<i>CYP2D6</i> Copy Number Variations Identified by KASP PCR

CYP2D6 is one of the most interesting pharmacogenetic genes. Over 100 different alleles have been identified, some of which exhibit distinct geographical and ethnic distributions [1, 2]. In addition to single nucleotide polymorphisms (SNPs), partial or whole gene deletions, as well as duplications are common. In humans, CYP2D6 is accompanied by two pseudogenes, CYP2D7P and CYP2D8P, and hybrid crossovers between CYP2D6 and CYP2D7P have also been observed [3]. Since it is estimated that approximately 25% of all drugs are metabolized, at least in part, by CYP2D6, genetic testing has become an important tool for predicting the metabolic phenotype of this enzyme [4, 5]. Although next-generation sequencing techniques are beginning to be applied, the complex genomic organization and polymorphic nature of the CYP2D6 gene make data analysis challenging. As a result, routine genotyping still largely relies on traditional polymerase chain reaction (PCR) methods, such as real-time PCR or probe-/bead chip-based detection, especially when it comes to the identification of copy number variations (CNV) [6, 7]. In a recent publication, we developed a novel method for identifying duplications and deletions of the CYP2D6 gene [8]. This approach utilizes CYP2D8P as a reference and employs a single homologous PCR primer set, which amplifies a sequence of both CYP2D6 and CYP2D8P, but not the CYP2D7P pseudogene. This removes any bias, which is common when using traditional multiplex PCR. It ensures that both targets are amplified with equal affinity, with CNVs being detected using either real-time PCR, TaqMan/5′-nuclease assays, or high-resolution melting (HRM) PCR. In the current study, we have expanded this methodology to include Competitive Allele-Specific PCR (KASP), enabling the detection of CNV based on a qualitative end point PCR method. The Danish Blood Donor Study (DBDS) was approved by Zealand (SJ-740) and Central Denmark (1–10-72–95-13) Regional Committees on Health Research Ethics and the Data Protection Agency (P-2019–99). The study was conducted in accordance with the Basic and Clinical Pharmacology and Toxicology policy for experimental and clinical studies [9]. Genomic DNA from eight individuals with known CYP2D6 copy number was obtained from Coriell Institute for Medical Research (Camden, New Jersey, USA) and is listed in Table 1. Anonymous blood samples were randomly collected in EDTA tubes from 19 Danish donors from the Region Zealand Blood Bank and subsequently stored at −80°C until further processing. DNA was extracted from 100-μL whole blood, using the MagNA Pure Compact Nucleic Acid Isolation Kit 1 and MagNA Pure Compact (Roche, Mannheim, Germany) according to the manufacturer's instructions. DNA concentrations were determined using Qubit 2.0 and dsDNA BR reagent (Thermo Fisher Scientific, Waltham, Massachusetts, USA). DNA was stored at −20°C until further analysis. The CYP2D6 genotype of each donor sample was determined using xTAG CYP2D6 Kit v3 (Luminex Corp, Austin, Texas, USA) according to the manufacturer's instructions and data was analysed using the TDAS CYP2D6 v 1.01 software (Luminex Corp, Austin, Texas, USA) (Table 1). Primer sequences for the competitive allele-specific PCR assay were adapted from the high-resolution melt curve primers published by Larsen and Jørgensen and synthesized by LGC Biosearch (Lystrup, Denmark) [8]. Sequences are listed in Table 2. The CNV assay consists of one universal CYP2D6/CYP2D8P forward primer and two gene-specific reverse primers targeting either CYP2D6 or CYP2D8P. Primer specificity was confirmed by BLAST against GRCh38.p13 and validated using HRM analysis as previously described [8]. KASP-PCR was performed in a total volume of 10-μL, consisting of 5-μL KASP-TF V4.0 (LGC Biosearch, Hoddesdon, Herts, United Kingdom), 0.14-μL KASP Assay mix, 1-μL genomic DNA (10 ng/μL) and 3.86-μL H2O. PCR amplification was performed using a CFX96 Real-Time PCR system (Bio-Rad, Hercules, California, USA) programmed with the following conditions: 94°C for 15 min, 10 cycles of touch down PCR, 94°C for 20 s, 61°C for 1 min, with 0.6°C decrement per cycle followed by 25 cycles of 94°C for 20 s, 55°C for 1 min, with cooling to 37°C followed by plate read. Data were analysed with CFX Maestro 2.3 software, using Allelic Discrimination (Bio-Rad, Hercules, California, USA). Negative controls were included in each PCR setup. Analysis separated the Coriell samples into four groups, based on the signal generated by the CYP2D6 probe; samples with deletion 5*, wildtype, a single duplication and samples containing