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CYP2D7-2D6 hybrid tandems: identification of novel CYP2D6 duplication arrangements and implications for phenotype prediction.

ABSTRACT: AIMS:Allelic variants of cytochrome P450 CYP2D6 (CYP2D6), such as gene deletion, duplication, multiplication and conversion, contribute to the wide range of CYP2D6 activity. Novel gene arrangements were discovered and characterized. MATERIALS & METHODS:DNA from 32 Caucasian and 59 African-American duplication-positive subjects were analyzed by long-range PCR and genotyping to detect CYP2D7-2D6 hybrid tandem alleles. Novel allelic variants were sequenced and a strategy for the detection and analysis of hybrid genes was refined. RESULTS:CYP2D7-2D6 hybrid tandem alleles were identified in one African-American and four Caucasian subjects. Three novel hybrid genes were found on CYP2D6*1 and CYP2D6*2 duplication backgrounds and designated CYP2D6*76, *77 and *78. CYP2D7 to 2D6 conversion occurred in introns 1 and 4, and exon 9. All carried a T-insertion in exon 1 abolishing activity. In Caucasians, four out of 33 (12%) of the duplication-positive alleles were hybrid tandems, three CYP2D6*77 + *2 and one CYP2D6*78 + *2. By contrast, in African-Americans only one of 60 duplication-positive alleles was identified as a hybrid tandem. This allele was designated CYP2D6*76 + *1. CONCLUSION:Hybrid tandem alleles occur infrequently (<0.25%) in Caucasians, but may explain why not every subject with a CYP2D6 duplication presents with an ultrarapid metabolizer phenotype.

SUBMITTER: Gaedigk A

PROVIDER: S-EPMC6556886 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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CYP2D7-2D6 hybrid tandems: identification of novel CYP2D6 duplication arrangements and implications for phenotype prediction.

Gaedigk Andrea A Fuhr Uwe U Johnson Charlene C Bérard L Anick LA Bradford Dianne D Leeder J Steven JS

Pharmacogenomics 20100101 1

<h4>Aims</h4>Allelic variants of cytochrome P450 CYP2D6 (CYP2D6), such as gene deletion, duplication, multiplication and conversion, contribute to the wide range of CYP2D6 activity. Novel gene arrangements were discovered and characterized.<h4>Materials & methods</h4>DNA from 32 Caucasian and 59 African-American duplication-positive subjects were analyzed by long-range PCR and genotyping to detect CYP2D7-2D6 hybrid tandem alleles. Novel allelic variants were sequenced and a strategy for the dete ...[more]

PMID: 20017671

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Project 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.

Project description:BackgroundPrimaquine and tafenoquine are the only licensed drugs that effectively kill the hypnozoite stage and are used to prevent Plasmodium vivax malaria relapse. However, both primaquine and tafenoquine can cause acute hemolysis in glucose-6-phosphate dehydrogenase (G6PD)-deficient people with varying degrees of severity depending on G6PD variants. Additionally, primaquine efficacy against malaria parasites was decreased in individuals with impaired cytochrome P450 2D6 (CYP2D6) activity due to genetic polymorphisms. This study aimed to characterize G6PD and CYP2D6 genetic variations in vivax malaria patients from Yala province, a malaria-endemic area along the Thai-Malaysian border, and determine the biochemical properties of identified G6PD variants.Methodology/principle findingsMultiplexed high-resolution melting assay and DNA sequencing detected five G6PD variants, including G6PD Kaiping, G6PD Vanua Lava, G6PD Coimbra, G6PD Mahidol, and G6PD Kerala-Kalyan. Biochemical and structural characterization revealed that G6PD Coimbra markedly reduced catalytic activity and structural stability, indicating a high susceptibility to drug-induced hemolysis. While Kerala-Kalyan had minor effects, it is possible to develop mild adverse effects when receiving radical treatment. CYP2D6 genotyping was performed using long-range PCR and DNA sequencing, and the phenotypes were predicted using the combination of allelic variants. Decreased and no-function alleles were detected at frequencies of 53.4% and 14.2%, respectively. The most common alleles were CYP2D6*36+*10 (25.6%), *10 (23.9%), and *1 (22.2%). Additionally, 51.1% of the intermediate metabolizers showed CYP2D6*10/*36+*10 as the predominant genotype (15.9%).Conclusions/significanceOur findings provide insights about genetic variations of G6PD and CYP2D6 in 88 vivax malaria patients from Yala, which may influence the safety and effectiveness of radical treatment. Optimization of 8-aminoquinoline administration may be required for safe and effective treatment in the studied population, which could be a significant challenge in achieving the goal of eliminating malaria.

Dataset Information

CYP2D7-2D6 hybrid tandems: identification of novel CYP2D6 duplication arrangements and implications for phenotype prediction.

Publications

CYP2D7-2D6 hybrid tandems: identification of novel CYP2D6 duplication arrangements and implications for phenotype prediction.

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