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.

Differential Allelic Expression in Leukoblast from Patients with Acute Myeloid Leukemia Suggests Genetic Regulation of CDA, DCK, NT5C2, NT5C3, and TP53

“…We did not observe higher expression for SLC29A1 in samples with C alleles for rs747199 as previously reported (Myers et al, 2006). Lower promoter activity has been shown for T alleles of rs532545 in CDA, which is consistent with our mRNA data in TTsamples for this variant (Fitzgerald et al, 2006;Gilbert et al, 2006). When subgroups of samples were compared for their correlation between genotypes and mRNA expression, statistically significant differences were observed in some cases.…”

“…Eighteen putative rSNPs in the 5Ј-region of our genes of interest were identified by bioinformatics tools or selected from the literature (Shi et al, 2004;Fitzgerald et al, 2006;Gilbert et al, 2006;Joerger et al, 2006;Myers et al, 2006;Sugiyama et al, 2007). Compared with sequencing of large upstream regions of genes, this method allows screening of thousands of kb in silico to make a selection of potential rSNPs in either proximal promoters or more distant candidate enhancers (Wasserman and Sandelin, 2004).…”

Pyrimidine pool imbalance induced by BLM helicase deficiency contributes to genetic instability in Bloom syndrome

“…CDA promoter cloning and luciferase reporter assays. The CDA promoter 33 was inserted into a pGL4.14 vector (Promega), upstream from the firefly luciferase gene. Cells were transfected by incubation for 72 h with 2.5 µg of the CDA promoter-containing plasmid and 0.25 µg of pGL4.74 vector (Promega, internal control, Renilla luciferase) in the presence of JetPEI reagent.…”

Pharmacogenomics of gemcitabine: can genetic studies lead to tailor-made therapy?

“…Remarkable reduction in activity of 70Thr CDA was reported in vitro (Yue et al, 2003) and in vivo (Sugiyama et al, 2007), while only marginal reduction in activity of 27Gln CDA was observed in vitro (Yue et al, 2003;Gilbert et al, 2006). On the other hand, Fitzgerald et al (2006) investigated SNPs in the promoter region of CDA in vitro and in vivo, and found that some promoter CDA haplotypes might affect CDA activity.…”