Cytosolic 5′-nucleotidase III (NT5C3): gene sequence variation and functional genomics

• Published in: Pharmacogenetics and genomics Vol. 19; no. 8; pp. 567 - 576
• Main Authors: Aksoy, Pinar, Zhu, Min Jia, Kalari, Krishna R, Moon, Irene, Pelleymounter, Linda L, Eckloff, Bruce W, Wieben, Eric D, Yee, Vivien C, Weinshilboum, Richard M, Wang, Liewei
• Format: Journal Article
• Language: English
• Published: Hagerstown, MD Lippincott Williams & Wilkins, Inc 01-08-2009; Lippincott Williams & Wilkins
• Subjects: 5'-Nucleotidase - genetics; Animals; Antimetabolites, Antineoplastic - pharmacology; Biological and medical sciences; Cell Line, Tumor; Cercopithecus aethiops; COS Cells; Cytarabine - pharmacology; Deoxycytidine - analogs & derivatives; Deoxycytidine - pharmacology; DNA - chemistry; General pharmacology; Genetic Variation; Glycoproteins - genetics; Haplotypes; Humans; Kinetics; Medical sciences; Models, Genetic; Pharmacogenetics - methods; Pharmacokinetics. Pharmacogenetics. Drug-receptor interactions; Pharmacology. Drug treatments; Antineoplastic agent; Pharmacogenetics; 5'-Nucleotidase; Gemcitabine; Enzyme; cytosine arabinoside; Phosphoric monoester hydrolases; cytosolic 5'-nucleotidase III; Esterases; Gene; Hydrolases; Cytosine; Single nucleotide polymorphism; single nucleotide polymorphisms; Functional genomics; Pharmacogenomics
• ISSN: 1744-6872; 1744-6880
• DOI: 10.1097/FPC.0b013e32832c14b8

BACKGROUND5′-Nucleotidases play a critical role in nucleotide pool balance and in the metabolism of nucleoside analogs such as gemcitabine and cytosine arabinoside (AraC). We previously performed an expression array association study with gemcitabine and AraC cytotoxicity using 197 human lymphoblastoid cell lines. One gene that was significantly associated with gemcitabine cytotoxicity was a nucleotidase family member, NT5C3. Very little is known with regard to the pharmacogenomics of this family of enzymes.
METHODSWe set out to identify common genetic variation in NT5C3 by resequencing the gene and to determine the effect of that variation on NT5C3 protein function and potential effect on response to cytidine analogs. We identified 61 NT5C3 polymorphisms, 48 of which were novel, by resequencing 240 ethnically defined DNA samples. Functional studies were performed with one nonsynonymous (G847C, Asp283His) and four synonymous cSNPs (T9C, C276T, T306C, and G759A),as well as three combined variants (T276/His283, T276/C306, T276/C9).
RESULTSThe His283 and T276/His283 constructs showed decreased levels of enzyme activity and protein. Substrate kinetic analysis showed no significant differences in Km values between wild type and His283 when cytidine monophosphate, AraCMP, and GemMP were used as substrates. An association study between single nucleotide polymorphisms (SNPs) and NT5C3 expression in the 240 cell lines from which DNA was extracted to resequence NT5C3 identified four SNPs that were significantly associated with NT5C3 expression. Electrophoretic mobility shift assays showed that two of those SNPs, I4(−114) and I6(9), altered DNA–protein binding patterns. These findings suggest that genetic variation in NT5C3 might affect protein function and potentially influence drug response.