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Validation of an Extensive CYP2D6 Assay Panel Based on Invader and TaqMan Copy Number Assays
Edward Ki Yun Leung1, Emanuele Agolini1, Xun Pei1
1Department of Pathology, UChicago Advanced Technology Clinical Laboratory, The University of Chicago, Chicago, IL.
Background:
CYP2D6 is involved in the oxidative metabolism of approximately 20% of all clinically used medications. Genotyping cytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6), is a challenge because of the high complexity of the locus.
Methods:
Twenty-nine CYP2D6 sequence variants were genotyped in 50 deidentified patient samples and 29 Coriell DNAs by Invader assay, and results were compared with Infiniti assay and Sanger sequencing. To determine CYP2D6 copy number, 3 TaqMan real-time hydrolysis probes were used and results were compared with long-range PCR. Discrimination of the duplicated alleles was done on 17 DNA samples with 3 copies of CYP2D6 by long-range PCR followed by Invader genotyping and single nucleotide extension for the comparison.
Results:
Complete concordance was observed for all samples between platforms except for 2 samples due to the lack of the *45 allele in the Infiniti panel. Reproducibility with the Invader assay and TaqMan copy number was 100%. Analytical sensitivity using DNA with 2 copies was determined to be 10 ng DNA for the Invader assay and 1 ng/μL DNA for the TaqMan assay, respectively. Complete concordance and reproducibility were observed for duplicated allele discrimination with the exception of 1 sample, determined to be *29/*43X2 by the Invader test and *1X2/*29 by the Infiniti method, which did not test for *43.
Conclusions:
This validation study showed that Invader and TaqMan assay combined panel provides an attractive, valid, highly accurate, and reproducible approach for CYP2D6 genotyping for clinical implementation.
Insights
This study validates the Invader and TaqMan assays for accurate and reproducible cytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6) genotyping. The combined panel offers a reliable method for clinical implementation of CYP2D6 genetic testing.
Area of Science:
- Pharmacogenomics
- Molecular Diagnostics
- Genetic Analysis
Background:
- Cytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6) is crucial for metabolizing 20% of clinical drugs.
- CYP2D6 genotyping is complex due to the locus's intricate nature.
Purpose of the Study:
- To validate the Invader and TaqMan assays for accurate CYP2D6 genotyping.
- To assess the reproducibility and clinical applicability of a combined assay panel.
Main Methods:
- Genotyped 29 CYP2D6 variants using Invader assay, compared with Infiniti and Sanger sequencing.
- Determined CYP2D6 copy number with TaqMan probes, compared with long-range PCR.
- Discriminated duplicated alleles using long-range PCR, Invader, and single nucleotide extension.
Main Results:
- Achieved complete concordance between platforms, with minor exceptions.
- Demonstrated 100% reproducibility for Invader assay and TaqMan copy number determination.
- Established analytical sensitivity and validated duplicated allele discrimination.
Conclusions:
- The combined Invader and TaqMan assay panel is a valid, accurate, and reproducible method for CYP2D6 genotyping.
- This approach is suitable for clinical implementation, enhancing pharmacogenetic testing.
