Many antidepressants, atomoxetine, and several antipsychotics are metabolized by the cytochrome P450 enzymes CYP2D6 and CYP2C19, and guidelines for prescribers based on genetic variants exist. Although some laboratories offer such testing, there is no consensus regarding validated methodology for clinical genotyping of CYP2D6 and CYP2C19. The aim of this paper was to cross-validate multiple technologies for genotyping CYP2D6 and CYP2C19 against each other, and to contribute to feasibility for clinical implementation by providing an enhanced range of assay options, customizable automated translation of data into haplotypes, and a workflow algorithm. AmpliChip CYP450 and some TaqMan single nucleotide variant (SNV) and copy number variant (CNV) data in the Genome-based therapeutic drugs for depression (GENDEP) study were used to select 95 samples (out of 853) to represent as broad a range of CYP2D6 and CYP2C19 genotypes as possible. These 95 included a larger range of CYP2D6 hybrid configurations than have previously been reported using inter-technology data. Genotyping techniques employed were: further TaqMan CNV and SNV assays, xTAGv3 Luminex CYP2D6 and CYP2C19, PharmacoScan, the Ion AmpliSeq Pharmacogenomics Panel, and, for samples with CYP2D6 hybrid configurations, long-range polymerase chain reactions (L-PCRs) with Sanger sequencing and Luminex. Agena MassARRAY was also used for CYP2C19. This study has led to the development of a broader range of TaqMan SNV assays, haplotype phasing methodology with TaqMan adaptable for other technologies, a multiplex genotyping method for efficient identification of some hybrid haplotypes, a customizable automated translation of SNV and CNV data into haplotypes, and a clinical workflow algorithm.

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CYP2D6 is a widely expressed human xenobiotic metabolizing enzyme, best known for its role in the hepatic phase I metabolism of up to 25% of prescribed medications, which is also expressed in other organs including the brain, where its potential role in physiology and mental health traits and disorders is under further investigation. Owing to the presence of homologous pseudogenes in the CYP2D locus and transposable repeat elements in the intergenic regions, the gene encoding the CYP2D6 enzyme, CYP2D6, is one of the most hypervariable known human genes - with more than 140 core haplotypes. Haplotypes include structural variants, with a subtype of these known as fusion genes comprising part of CYP2D6 and part of its adjacent pseudogene, CYP2D7. The fusion genes are particularly challenging to identify. The CYP2D6 enzyme activity corresponding to some of these fusion genes is known, while for others it is unknown. The most recent (high fidelity, or HiFi) version of single molecule real-time (SMRT) long-read sequencing can cover whole CYP2D6 haplotypes in a single continuous sequence read, ideal for structural variant detection. In addition, the accuracy of base calling has increased to a level sufficient for accurate characterization of single nucleotide variants. As new CYP2D6 haplotypes are continuously being discovered, and likely many more remain to be identified in populations that are relatively understudied to date, a method of characterization that employs sequencing with at least this degree of accuracy is required. The aim of the work reported herein was to develop an efficient and accurate HiFi SMRT amplicon-based method capable of detecting the full range of CYP2D6 haplotypes including fusion genes. We report proof-of-concept for 20 amplicons, aligned to fusion gene haplotypes, with prior cross-validation data. Amplicons with CYP2D6-D7 fusion genes aligned to *36, *63, *68, and *4 (*4-like; *4N, or *4.013) hybrid haplotypes. Amplicons with CYP2D7-D6 fusion genes aligned to the *13 subhaplotypes predicted (e.g., *13F, *13A2). Data analysis was efficient, and further method development indicates that this technique could suffice for the characterization of the full range of CYP2D6 haplotypes. Although included in drug labelling by regulatory bodies (the U.S. Food and Drug Administration, the European Medicines Agency, the Pharmaceuticals and Medical Devices Agency) and prescribing recommendations by consortia (Clinical Pharmacogenetics Implementation Consortium and the Dutch Pharmacogenetics Working Group), the identification of CYP2D6 variants is not yet routine in clinical practice. The HiFi sequencing method reported herein is suitable for high throughput, efficient, identification of the full range of known CYP2D6 haplotypes and novel haplotypes, and can be completed in a week or less. Moreover, the method that we have developed could be extended to other complex loci and to other species in a multiplexed high throughput assay.

The human leukocyte antigen haplotypes HLA-B*15:02 and HLA-A*31:01 have been linked to life-threatening adverse drug reactions to the anticonvulsants carbamazepine and oxcarbazepine. Identification of these haplotypes via pharmacogenetic techniques facilitates implementation of precision medicine to prevent such reactions. Using reference samples from diverse ancestral origins, we investigated the test analytical validity (i.e., ability to detect whether or not the haplotypes were present or absent) of TaqMan assays for single nucleotide variants previously identified as potentially being able to “tag” these haplotypes. A TaqMan custom assay for rs10484555 and an inventoried assay for rs17179220 and were able to identify with 100% sensitivity and 100% specificity HLA-B*15:02 and HLA-A*31:01 respectively. A custom assay for rs144012689 that takes into account a neighboring single nucleotide variant with manual calling was also able to identify HLA-B*15:02 with 100% sensitivity and 100% specificity. A custom assay for rs106235 identified HLA-A*31:01 with 100% sensitivity and 95% specificity. The slight reduction in specificity for the latter was owing to another haplotype (HLA-A*33:03) also being detected. While any positive call using the rs106235 assay could therefore be further investigated, as the presence of the HLA-A*31:01 haplotype confers adverse drug reaction risk, the absence of false negatives (indexed by sensitivity) is more important than false positives. In summary, we present validated TaqMan assay methodology for efficient detection of HLA haplotypes HLA-B*15:02 and HLA-A*31:01. Our data are relevant for other genotyping technologies that identify, or have the potential to identify, these haplotypes using single nucleotide variants.

There are some data associating variants in the CYP2D6 and/or CYP2C19 genes with concentration-to-dose ratios, efficacy, and retention in treatments. However, much of the above arises from relatively small studies or large datasets with limited genotyping methodologies. Our aim was to develop and validate comprehensive and accurate genotyping methodology for these two genes to facilitate regenotyping in large datasets and hence the generation of more accurate clinical associations. TaqMan copy number variant (CNV) assays for CYP2D6 were used to identify samples from a relevant large dataset (GENDEP study, N = 853) with particularly challenging genotypes to call. These and those representing as broad a range of CYP2D6 and CYP2C19 genotypes as possible by prior available data (AmpliChip CYP450 and TaqMan CYP2C19*17) were chosen for further analysis (N = 96). Genotyping techniques employed were: Luminex CYP2D6 xTAGv3 and Luminex CYP2C19 xTAGv3, PharmacoScan, the Ion S5 AmpliSeq Pharmacogenomics Panel, TaqMan single nucleotide variant (SNV) assays, and, for the CYP2D6 hybrids, long-range polymerase chain reactions (L-PCRs) with Sanger sequencing. Agena was also used for CYP2C19. The TaqMan SNV assays were able to assist with identifying which gene was duplicated or in tandem for multiple copy variants. A multiplex assay was adaptable for analysis of CYP2D6 hybrid genes, with Sanger sequencing data being consistent with the data arising; we provide these data for efficient genotyping of such CYP2D6 hybrid genes with adaptable multiplex methods. Consensus genotypes generated to date resulted in revision of assigned enzyme activity score for 28/96(29%) and 2/93 samples (2.2%) for CYP2D6 and CYP2C19, respectively.

A Corrigendum on Validation of single nucleotide variant assays for human leukocyte antigen haplotypes HLA-B*15:02 and HLA-A*31:01 across diverse ancestral backgrounds