Abstract
The human concentrative nucleoside transporter 2 (CNT2) plays an important role in the absorption, disposition, and biological effects of endogenous nucleosides and nucleoside analog drugs. We identified genetic variation in the basal promoter region of CNT2 and characterized the function of the variants. We screened DNA from an ethnically diverse population and identified five basal promoter variants in CNT2. Three major haplotypes in the CNT2 basal promoter region were identified and were found at different allele frequencies in various ethnic groups. The common promoter variants and haplotypes were constructed and characterized for their promoter activity using luciferase reporter assays. One polymorphic variant, rs2413775 (-146T>A), with an allele frequency >20% in all populations, showed a gain of function in luciferase activity. Furthermore, in vivo mouse promoter assays of these nucleotide variants using the hydrodynamic tail vein injection, leading to their expression in the liver, demonstrated similar results. Transcription factor binding site (TFBS) analysis indicated this variant alters a hepatic nuclear factor (HNF) 1 TFBS. Electrophoretic mobility shift assay demonstrated stronger binding of HNF1α and weaker binding of HNF1β to the -146T and -146A regions, whereas the single nucleotide polymorphism (SNP), -146A, exhibited enhanced binding to both HNF1α and HNF1β, consistent with its greater activity in reporter assays. The data collectively suggest that the common variant, -146T>A, in the proximal promoter of CNT2 may result in an enhanced transcription rate of the gene and, thus, expression levels of CNT2. This SNP may play a role in variation in the pharmacokinetics and pharmacological effects of nucleoside analogs.
Footnotes
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This work was supported in part by the National Institutes of Health National Institute of General Medical Sciences [Grant GM61390]; by the National Institutes of Health National Cancer Institute [Grant P50-CA58207]; and by the Sandler Family Supporting Foundation.
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A grant from the New Energy and Industrial Technology Development Organization supported some of the methods of development in this study.
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Data showing allele frequencies of CNT2 promoter variants are available at http://www.pharmgkb.org.
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doi:10.1124/jpet.108.147207.
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ABBREVIATIONS: CNT, concentrative nucleoside transporter; SNP, single nucleotide polymorphism; bp, base pair; HNF, hepatic nuclear factor; SOPHIE, Studies of Pharmacogenetics in Ethnically Diverse Populations; PCR, polymerase chain reaction; EMSA, electrophoretic mobility shift assay; TSS, transcription start site; LD, linkage disequilibrium.
- Received October 8, 2008.
- Accepted December 16, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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