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METHODS article

Front. Pharmacol.
Sec. Pharmacogenetics and Pharmacogenomics
Volume 15 - 2024 | doi: 10.3389/fphar.2024.1429286
This article is part of the Research Topic Advances in Pharmacogenomics: Basic, Translational, and Clinical View all 3 articles

CYP2D6 Copy Number Determination using Digital PCR

Provisionally accepted
Wendy Y. Wang Wendy Y. Wang 1Lancy Lin Lancy Lin 2Erin C. Boone Erin C. Boone 1Junko Stevens Junko Stevens 2Andrea Gaedigk Andrea Gaedigk 1,3*
  • 1 Division of Clinical Pharmacology, Toxicology & Therapeutic Innovation, Children's Mercy Kansas City, Kansas City, United States
  • 2 Thermo Fisher Scientific (United States), Waltham, United States
  • 3 School of Medicine, University of Missouri–Kansas City, Kansas City, Kansas, United States

The final, formatted version of the article will be published soon.

    Background: CYP2D6 testing is increasingly used to guide drug therapy and thus, reliable methods are needed to test this complex and polymorphic gene locus. A particular challenge arises from the detection and interpretation of structural variants (SVs) including gene deletions, duplications, and hybrids with the CYP2D7 pseudogene. This study validated the Absolute Q platform for digital PCR-based CYP2D6 copy number variation (CNV) determination by comparing results to those obtained with a previously established method using the QX200 platform. In addition, protocols for streamlining CYP2D6 CNV testing were established and validated including the “One-pot” single-step restriction enzyme digestion and a multiplex assay simultaneously targeting the CYP2D6 5’UTR, intron 6, and exon 9 regions. Methods: Genomic DNA (gDNA) samples from Coriell (n=13) and from blood, saliva, and liver tissue (n=17) representing 0 – 6 copies were tested on the Absolute Q and QX200 platforms. Custom TaqMan™ copy number (CN) assays targeting CYP2D6 the 5’UTR, intron 6, and exon 9 regions and a reference gene assay (TERT or RNaseP) were combined for multiplexing by optical channel. In addition, two digestion methods (One-pot digestion and traditional) were assessed. Inconclusive CN values on the Absolute Q were resolved using an alternate reference gene and/or diluting gDNA. Results: Overall, results between the two platforms and digestions methods were consistent. The “One-pot” digestion method and optically multiplexing up to three CYP2D6 regions yielded consistent result across DNA sample types and diverse SVs, reliably detecting up to 6 gene copies. Rare variation in reference genes were found to interfere with results and interpretation, which were resolved by using a different reference. Conclusions: The Absolute Q produced accurate and reliable CYP2D6 copy number results allowing for a streamlined and economical protocol using One-pot digestion and multiplexing three target regions. Protocols are currently being expanded to other pharmacogenes presenting with SVs/CNVs.

    Keywords: CYP2D6, copy number variation, digital PCR, Multiplex, One-pot, Absolute Q, quantitative PCR, reference gene

    Received: 07 May 2024; Accepted: 22 Jul 2024.

    Copyright: © 2024 Wang, Lin, Boone, Stevens and Gaedigk. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence: Andrea Gaedigk, Division of Clinical Pharmacology, Toxicology & Therapeutic Innovation, Children's Mercy Kansas City, Kansas City, United States

    Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.