Functional characterization of CYP2D6 enhancer polymorphisms.
CYP2D6 metabolizes nearly 25% of clinically used drugs. Genetic polymorphisms cause large inter-individual variability in CYP2D6 enzyme activity and are currently used as biomarker to predict CYP2D6 metabolizer phenotype. Previously, we had identified a region 115 kb downstream of CYP2D6 as enhancer for CYP2D6, containing two completely linked single nucleotide polymorphisms (SNPs), rs133333 and rs5758550, associated with enhanced transcription. However, the enhancer effect on CYP2D6 expression, and the causative variant, remained to be ascertained. To characterize the CYP2D6 enhancer element, we applied chromatin conformation capture combined with the next-generation sequencing (4C assays) and chromatin immunoprecipitation with P300 antibody, in HepG2 and human primary culture hepatocytes. The results confirmed the role of the previously identified enhancer region in CYP2D6 expression, expanding the number of candidate variants to three highly linked SNPs (rs133333, rs5758550 and rs4822082). Among these, only rs5758550 demonstrated regulating enhancer activity in a reporter gene assay. Use of clustered regularly interspaced short palindromic repeats mediated genome editing in HepG2 cells targeting suspected enhancer regions decreased CYP2D6 mRNA expression by 70%, only upon deletion of the rs5758550 region. These results demonstrate robust effects of both the enhancer element and SNP rs5758550 on CYP2D6 expression, supporting consideration of rs5758550 for CYP2D6 genotyping panels to yield more accurate phenotype prediction.
Center for Pharmacogenomics, School of Medicine, The Ohio State University, Columbus, OH 43210, USA wang.808@osu.edu.
Danxin
D
Wang
Center for Pharmacogenomics, School of Medicine, The Ohio State University, Columbus, OH 43210, USA.
Audrey C
AC
Papp
Center for Pharmacogenomics, School of Medicine, The Ohio State University, Columbus, OH 43210, USA.
Xiaochun
X
Sun
D000328:Adult; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D005260:Female; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D008297:Male; D008875:Middle Aged; D020641:Polymorphism, Single Nucleotide; D014158:Transcription, Genetic
D019389:Cytochrome P-450 CYP2D6