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.

Many genetic variants in drug metabolizing enzymes and transporters have been shown to be relevant for treating psychiatric disorders. Associations are strong enough to feature on drug labels and for prescribing guidelines based on such data. A range of commercial tests are available; however, there is variability in included genetic variants, methodology, and interpretation. We herein provide relevant background for understanding clinical associations with specific variants, other factors that are relevant to consider when interpreting such data (such as age, gender, drug–drug interactions), and summarize the data relevant to clinical utility of pharmacogenetic testing in psychiatry and the available prescribing guidelines. We also highlight areas for future research focus in this field.

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AbstractBackgroundCYP2D6 and CYP2C19 are cytochrome P450 enzymes involved in the metabolism of many medications from multiple therapeutic classes. The genes encoding them, CYP2D6 and CYP2C19, have multiple functional variants that have been associated with different levels of enzyme activity. There is evidence that methodology for identifying such variants (“genotyping”) effectively and efficiently could lead to substantial health care costs savings. There are various genotyping technologies available; we cross-validated several against each other.MethodsNinety-six samples with a variety of CYP2D6 and CYP2C19 genotypes according to prior AmpliChip CYP450 and TaqMan CYP2C19*17 data were selected from the Genome-based therapeutic drugs for depression (GENDEP) study. Genotyping was performed with TaqMan copy number variant (CNV) analysis using probes specific for three different regions (CYP2D6 only), the next generation sequencing-based Ion S5 AmpliSeq Pharmacogenomics Panel, PharmacoScan, and long-range polymerase chain reaction (XL-PCR) followed by TaqMan single nucleotide variant (SNV) analysis or the Agena MassARRAY. Variant to “star allele” translation was automated.ResultsThe inter-platform concordance for CYP2C19 was high (94-98%). For CYP2D6, the IonS5-PharmacoScan concordance was 94% for a range of variants tested apart from those with at least one extra copy of a CYP2D6 gene (occurring at a frequency of 3.8%, 33/853), or a pseudogene-gene conversion (2%, 18/853).ConclusionsInter-platform concordance for CYP2C19 was high, and, moreover, the Ion S5 and PharmacoScan data were 100% concordant with that from a TaqMan CYP2C19*2 assay. The level of concordance was also good for CYP2D6, apart from those with at least one extra copy of a CYP2D6 gene or a pseudogene-gene conversion. We have also demonstrated feasibility of using a NGS platform for genotyping CYP2D6 and CYP2C19, with automated data interpretation methodology. CYP2D6 and CYP2C19 genotyping may thereby become much more readily accessible, with potential substantial savings in health care costs and public benefit.

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.