CYP2D7 Sequence Variation Interferes with TaqMan CYP2D6 (*) 15 and (*) 35 Genotyping

Abstract

TaqMan™ genotyping assays are widely used to genotype CYP2D6, which encodes a major drug metabolizing enzyme. Assay design for CYP2D6 can be challenging owing to the presence of two pseudogenes, CYP2D7 and CYP2D8, structural and copy number variation and numerous single nucleotide polymorphisms (SNPs) some of which reflect the wild-type sequence of the CYP2D7 pseudogene. The aim of this study was to identify the mechanism causing false-positive CYP2D6 (*) 15 calls and remediate those by redesigning and validating alternative TaqMan genotype assays. Among 13,866 DNA samples genotyped by the CompanionDx® lab on the OpenArray platform, 70 samples were identified as heterozygotes for 137Tins, the key SNP of CYP2D6 (*) 15. However, only 15 samples were confirmed when tested with the Luminex xTAG CYP2D6 Kit and sequencing of CYP2D6-specific long range (XL)-PCR products. Genotype and gene resequencing of CYP2D6 and CYP2D7-specific XL-PCR products revealed a CC>GT dinucleotide SNP in exon 1 of CYP2D7 that reverts the sequence to CYP2D6 and allows a TaqMan assay PCR primer to bind. Because CYP2D7 also carries a Tins, a false-positive mutation signal is generated. This CYP2D7 SNP was also responsible for generating false-positive signals for rs769258 (CYP2D6 (*) 35) which is also located in exon 1. Although alternative CYP2D6 (*) 15 and (*) 35 assays resolved the issue, we discovered a novel CYP2D6 (*) 15 subvariant in one sample that carries additional SNPs preventing detection with the alternate assay. The frequency of CYP2D6 (*) 15 was 0.1% in this ethnically diverse U.S. population sample. In addition, we also discovered linkage between the CYP2D7 CC>GT dinucleotide SNP and the 77G>A (rs28371696) SNP of CYP2D6 (*) 43. The frequency of this tentatively functional allele was 0.2%. Taken together, these findings emphasize that regardless of how careful genotyping assays are designed and evaluated before being commercially marketed, rare or unknown SNPs underneath primer and/or probe regions can impact the performance of PCR-based genotype assays, including TaqMan. Regardless of the test platform used, it is prudent to confirm rare allele calls by an independent method.

Keywords: CYP2D6; CYP2D6*15; CYP2D6*35; CYP2D6*43; CYP2D7; TaqMan; genotyping.

Figure 1

Interference of CYP2D7 variation on CYP2D6^*15 and ^*35 TaqMan genotype assay results. (A) Shows the exon 1 region of CYP2D6 and the CYP2D7 pseudogene. Sequence differences at the −1 and −2 positions are highlighted in green and the ATG translation start codon is underlined. CYP2D7 carries a CC at positions −1 and −2 while CYP2D6 has GT. This difference was employed for primer design for the initial CYP2D6^*15 TaqMan genotyping assay. SNPs defining allelic variants are shown in red; nucleotide positions are as indicated. The primer location is provided by the arrow. An “X” indicates that the primer does not generate PCR product. (B) Details the mechanism by which false-positive assay results are generated. The first example shows a correct CYP2D6^*15 call; the TaqMan primer only binds to CYP2D6. In the second example, one CYP2D7 allele carries GT allowing primer binding and PCR product amplification. Since the CYP2D7 derived amplicon has the T-insertion and 31A, the assay yields a false-positive results for CYP2D6^*15 and ^*35. In the third example, CYP2D6^*15 will also be false-positive. Regarding 31G>A, fluorescent signals will be generated from the variant CYP2D7 allele as well as the CYP2D6^*35 allele which may lead to a shift in cluster position.

Figure 2

High Resolution Melt (HRM) assay detecting variation on CYP2D7-specific XL-PCR template. Blue indicates wt, green heterozygous and red variant.

Figure 3

TaqMan scatter plots generated with the initial (A) and alternate (B) CYP2D6^*15 TaqMan genotyping assay for false-positive and true positive samples. Profiles shown were generated on WGA-DNA. Boxed samples are false-positives with the initial assay (A) that have been resolved with the alternate TaqMan genotyping assay. (B) Black dot in symbol indicates sample 13 carrying the novel CYP2D6^*15var. This subject was false-negative with the alternate CYP2D6^*15 genotyping assay.

Figure 4

TaqMan amplification profiles generated with the initial (A) and alternate (B) CYP2D6^*35 TaqMan genotyping assay for false-positive and true positive samples. Profiles shown were generated on WGA-DNA. Boxed samples are false-positives (A) that have been resolved with the alternate TaqMan genotyping assay (B).