right Patient Right Drug) Diagnostics, and Icahn School of Medicine at Mount Sinai, used a variety of genotyping and sequencing platforms. DNA was prepared from each of the selected cell lines by the Coriell Cell Repositories using Gentra/Qiagen Autopure (Valencia, CA) per the manufacturer's instructions. Each of the testing laboratories received one 10-μg aliquot of DNA from each of the cell lines that they volunteered to test. Each laboratory tested the samples using their standard methods and/or additional methods needed to resolve inconclusive genotype calls. The test platforms and genotyping assays used in the study, and the alleles detected by each method, are shown in Supplemental Table S1 and are described in the following sections. The results were submitted to two researchers (A.G. and V.M.P.), who examined the data for quality and discordances, and determined the consensus genotype. If discordances were noted, the participating laboratories were asked to re-evaluate their data for the sample(s) in question to determine the cause of the inconsistency. Supplementary testing was performed if required. Following the manufacturer's instructions, genomic DNA first was amplified (DNA amplification and multiplex PCR). The amplified products were pooled, purified, fragmented, labeled, and hybridized to the PharmacoScan Array (Thermo Fisher Scientific, Waltham, MA) per the manufacturer's recommendations. Arrays were stained with a fluorescent antibody and scanned on the GeneTitan Multi-Channel Instrument (Thermo Fisher Scientific). Data were analyzed using the Axiom Analysis Suite 3.1 (Thermo Fisher Scientific). Analysis was performed using either the commercially released allele translation table (version r6; Thermo Fisher Scientific) or a custom translation table that includes additional CYP2D6 star alleles for translation (version v.r6+20180103). The PharmacoScan Array detects nucleotide variants and copy number state (0, 1, 2, ≥3) on a subset of genes. For CYP2D6, three gene regions (5′ flanking, 3′ flanking, and exon 9) are interrogate by the array (Figure 1 and Table 1).Table 1Copy Number Variation Calls from Different Platforms for Examples Provided in Figure 1Example 15′UTRExon 1Intron 2Intron 4Intron 5Intron 6Exon 93′UTRTaqMan––3––33–TaqMan–––––33–ddPCR3––––33–MPA–3––333–PharmacoScan3–––––33VeriDose3333–33–Example 25′UTRExon 1Intron 2Intron 4Intron 5Intron 6Exon 93′UTRTaqMan––3––32–TaqMan–––––32–ddPCR3––––32–MPA–3––332–PharmacoScan3–––––22VeriDose3333–32–Example 35′UTRExon 1Intron 2Intron 4Intron 5Intron 6Exon 93′UTRTaqMan––1––22–TaqMan–––––22–ddPCR1––––22–MPA–1––222–PharmacoScan1–––––22VeriDose1112–22–Different platforms or methods may target different gene regions [5′ or 3′ untranslated regions (UTR), introns, or exons]. Examples 1, 2, and 3 provide the calls for those shown graphically in Figure 1. Example 1 has three-copy calls across all regions tested. Example 2 has a CYP2D6*36 gene copy that generates three-copy calls for the 5′UTR through intron 6 regions and two-copy calls for exon 9. Example 3 shows a sample with a CYP2D7–CYP2D6 hybrid that generates one-copy calls for the 5′UTR through intron 2 regions and two-copy calls for the intron 4 through exon 9 regions.–, indicated genomic region was not amplified by the method used; ddPCR, droplet digital PCR; MPA, multiplex PCR amplification. Open table in a new tab Different platforms or methods may target different gene regions [5′ or 3′ untranslated regions (UTR), introns, or exons]. Examples 1, 2, and 3 provide the calls for those shown graphically in Figure 1. Example 1 has three-copy calls across all regions tested. Example 2 has a CYP2D6*36 gene copy that generates three-copy calls for the 5′UTR through intron 6 regions and two-copy calls for exon 9. Example 3 shows a sample with a CYP2D7–CYP2D6 hybrid that generates one-copy calls for the 5′UTR through intron 2 regions and two-copy calls for the intron 