This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Background: Deficiencies in dihydropyrimidine dehydrogenase (DPD) and other enzymes of the pyrimidine catabolic pathway is critical in the predisposition to severe, unanticipated 5-fluorouracil (5-FU) toxicity. Rationale: We, and others, have linked molecular defects in the DPYD gene (the gene that encodes the DPD enzyme) to reduced enzyme activity. This decreased enzyme activity has been shown by our laboratory and others to result in decreased 5-FU clearance and increased 5-FU exposure. Other investigators have reported similar observations with the other enzymes of the pyrimidine catabolic pathway. Specifically, life-threatening 5-FU toxicities have been observed in cancer patients with dihydropyrimidinase (DPYS) and beta-ureidopropionate (BUP) deficiencies. Study Objectives: The long-term objective of this research project is to better understand the genetic basis for severe, potentially life-threatening toxicity secondary to treatment with 5-Fluorouracil (5-FU). To accomplish this goal, we will a) continue to further characterize the pharmacogenetic syndrome of DPD deficiency and b) characterize DPYD and BUP deficiencies. To accomplish these goals we will continue to obtain and utilize biochemical and molecular data (e.g., enzyme activity, mRNA levels, and analysis for mutations in pyrimidine catabolic genes) from patients presenting with grade IV toxicity after 5-FU therapy. In particular, we will examine: 1) the role of additional transcriptional regulatory elements affecting DPYD gene expression including identification of a) transcription factor(s) that bind to regulatory elements I and II in the previously identified promoter, and b) additional potential regulatory regions in introns 1 and the 3'-untranslated region; 2)-Determine the role of the ubiquitin (Ub)-proteasome system in the regulation of DPD protein- a) determine DPD protein half-life for wild type and mutant DPD protein, and b) identify putative destabilizing element(s) of DPD protein; 3) Develop user-friendly diagnostic tests for DPD deficiency and other deficiencies of the pyrimidine catabolic pathway including a) phenotypic tests and b) genotypic tests; 4) the role of other factors which may contribute to severe 5-FU toxicity including a} altered gene expression of the 5-FU site of action - thymidylate synthase, b) altered gene expression of anabolic enzymes, e.g. uridine and thymidine phosphorylases and kinases and orotate phosphoribosyltransferase, and c) altered gene expression of other catabolic enzymes, e.g. dihydropyrimidinase and BUP. Utilization of GCRC Resources: The nursing and laboratory staff has been utilized to 1) perform blood collection from 5-FU toxic cancer patients (over 350 to date) 2) perform pharmacokinetic studies which examine pyrimidine catabolism in 5-FU toxic cancer patients (8 hours per patient; over 25 patients to date) 3) isolation of plasma by laboratory staff from 5-FU toxic cancer patients who have performed the pharmacokinetic study, 4) culturing and maintainence of fibroblasts from 5-FU toxic, DPD deficient, DPYS deficiency, and BUP deficient subjects/cancer patients. Study Population and Outcome: Successful progress on this research project should translate into improved care for patients receiving fluoropyrimidine drugs in the future.
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