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Identification of Novel CYP2D7-2D6 Hybrids: Non-Functional and Functional Variants.

ABSTRACT: Polymorphic expression of CYP2D6 contributes to the wide range of activity observed for this clinically important drug metabolizing enzyme. In this report we describe novel CYP2D7/2D6 hybrid genes encoding non-functional and functional CYP2D6 protein and a CYP2D7 variant that mimics a CYP2D7/2D6 hybrid gene. Five-kilobyte-long PCR products encompassing the novel genes were entirely sequenced. A quantitative assay probing in different gene regions was employed to determine CYP2D6 and 2D7 copy number variations and the relative position of the hybrid genes within the locus was assessed by long-range PCR. In addition to the previously known CYP2D6*13 and *66 hybrids, we describe three novel non-functional CYP2D7-2D6 hybrids with gene switching in exon 2 (CYP2D6*79), intron 2 (CYP2D6*80), and intron 5 (CYP2D6*67). A CYP2D7-specific T-ins in exon 1 causes a detrimental frame shift. One subject revealed a CYP2D7 conversion in the 5'-flanking region of a CYP2D6*35 allele, was otherwise unaffected (designated CYP2D6*35B). Finally, three DNAs revealed a CYP2D7 gene with a CYP2D6-like region downstream of exon 9 (designated CYP2D7[REP6]). Quantitative copy number determination, sequence analyses, and long-range PCR mapping were in agreement and excluded the presence of additional gene units. Undetected hybrid genes may cause over-estimation of CYP2D6 activity (CYP2D6*1/*1 vs *1/hybrid, etc), but may also cause results that may interfere with the genotype determination. Detection of hybrid events, "single" and tandem, will contribute to more accurate phenotype prediction from genotype data.

SUBMITTER: Gaedigk A

PROVIDER: S-EPMC3153001 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Identification of Novel CYP2D7-2D6 Hybrids: Non-Functional and Functional Variants.

Gaedigk Andrea A Jaime Lazara Karelia Montane LK Bertino Joseph S JS Bérard Anick A Pratt Victoria M VM Bradfordand L Dianne LD Leeder J Steven JS

Frontiers in pharmacology 20101004

Polymorphic expression of CYP2D6 contributes to the wide range of activity observed for this clinically important drug metabolizing enzyme. In this report we describe novel CYP2D7/2D6 hybrid genes encoding non-functional and functional CYP2D6 protein and a CYP2D7 variant that mimics a CYP2D7/2D6 hybrid gene. Five-kilobyte-long PCR products encompassing the novel genes were entirely sequenced. A quantitative assay probing in different gene regions was employed to determine CYP2D6 and 2D7 copy num ...[more]

PMID: 21833166

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Project description:BACKGROUND: Peptidase inhibitor 3 (PI3) inhibits neutrophil elastase and proteinase-3, and has a potential role in skin and lung diseases as well as in cancer. Genome-wide expression profiling of chorioamniotic membranes revealed decreased expression of PI3 in women with preterm premature rupture of membranes. To elucidate the molecular mechanisms contributing to the decreased expression in amniotic membranes, the PI3 gene was searched for sequence variations and the functional significance of the identified promoter variants was studied. METHODS: Single nucleotide polymorphisms (SNPs) were identified by direct sequencing of PCR products spanning a region from 1,173 bp upstream to 1,266 bp downstream of the translation start site. Fourteen SNPs were genotyped from 112 and nine SNPs from 24 unrelated individuals. Putative transcription factor binding sites as detected by in silico search were verified by electrophoretic mobility shift assay (EMSA) using nuclear extract from Hela and amnion cell nuclear extract. Deviation from Hardy-Weinberg equilibrium (HWE) was tested by chi2 goodness-of-fit test. Haplotypes were estimated using expectation maximization (EM) algorithm. RESULTS: Twenty-three sequence variations were identified by direct sequencing of polymerase chain reaction (PCR) products covering 2,439 nt of the PI3 gene (-1,173 nt of promoter sequences and all three exons). Analysis of 112 unrelated individuals showed that 20 variants had minor allele frequencies (MAF) ranging from 0.02 to 0.46 representing "true polymorphisms", while three had MAF < or = 0.01. Eleven variants were in the promoter region; several putative transcription factor binding sites were found at these sites by database searches. Differential binding of transcription factors was demonstrated at two polymorphic sites by electrophoretic mobility shift assays, both in amniotic and HeLa cell nuclear extracts. Differential binding of the transcription factor GATA1 at -689C>G site was confirmed by a supershift. CONCLUSION: The promoter sequences of PI3 have a high degree of variability. Functional promoter variants provide a possible mechanism for explaining the differences in PI3 mRNA expression levels in the chorioamniotic membranes, and are also likely to be useful in elucidating the role of PI3 in other diseases.

Dataset Information

Identification of Novel CYP2D7-2D6 Hybrids: Non-Functional and Functional Variants.

Publications

Identification of Novel CYP2D7-2D6 Hybrids: Non-Functional and Functional Variants.

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