title: Identification of functional single nucleotide polymorphism haplotypes in the cytidine deaminase promoter.
abstract: Cytidine deaminase (CDA) hydrolytically deaminates and irreversibly deactivates the chemotherapeutic agent cytosine arabinoside (Ara-C), a deoxycytidine analog used for treatment of acute leukemias and lymphomas. To determine if single nucleotide polymorphisms (SNPs) in the promoter region of CDA affected gene expression, we sequenced approximately 1.6 Kb upstream of the CDA translation initiation site and containing the proximal promoter of CDA. We identified 6 SNPs; -92A>G, -205C>G, -451C>T, -897C>A, -1075A>G and -1181G>A. Based on predicted changes in transcription factor binding sites, three SNPs (-92A>G, -451C>T and -897C>A) were chosen for further investigation. The five haplotypes segregating in the population were cloned into a luciferase expression plasmid, transfected into Cos-1 cells and reporter activity measured at 24 and 48 h. Four haplotypes showed an average expression which was 2.5-fold higher at 24 h (P<0.0001) and 3.3-fold higher at 48 h (P<0.0001) than the lowest expressing haplotype. When reanalyzed as single SNP genotypes, the differences in expression were significant, except for -897 C/A, at 24 h, but the magnitude of difference was reduced, suggesting that no single SNP completely accounts for the expression differences observed at the haplotype level. As predicted from the in vitro analysis, individuals homozygous for common haplotype (ACC/ACC) showed higher levels of CDA enzymatic activity as individuals heterozygous for the wild type and low expressing haplotype (ACC/ATC). As CDA promoter region haplotypes may influence Ara-C chemosensitivity, shown here in in vitro and in vivo studies, the clinical relevance of these findings should be examined.
authors.affiliation: Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261, USA. smfitzge@hotmail.com
authors.forename: Sara M
authors.initials: SM
authors.lastname: Fitzgerald
authors.forename: Rakesh K
authors.initials: RK
authors.lastname: Goyal
authors.forename: William R A
authors.initials: WR
authors.lastname: Osborne
authors.forename: Jennifer D
authors.initials: JD
authors.lastname: Roy
authors.forename: John W
authors.initials: JW
authors.lastname: Wilson
authors.forename: R E
authors.initials: RE
authors.lastname: Ferrell
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003564:Cytidine Deaminase / Q000235:genetics*; D005786:Gene Expression Regulation; D006239:Haplotypes; D006801:Humans; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D014162:Transfection
chemical_list: D003564:Cytidine Deaminase