CYP2D7 sequence variation interferes with TaqMan CYP2D6*15 and *35 genotyping
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 (SNP...
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doaj-0106e0dda02048d290ba46d3d451a9bd2020-11-25T02:07:11ZengFrontiers Media S.A.Frontiers in Pharmacology1663-98122016-01-01610.3389/fphar.2015.00312174129CYP2D7 sequence variation interferes with TaqMan CYP2D6*15 and *35 genotypingAmanda K Riffel0Mehdi eDehghani1Mehdi eDehghani2Toinette eHartshorne3Kristen eFloyd4J Steven eLeeder5J Steven eLeeder6Kevin P Rosenblatt7Kevin P Rosenblatt8Andrea eGaedigk9Andrea eGaedigk10Children's Mercy Kansas CityCompanionDx® Reference LabUniversity of Texas Health Science Center at HoustonThermo Fisher ScientificCompanionDx® Reference LabChildren's Mercy Kansas CityUniversity of Missouri-Kansas CityCompanionDx® Reference LabUniversity of Texas Health Science Center at HoustonChildren's Mercy Kansas CityUniversity of Missouri-Kansas CityTaqMan™ 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.http://journal.frontiersin.org/Journal/10.3389/fphar.2015.00312/fullCYP2D6genotypingTaqManCYP2D7CYP2D6*15CYP2D6*35 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Amanda K Riffel Mehdi eDehghani Mehdi eDehghani Toinette eHartshorne Kristen eFloyd J Steven eLeeder J Steven eLeeder Kevin P Rosenblatt Kevin P Rosenblatt Andrea eGaedigk Andrea eGaedigk |
spellingShingle |
Amanda K Riffel Mehdi eDehghani Mehdi eDehghani Toinette eHartshorne Kristen eFloyd J Steven eLeeder J Steven eLeeder Kevin P Rosenblatt Kevin P Rosenblatt Andrea eGaedigk Andrea eGaedigk CYP2D7 sequence variation interferes with TaqMan CYP2D6*15 and *35 genotyping Frontiers in Pharmacology CYP2D6 genotyping TaqMan CYP2D7 CYP2D6*15 CYP2D6*35 |
author_facet |
Amanda K Riffel Mehdi eDehghani Mehdi eDehghani Toinette eHartshorne Kristen eFloyd J Steven eLeeder J Steven eLeeder Kevin P Rosenblatt Kevin P Rosenblatt Andrea eGaedigk Andrea eGaedigk |
author_sort |
Amanda K Riffel |
title |
CYP2D7 sequence variation interferes with TaqMan CYP2D6*15 and *35 genotyping |
title_short |
CYP2D7 sequence variation interferes with TaqMan CYP2D6*15 and *35 genotyping |
title_full |
CYP2D7 sequence variation interferes with TaqMan CYP2D6*15 and *35 genotyping |
title_fullStr |
CYP2D7 sequence variation interferes with TaqMan CYP2D6*15 and *35 genotyping |
title_full_unstemmed |
CYP2D7 sequence variation interferes with TaqMan CYP2D6*15 and *35 genotyping |
title_sort |
cyp2d7 sequence variation interferes with taqman cyp2d6*15 and *35 genotyping |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Pharmacology |
issn |
1663-9812 |
publishDate |
2016-01-01 |
description |
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. |
topic |
CYP2D6 genotyping TaqMan CYP2D7 CYP2D6*15 CYP2D6*35 |
url |
http://journal.frontiersin.org/Journal/10.3389/fphar.2015.00312/full |
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