4 through exon 9 regions. –, indicated genomic region was not amplified by the method used; ddPCR, droplet digital PCR; MPA, multiplex PCR amplification. For a subset of samples analyzed with the PharmacoScan Array (HG00463, HG01190, HG02373, NA18545, and 96 Tier 1 samples from the PGX2 study13Pratt 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 (83) Google Scholar), TaqMan copy number assays were run as per the manufacturer's instructions on a CFX384 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA) to interrogate copy number state in intron 2, intron 6, and exon 9 (assay IDs: Hs04083572_cn, Hs04502391_cn, and Hs00010001_cn; Thermo Fisher Scientific) (Figure 1 and Table 1). The copy number data were analyzed using CopyCaller software version 2.0 (Thermo Fisher Scientific). Seven two-copy control samples were run on each plate; the sample that showed the median ΔCT within the two-copy samples was selected as the calibrator. In addition, a one-copy and a three-copy sample were included on each plate. The manual CT was 0.2 and the baseline was set on automatic. A confidence level of 95% and z-score value of less than 1.75 was applied to call the copy number. Two TaqMan real-time PCR assays targeting intron 6 and exon 9 were performed (assay IDs: Hs04502391_cn and Hs00010001_cn) (Figure 1 and Table 1). All assays were performed on a QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific) using 96-well plates, in four replicates along with RNase P (assay ID 4403326; Thermo Fisher Scientific) as a reference gene, according to the manufacturer's protocol. A two-gene copy calibrator, a sample with one gene copy, and a sample with three gene copies were included with each run. Relative quantification of the CYP2D6 copy number was performed using CopyCaller (Thermo Fisher Scientific). If the calculated copy number value for a given probe was N ± 0.4 gene copies (where N is a whole number), it was predicted to be that whole number; however, if it was greater than ±0.4 gene copies the assay was repeated. A confidence level of 95% and z-score value of less than 1.75 was applied to call the copy number. By using the CYP2D6 panel, specific DNA fragments were amplified from genomic DNA in three PCR reactions and 35 of the most clinically relevant variants subsequently were interrogated using iPLEX PRO (Agena Bioscience, San Diego, CA) single-base extension biochemistry. Genotypes were detected using a MassARRAY Analyzer 4 system and haplotypes were assigned using an ADME CYP2D6 Reporter plugin for the Typer Analyzer software version 4.1.183 (Agena Bioscience). A set of 13 additional variant genotyping assays was designed and run using the same single-base extension protocol to distinguish rare haplotypes. These were interpreted manually in addition to the standard analysis. By using the VeriDose (Agena Bioscience) CYP2D6 copy number variation (CNV) panel for copy number determination, 11 assays in six target regions within the CYP2D6 gene (5′ untranslated region, exon 1, intron 2, intron 4, intron 6, and exon 9) (Figure 1 and Table 1) were amplified from genomic DNA in one PCR reaction together with a control region in either CYP2D7 or CYP2D8. The 11 assays interrogating single-base differences subsequently were amplified using single-base extension. Data for these assays were analyzed using a MassARRAY Analyzer 4 system and the gene copy number was calculated using a CYP2D6 PGx Reporter plugin for the Typer Analyzer software (Agena Bioscience). The CNV software (PGx Reporter software version 3.51; Agena Bioscience) calculates the copy number for the CYP2D6 gene as well as indicates the presence of CYP2D6*13 (CYP2D7–CYP2D6 hybrid genes), CYP2D6*68 (CYP2D6–CYP2D7 hybrid genes), or genes carrying a CYP2D7-derived exon 9 conversion including *36 and *4N. An aggregate CYP2D6 copy number is reported in the main output, but individual calls per assay are reported in a separate