Establishment of CYP2D6 reference samples by multiple validated genotyping platforms

Fang, Hua; Liu, Xiao; Ramı́rez, Jacqueline; Kubo, Michiaki; Im, Hae Kyung; Konkashbaev, Anuar; Cox, Nancy J.; Ratain, Mark J.; Nakamura, Yusuke; O’Donnell, Peter H.

doi:10.1038/tpj.2014.27

Cited by 27 publications

(29 citation statements)

References 27 publications

(33 reference statements)

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“…SMRT sequencing of upstream and downstream CYP2D6 gene copies identified the haplotypes of the duplicated alleles in all samples (Table ), which were consistent with the previously reported commercially available CNV controls [Pratt et al., ; Fang et al., ] with copy number gains by qPCR. Moreover, when coupled with the qPCR results, duplication SMRT sequencing could refine the CYP2D6 diplotypes with suballele resolution and exact number of allele copies (e.g., *1Ax2/*2M , *1A/*4×3 ).…”

Section: Resultssupporting

confidence: 87%

“…CYP2D6 copy number was interrogated using commercially available TaqMan® real‐time qPCR Copy Number Assays (Applied Biosystems, Carlsbad, CA) as per the manufacturer's instructions and described previously [Martis et al., ; Fang et al., ]. In brief, FAM ™ ‐labeled TaqMan® minor groove binding probe and unlabeled PCR primer assays [Hs04083572_cn (intron 2), Hs00010001_cn (exon 9)] were individually run in a duplex qPCR with a VIC ™ ‐labeled RNase P TaqMan® Copy Number Reference Assay (catalog number: 4403326; Applied Biosystems).…”

Section: Methodsmentioning

confidence: 99%

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Long-Read Single Molecule Real-Time Full Gene Sequencing of Cytochrome P450-2D6

et al. 2015

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The CYP2D6 enzyme metabolizes ~25% of common medications, yet homologous pseudogenes and copy-number variants (CNVs) make interrogating the polymorphic CYP2D6 gene with short-read sequencing challenging. Therefore, we developed a novel long-read, full gene CYP2D6 single-molecule real-time (SMRT) sequencing method using the Pacific Biosciences platform. Long-range PCR and CYP2D6 SMRT sequencing of 10 previously genotyped controls identified expected star (*) alleles, but also enabled suballele resolution, diplotype refinement, and discovery of novel alleles. Coupled with an optimized variant calling pipeline, CYP2D6 SMRT sequencing was highly reproducible as triplicate intra- and inter-run non-reference genotype results were completely concordant. Importantly, targeted SMRT sequencing of upstream and downstream CYP2D6 gene copies characterized the duplicated allele in 15 control samples with CYP2D6 CNVs. The utility of CYP2D6 SMRT sequencing was further underscored by identifying the diplotypes of 14 samples with discordant or unclear CYP2D6 configurations from previous targeted genotyping, which again included suballele resolution, duplicated allele characterization, and discovery of a novel allele and tandem arrangement (CYP2D6*36+*41). Taken together, long-read CYP2D6 SMRT sequencing is an innovative, reproducible, and validated method for full-gene characterization, duplication allele-specific analysis and novel allele discovery, which will likely improve CYP2D6 metabolizer phenotype prediction for both research and clinical testing applications.

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Section: Resultssupporting

confidence: 87%

Section: Methodsmentioning

confidence: 99%

Long-Read Single Molecule Real-Time Full Gene Sequencing of Cytochrome P450-2D6

et al. 2015

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“…There are software tools that can be used to enhance the performance of genome sequencing for CYP2D6 . Additional standard samples with known allele compositions would be needed to validate genome sequencing as a method to assess CYP2D6 copy number …”

Section: Resultsmentioning

confidence: 99%

Comparison of genome sequencing and clinical genotyping for pharmacogenes

Yang

Broeckel

et al. 2016

Clin Pharma and Therapeutics

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We compared whole exome sequencing (WES, n = 176 patients) and whole genome sequencing (WGS, n = 68) and clinical genotyping (DMET array-based approach) for interrogating 13 genes with Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines. We focused on 127 CPIC important variants: 103 single nucleotide variations (SNV), 21 insertion/deletions (Indel), HLA-B alleles, and two CYP2D6 structural variations. WES and WGS provided interrogation of nonoverlapping sets of 115 SNV/Indels with call rate >98%. Among 68 loci interrogated by both WES and DMET, 64 loci (94.1%, confidence interval [CI]: 85.6–98.4%) showed no discrepant genotyping calls. Among 66 loci interrogated by both WGS and DMET, 63 loci (95.5%, CI: 87.2–99.0%) showed no discrepant genotyping calls. In conclusion, even without optimization to interrogate pharmacogenetic variants, WES and WGS displayed potential to provide reliable interrogation of most pharmacogenes and further validation of genome sequencing in a clinical lab setting is warranted.

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“…Sequenom custom MassARRAYs (Agena Bioscience, San Diego, CA) were used prior to May 5, 2015, while custom OpenArrays from Life Technologies (ThermoFisher, Waltham, MA) were used thereafter. All patients were additionally genotyped across a custom CYP2D6 panel developed in conjunction with Hologic(45) with supplemental CYP2D6 Taqman copy number assessment. All genotyping was conducted in Clinical Laboratory Improvement Amendments (CLIA)-certified and College of American Pathologists (CAP)-accredited laboratories.…”

Section: Methodsmentioning

confidence: 99%

Pharmacogenomics‐Based Point‐of‐Care Clinical Decision Support Significantly Alters Drug Prescribing

O'Donnell

Wadhwa

Danahey

et al. 2017

Clin Pharma and Therapeutics

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Changes in behavior are necessary to apply genomic discoveries to practice. We prospectively studied medication changes made by providers representing eight different medicine specialty clinics whose patients had submitted to preemptive pharmacogenomic genotyping. An institutional clinical decision support (CDS) system provided pharmacogenomic results using traffic light alerts: green/genomically favorable, yellow/genomic caution, red/high risk. The influence of pharmacogenomic alerts on prescribing behaviors was the primary endpoint. 2279 outpatient encounters were analyzed. Independent of other potential prescribing mediators, medications with high pharmacogenomic risk were changed significantly more often than prescription drugs lacking pharmacogenomic information (odds ratio [OR]=26.2 [9.0–75.3], p<0.0001). Medications with cautionary pharmacogenomic information were also changed more frequently (OR=2.4 [1.7–3.5], p<0.0001). No pharmacogenomically high-risk medications were prescribed during the entire study when physicians consulted the CDS tool. Pharmacogenomic information improved prescribing in patterns aimed at reducing patient risk, demonstrating that enhanced prescription decision-making is achievable through clinical integration of genomic medicine.

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Establishment of CYP2D6 reference samples by multiple validated genotyping platforms

Cited by 27 publications

References 27 publications

Long-Read Single Molecule Real-Time Full Gene Sequencing of Cytochrome P450-2D6

Long-Read Single Molecule Real-Time Full Gene Sequencing of Cytochrome P450-2D6

Comparison of genome sequencing and clinical genotyping for pharmacogenes

Pharmacogenomics‐Based Point‐of‐Care Clinical Decision Support Significantly Alters Drug Prescribing

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