CYP2D6 copy number determination using digital PCR

Abstract

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.

Conclusion: 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: Absolute Q; CYP2D6; One-pot; copy number variation; digital PCR; multiplex; quantitative PCR; reference gene.

FIGURE 1

Overview of the traditional vs. One-pot workflows for dPCR copy number testing. In the traditional workflow, gDNA samples undergo a pre-work step via restriction enzyme (RE) digestion at 37°C. Digested gDNA is then incorporated into a reaction mixture containing the dPCR master mix and multiplex assay. The assembled mixture and isolation buffer are loaded onto a MAP16 plate and subsequently run on the Absolute Q dPCR instrument. The One-pot workflow contrasts by skipping the pre-work step and directly incorporating the RE into the reaction mixture. After loading the assembled mixture and isolation buffer, the MAP16 plate incubates at ambient laboratory conditions before being transferred to the dPCR instrument.

FIGURE 2

Panel (A) depicts an example of a 2D scatter plot when multiplexing by amplitude using the QX200 ddPCR system. Individual clusters are well-defined. “Channel 1” plots FAM reactions and “Channel 2” plots VIC reactions. The two CYP2D6 targets are labelled with FAM, and the reference gene target is labelled with VIC. Panel (B) represents a 2D scatter plot when multiplexing by amplitude under suboptimal conditions (likely due to degraded QX200 ddPCR reagents). Amplitudes of all targets are lower and VIC+/Target + clusters (orange, brown, and pink) are not well-defined making data interpretation difficult. Panel (C) shows four 1D plots when multiplexing by optical channel using the Absolute Q dPCR system. CYP2D6 targets and the TERT reference gene target are each labelled with unique florescent dyes (FAM, VIC, JUN, ABY). While the amplitude values of the positive reactions may overlap (FAM and ABY positive reactions cluster around 10,000 to 16,000 and VIC and JUN cluster around 2,000 to 5,000), the positive signals from each channel are able to be individually gated (blue background).