CNV output file. Of the samples tested with the TaqMan-based genotyping assays (Thermo Fisher Scientific) at Children's Mercy Kansas City, 72 were run on the OpenArray platform (Thermo Fisher Scientific) using a custom-designed array panel. OpenArrays were performed as prescribed by the manufacturer. The remaining samples were genotyped using single-tube TaqMan genotyping assays in a 96-well reaction format. OpenArray no-calls or uncertain calls were repeated using the single-tube assay as follows. Each 8-μL reaction contained 0.8 μL DNA (15 ng/μL) and 1× Probe Force qPCR Master Mix (KAPA Biosystems, Wilmington, MA). The initial denaturation was 95°C as recommended for the KAPA mastermix, and cycling parameters were as recommended by the provided TaqMan assay protocols. All assays and OpenArrays were run on QuantStudio 12K Flex Real-Time PCR System version 1.2.2 (Thermo Fisher Scientific). Data were analyzed with the TaqMan Genotyper software version 1.4.0 (Thermo Fisher Scientific); each call was inspected manually. Children's Mercy Kansas City also tested the samples using long-range (XL) PCR as previously described.14Gaedigk A. Ndjountche L. Divakaran K. Dianne Bradford L. Zineh I. Oberlander T.F. Brousseau D.C. McCarver D.G. Johnson J.A. Alander S.W. Wayne Riggs K. Steven Leeder J. Cytochrome P4502D6 (CYP2D6) gene locus heterogeneity: characterization of gene duplication events.Clin Pharmacol Ther. 2007; 81: 242-251Crossref PubMed Scopus (133) Google Scholar, 15Gaedigk A. Fuhr U. Johnson C. Berard L.A. Bradford D. Leeder J.S. CYP2D7-2D6 hybrid tandems: identification of novel CYP2D6 duplication arrangements and implications for phenotype prediction.Pharmacogenomics. 2010; 11: 43-53Crossref PubMed Scopus (54) Google Scholar, 16Gaedigk A. Jaime L.K. Bertino Jr., J.S. Berard A. Pratt V.M. Bradfordand L.D. Leeder J.S. Identification of novel CYP2D7-2D6 hybrids: non-functional and functional variants.Front Pharmacol. 2010; 1: 121Crossref PubMed Scopus (54) Google Scholar Briefly, a series of XL-PCR fragments was amplified to show or validate the presence of a CNV event or the presence of hybrid gene copies (Table 2). For example, the presence of a CYP2D6*2×2 duplication on one of the chromosomes generates fragment B and an approximately 8-Kb long fragment D, whereas the presence of a CYP2D6*36+*10 tandem generates an approximately 10-Kb long fragment D (the longer size is because of a CYP2D7-derived region downstream of *36), but not fragment B. An approximately 10-Kb long fragment D also is generated from CYP2D6*68+*4 (*68 is a CYP2D6–CYP2D7 hybrid gene), and CYP2D7–CYP2D6 hybrid genes amplify an approximately 5-Kb long fragment H. To determine which CYP2D6 alleles are present in a gene duplication (two identical or near-identical gene copies such as CYP2D6*1×2, *2×2, or *4×2), fragment D was genotyped for one or more single-nucleotide polymorphisms (SNPs) to discriminate (eg, a CYP2D6*1×2 from a CYP2D6*4×2). For example, if fragment D was generated from a duplicated CYP2D6*4×2 allele, the genotype calls for rs1065852 (c.100C>T) and rs1065852 (c.1847G>A) (positions according to the CYP2D6 RefSeq NG_008376.3) appeared homozygous; if the duplication is on the CYP2D6*1×2 allele, fragment D is negative for these SNPs. The presence of the CYP2D6*5 gene deletion was detected by XL-PCR, as previously described.14Gaedigk A. Ndjountche L. Divakaran K. Dianne Bradford L. Zineh I. Oberlander T.F. Brousseau D.C. McCarver D.G. Johnson J.A. Alander S.W. Wayne Riggs K. Steven Leeder J. Cytochrome P4502D6 (CYP2D6) gene locus heterogeneity: characterization of gene duplication events.Clin Pharmacol Ther. 2007; 81: 242-251Crossref PubMed Scopus (133) Google ScholarTable 2Summary of Long-Range (XL)-PCR Products Generated for Gene Locus Characterization and/or SequencingFragment IDPrimer sequencesCYP2D6 XL-PCR ampliconsProduct size, KbSamples∗Samples listed only when sequenced; some XL-PCRs