two duplications of the CYP2D6 gene (Figure 1). The separation of the groups depended on the relative difference in the fluorescence generated by the CYP2D6 probes, with the *5 deletion having the largest separation compared to wildtype samples and a single duplication the least. The KASP assay was used to verify the genotype of the 19 donor samples genotyped using the Luminex xTAG CYP2D6 kit (Table 2). As the Luminex assay is not capable of discriminating between CNVs beyond 2 copies or identifying copied alleles, these results are reported as ‘xN’ (Table 1). The KASP assay confirmed the CNV result of all samples (Figure 2). In this study, an assay based on the KASP allele discrimination PCR technique was developed, to distinguish between CNV of the CYP2D6 gene. To our knowledge, this is the first of its kind eliminating the need for real-time PCR and enabling the use of KASP for both SNP and CNV detection. Utilizing a single, standardized technique for all allele assays within a test panel simplifies the workflow, allows for potential automation and reduces costs [14]. A crucial factor in genomic allele discrimination by PCR is achieving clear separation between the signals of homozygous and heterozygous samples. This also applies to the CYP2D6:2D8P KASP CNV assay. While deletions and multiple copies are relatively easy to identify, single duplications present a greater challenge. The difficulty arises from the small relative increase in template DNA compared to wildtype, which is a key distinguishing parameter in real-time PCR. A single extra gene copy results in only a 33% increase in starting material (a 2:3 ratio of wildtype to duplication), whereas a deletion (*5 allele) or multicopy duplication exhibits a 50% or more difference compared to the wildtype (a 2:1 ratio for deletions and a 2:≥ 4 ratio for multicopy duplications) (Figure 1). As PCR is extremely sensitive, commonly resulting in signal variations between runs, this makes it difficult to distinguish single-copy CNVs from the wildtype using standard allele discrimination methods [8]. One possible way to enhance separation in the KASP assay could be by optimizing the concentration of the universal CYP2D6:2D8P primers, making it a limiting factor in the PCR reaction. During cycling, this would ensure that the two gene-specific primers (added in excess) compete for a continuously diminishing pool of the reverse primer. Using this approach, any variations between target (CYP2D6) and reference gene (CYP2D8P), would be enlarged during the PCR cycles, as there with an increase in one template is a higher chance of the universal primer annealing. This approach could further improve resolution between different CNV genotypes, as previously observed developing an HRM assay [8]. However, since the KASP assay was provided as a standardized multiprimer mix, this hypothesis was not tested in the present study and warrants future investigation. Although the identification of duplications in a patient can be relevant when evaluating lack of clinical effect, which can be caused by either ultrarapid metabolization or non-compliance, the most clinically significant CNV to detect is that of the 5* deletion. This is because it, in combination with other nonfunctional alleles, may cause a poor metabolizer phenotype and hereby lead to adverse drug reactions at standard doses [15, 16]. In conclusion, this study presents a novel qualitative PCR assay for detecting CYP2D6 CNV using KASP technology. The assay offers a simplified setup when designing genotyping test panels. As KASP relies on a single common primer for allelic discrimination, this method may more easily be applied to target other genes/pseudogenes. All authors contributed to the study conception and design according to journal author guidelines and the ICMJE definition of authorship. This study was performed using The Danish Blood Donor study cohort, and we thank the staff and blood donors who participated in the study. The authors declare no conflicts of interest. Figure S1: Primer efficiency of the KASP PCR. Endpoint fluorescence (RFU) is plotted against input concentration (log10) for a duplex KASP assay. Each point represents technical replicates at three concentration levels. Dotted lines denote least-squares regressions. Fluorescence increases linearly with input DNA across the tested range; CYP2D6 (blue) consistently yields higher RFU than CYP2D8p (orange), and the approximately parallel slopes indicate comparable assay behaviour without evident target competition. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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