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BackgroundCYP2D6 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.MethodsGenomic 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.ResultsOverall, 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.ConclusionThe 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.

Objective: Hmong individuals represent a unique East Asian subpopulation in whom limited information concerning pharmacogenetic variation exists. The objectives of this study were to comprehensively characterize the highly polymorphic CYP2D6 gene in Hmong, estimate allele and phenotype frequencies and to compare results between two testing platforms.Methods: DNA from 48 self-identified Hmong participants were sequenced using a targeted next-generation sequencing (NGS) panel. Star allele calls were made using Astrolabe, manual inspection of NGS variant calls and confirmatory Sanger sequencing. Structural variation was determined by long-range (XL)-PCR and digital droplet PCR (ddPCR). The consensus diplotypes were subsequently translated into phenotype utilizing the activity score system. Clinical grade pharmacogenetic testing was obtained for 12 of the 48 samples enabling an assessment of concordance between the consensus calls and those determined by clinical testing platforms.Results: A total of 13 CYP2D6 alleles were identified. The most common alleles were CYP2D6*10 and its structural arrangements (37.5%, 36/96) and the *5 gene deletion (13.5%, 13/96). Three novel suballeles (*10.007, *36.004, and *75.002) were also identified. Phenotype frequencies were as follows: ultrarapid metabolizers (4.2%, 2/48), normal metabolizers (41.7%, 20/48) and intermediate metabolizers (52.1%, 25/48); none of the 48 participants were predicted to be poor metabolizers. Concordance of diplotype and phenotype calls between the consensus and clinical testing were 66.7 and 50%, respectively.Conclusion: Our study to explore CYP2D6 genotypes in the Hmong population suggests that this subpopulation is unique regarding CYP2D6 allelic variants; also, a higher portion of Hmong participants (50%) are predicted to have an intermediate metabolizer phenotype for CYP2D6 compared to other East Asians which range between 27 and 44%. Results from different testing methods varied considerably. These preliminary findings underscore the importance of thoroughly interrogating unique subpopulations to accurately predict a patient’s CYP2D6 metabolizer status.

BackgroundThe CYP2D6 gene locus has been extensively studied over decades, yet a portion of variability in CYP2D6 activity cannot be explained by known sequence variations within the gene, copy number variation, or structural rearrangements. It was proposed that rs5758550, located 116 kb downstream of the CYP2D6 gene locus, increases gene expression and thus contributes to variability in CYP2D6 activity. This finding has, however, not been validated. The purpose of the study was to address a major technological barrier, i.e., experimentally linking rs5758550, also referred to as the “enhancer” single-nucleotide polymorphism (SNP), to CYP2D6 haplotypes >100 kb away. To overcome this challenge is essential to ultimately determine the contribution of the “enhancer” SNP to interindividual variability in CYP2D6 activity.MethodsA large ethnically mixed population sample (n=3,162) was computationally phased to determine linkage between the “enhancer” SNP and CYP2D6 haplotypes (or star alleles). To experimentally validate predicted linkages, DropPhase2D6, a digital droplet PCR (ddPCR)-based method was developed. 10X Genomics Linked-Reads were utilized as a proof of concept.ResultsPhasing predicted that the “enhancer” SNP can occur on numerous CYP2D6 haplotypes including CYP2D6*1, *2, *5, and *41 and suggested that linkage is incomplete, i.e., a portion of these alleles do not have the “enhancer” SNP. Phasing also revealed differences among the European and African ancestry data sets regarding the proportion of alleles with and without the “enhancer” SNP. DropPhase2D6 was utilized to confirm or refute the predicted “enhancer” SNP location for individual samples, e.g., of n=3 samples genotyped as *1/*41, rs5758550 was on the *41 allele of two samples and on the *1 allele of one sample. Our findings highlight that the location of the “enhancer” SNP must not be assigned by “default.” Furthermore, linkage between the “enhancer” SNP and CYP2D6 star allele haplotypes was confirmed with 10X Genomics technology.ConclusionsSince the “enhancer” SNP can be present on a portion of normal, decreased, or no function alleles, the phase of the “enhancer” SNP must be considered when investigating the impact of the “enhancer” SNP on CYP2D6 activity.