Received for publication December 13, 2007.
Revised January 25, 2008.
Accepted for publication February 28, 2008.

Novel CYP2C9 Promoter Variants and Assessment of Their Impact on Gene Expression

Abstract

A considerable number of reports identifying and characterizing genetic variants within the CYP2C9 coding region have appeared. Much less is known about polymorphic promoter sequences that also might contribute to interindividual differences in CYP2C9 expression. To address this problem, approximately 10,000 bp of CYP2C9 upstream information was re-sequenced using 24 DNA samples from the Coriell Polymorphism Discovery Resource. Thirty-one single nucleotide polymorphisms (SNPs) were identified; nine were novel while 22 were previously reported. Using both sequencing and multiplex single-base extension, individual SNP frequencies were determined in 193 DNA samples obtained from unrelated, self-reported Hispanic Americans of Mexican descent and compared to similar data obtained from a non-Latino White cohort. Significant inter-ethnic differences were observed in several SNP frequencies, some of which appeared unique to the Hispanic population. Analysis using PHASE 2.1 inferred nine common (>1%) variant haplotypes, two of which included the g.3608C>T (R144C) CYP2C9*2 and two the g.42614A>C (I359L) CYP2C9*3 SNPs. Haplotype variants were introduced into a CYP2C9/luciferase reporter plasmid using site-directed mutagenesis and the impact of the variants on promoter activity assessed by transient expression in HepG2 cells. Both constitutive and PXR-mediated inducible activities were measured. Haplotypes 1B, 3A, and 3B each exhibited a 65% decrease in constitutive promoter activity relative to the reference haplotype. Haplotypes 1D and 3B exhibited a 50% decrease and a 40% increase in induced promoter activity, respectively. These data suggest that genetic variation within CYP2C9 regulatory sequences likely contributes to differences in CYP2C9 phenotype both within and among different populations.

Key words: Promoter analysis, Pharmacogenomic analyses, Mutagenesis/Chimeric approaches, Regulation of gene expression, Cytochrome P450, Genetics, Regulation - transcriptional