DIHYDROPYRIMIDINE DEHYDROGENASE (DPD) DEFICIENCY IN POPULATION STUDIES

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。背景资料：二氢嘧啶脱氢酶（DPD）和嘧啶分解代谢途径的其他酶的缺陷在重度、非预期5-氟尿嘧啶（5-FU）毒性的易感性中至关重要。基本原理：我们和其他人已经将DPYD基因（编码DPD酶的基因）中的分子缺陷与酶活性降低联系起来。我们的实验室和其他实验室已经证明，这种酶活性降低会导致5-FU清除率降低和5-FU暴露量增加。其他研究者报道了嘧啶分解代谢途径的其他酶的类似观察结果。具体而言，在二氢嘧啶酶（DPYS）和β-脲基丙酸酯（BUP）缺乏的癌症患者中观察到危及生命的5-FU毒性。研究目的：本研究项目的长期目标是更好地了解5-氟尿嘧啶（5-FU）治疗继发的严重、可能危及生命的毒性的遗传基础。为了实现这一目标，我们将a）继续进一步表征DPD缺乏的药物遗传学综合征，B）表征DPYD和BUP缺乏。为了实现这些目标，我们将继续获得和利用生物化学和分子数据（例如，酶活性、mRNA水平和嘧啶分解代谢基因突变的分析）。具体地说，我们将研究：1）影响DPYD基因表达的另外的转录调控元件的作用，包括鉴定a）与先前鉴定的启动子中的调控元件I和II结合的转录因子，和B）内含子1和3 '-非翻译区中的另外的潜在调控区; 2）-确定泛素（U B）-蛋白酶体系统在DPD蛋白调节中的作用- a）确定野生型和突变型DPD蛋白的DPD蛋白半衰期，和B）鉴定DPD蛋白的推定的不稳定元件; 3）开发用于DPD缺乏和嘧啶分解代谢途径的其他缺乏的用户友好的诊断测试，包括a）表型测试和B）基因型测试; 4）可能导致严重5-FU毒性的其他因素的作用，包括：a）改变5-FU作用位点-胸苷酸合酶的基因表达，B）改变的合成代谢酶的基因表达，例如尿苷和胸苷磷酸化酶和激酶以及乳清酸磷酸核糖基转移酶，和c）改变的其它分解代谢酶的基因表达，例如二氢嘧啶酶和BUP。GCRC资源的利用：护理和实验室人员已被用于1）从5-FU中毒的癌症患者中进行血液采集（迄今为止超过350例）2）进行药代动力学研究，检查5-FU毒性癌症患者中的嘧啶催化剂（每例患者8小时;迄今为止超过25名患者）3）由实验室工作人员从进行了药代动力学研究的5-FU毒性癌症患者中分离血浆，4）来自5-FU毒性、DPD缺陷、DPYS缺陷和BUP缺陷受试者/癌症患者的成纤维细胞的培养和传代。研究人群和结局：该研究项目的成功进展应转化为未来接受氟嘧啶药物的患者的护理改善。

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.