were also run on other samples to determine which allele is duplicated or to confirm structural variation.1 (A)†PCR as described.25F: 5′-GTCCCACACCAGGCACCTGTACT-3′Nonduplicated gene and gene deletion15.6 (nonduplicated) 3.5 (deletion)HG00337, NA18632, NA18642R: 5′-GAATTAGTGGTGGTGGGTGTTTG-3′2 (B)†PCR as described.25F: 5′-TCACCCCCAGCGGACTTATCA-3′5′ CYP2D6 gene in duplication12HG00337, NA18642R: 5′-CCACAGCCCTCAATAAGTGAA-3′3 (C)†PCR as described.25F: 5′-CCCTGGGAAGGCCCCATGGAAG-3′3′ CYP2D6 gene in duplication12HG00337, NA18642R: 5′-TAGGTAGCCCTGGCCTATAGCTCCCTGACGCC-3′4 (E)†PCR as described.25F: 5′-TCACCCCCAGCGGACTTATCA-3′CYP2D6–CYP2D7 hybrid gene6.7HG00337, NA18632, NA18642R: 5′-TACGGTGGGCTCCCTGCGAG-3′5 (D)†PCR as described.25F: 5′-TTGCCACATTATCGCCCGTGAAA-3′Full-length CYP2D6 in any arrangement8.4HG00337, HG04090, HG04206, NA18632, NA18642, NA18973, NA19908, NA20803, NA20875, NA21105R: 5′-TAGGTAGCCCTGGCCTATAGCTCCCTGACGCC-3′6 (A)‡XL-PCR performed with primers as described.18F: 5′-TCACCCCCAGCGGACTTATCAACC-3′Full-length CYP2D6 in any arrangement6.7HG00373, HG03225, HG03246, HG03259, HG03780, NA17128, NA18632, NA19777R: 5′-CGACTGAGCCCTGGGAGGTAGGTAG-3′6 (AS-A)‡XL-PCR performed with primers as described.18F: 5′-TGGAGAGAGGCCACCTGAGGTAGTC-3′ASXL-PCR of entire CYP2D6 gene (-2609C)7.4NA19180R: 5′-CGACTGAGCCCTGGGAGGTAGGTAG-3′6 (AS-A)‡XL-PCR performed with primers as described.18F: 5′-CGTCAAGCTTTCCGACATACACG-3′ASXL-PCR of entire CYP2D6 gene (-2532G)7.3NA23878R: 5′-CGACTGAGCCCTGGGAGGTAGGTAG-3′6 (AS-A)‡XL-PCR performed with primers as described.18F: 5′-CCTCCCAAATCTGATGAAAAATATTAATCC-3′ASXL-PCR of entire CYP2D6 gene (-2421C)7.2NA19917R: 5′-CGACTGAGCCCTGGGAGGTAGGTAG-3′6 (AS-A)§XL-PCR performed as described18 with newly designed primer.F: 5′-GAGGCAACCTGCTCGGG-3′ASXL-PCR of entire CYP2D6 gene (-2178G)6.8NA07439, NA12154, NA17137, NA18642, NA19174R: 5′-CGACTGAGCCCTGGGAGGTAGGTAG-3′6 (AS-A)§XL-PCR performed as described18 with newly designed primer.F: 5′-CTGTCCTCAGTGGATGATCCCG-3′ASXL-PCR of entire CYP2D6 gene (-1770G)6.5NA23877R: 5′-CGACTGAGCCCTGGGAGGTAGGTAG-3′6 (AS-A)‡XL-PCR performed with primers as described.18F: 5′-CCTGGACAACTTGGAAGAACCG-3′ASXL-PCR of entire CYP2D6 gene (-1584G)6.4HG00337, HG00436, HG00589, HG01086, HG01094, HG02373, HG03781, NA17448, NA18552, NA18973, NA20289, NA21105R: 5′-CGACTGAGCCCTGGGAGGTAGGTAG-3′6 (AS-A)‡XL-PCR performed with primers as described.18F: 5′-CCTGGACAACTTGGAAGAACCC-3′ASXL-PCR of entire CYP2D6 gene (-1584C)6.4HG00337, HG01806, HG03619, HG03781, NA20289R: 5′-CGACTGAGCCCTGGGAGGTAGGTAG-3′6 (AS-A)§XL-PCR performed as described18 with newly designed primer.F: 5′-CATGGTGAAACCCTATCTCTACTGAAAATAC-3′ASXL-PCR of entire CYP2D6 gene (-1426C)6.2NA17185R: 5′-CGACTGAGCCCTGGGAGGTAGGTAG-3′6 (AS-A)‡XL-PCR performed with primers as described.18F: 5′-TGTGTGTGAGAGAGAATGTGTGCC-3′ASXL-PCR of entire CYP2D6 gene (-740C)5.5NA19239, NA23877R: 5′-CGACTGAGCCCTGGGAGGTAGGTAG-3′6 (AS-A)§XL-PCR performed as described18 with newly designed primer.F: 5′-TCACCCCCAGCGGACTTATCAACC-3′ASXL-PCR of entire CYP2D6 gene (1023C)2.9NA19239R: 5′-CCCGAAACCCAGGATCTGGG-3′6 (AS-A)§XL-PCR performed as described18 with newly designed primer.F: 5′-TCACCCCCAGCGGACTTATCAACC-3′ASXL-PCR of entire CYP2D6 gene (4401C)6.3NA17185R: 5′-GACATCTGCTCAGCCTCAACG-3′6 (AS-A)§XL-PCR performed as described18 with newly designed primer.F: 5′-TCACCCCCAGCGGACTTATCAACC-3′ASXL-PCR of entire CYP2D6 gene (4723G)6.6NA17176, NA19908R: 5′-CTGGGAGGTAGGTAGCCCTGACC-3′7 (D)¶XL-PCR performed as described.14–16F: 5′-CCAGAAGGCTTTGCAGGCTTCAG-3′CYP2D6–CYP2D7 hybrid gene8.6 or 10.2‖This fragment is 8.6 Kb when the duplicated gene has a CYP2D6 (REP6) downstream region and 10.2 Kb if the duplicated gene has a CYP2D7 (REP7) downstream region (see Figure 2).NA18632, NA18642R: 5′-CGGCAGTGGTCAGCTAATGAC-3′8 (B)¶XL-PCR performed as described.14–16F: 5′-CCATGGAAGCCCAGGACTGAGC-3′5′ CYP2D6 intergenic region i
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