DIHYDROPYRIMIDINE DEHYDROGENASE (DPD) DEFICIENCY IN POPULATION STUDIES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 背景：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5-氟尿嘧啶(5-FU)严重毒性的易感性中起关键作用。原理：我们和其他人将DPYD基因(编码DPD酶的基因)的分子缺陷与酶活性降低联系在一起。我们的实验室和其他实验室已经证明，这种酶活性的降低会导致5-FU清除量的降低和5-FU暴露的增加。其他研究人员也报道了与嘧啶分解代谢途径的其他酶类似的观察结果。具体地说，在二氢嘧啶酶(DPYS)和β-尿苷丙酸(BUP)缺乏的癌症患者中观察到了危及生命的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)治疗后继发的严重、潜在威胁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缺乏的药物遗传综合征，b)表征DPYD和BUP缺陷。为了实现这些目标，我们将继续从5-FU治疗后出现IV级毒性的患者那里获得和利用生化和分子数据(例如，酶活性、mRNA水平和嘧啶分解代谢基因突变分析)。特别是，我们将研究：1)其他影响DPYD基因表达的转录调控元件的作用，包括a)与先前已确定的启动子中的调节元件I和II结合的转录因子(S)，以及b)内含子1和3‘-非翻译区中的额外潜在调节区；2)确定泛素(Ub)-蛋白酶体系统在调节Dpd蛋白中的作用-a)确定野生型和突变型Dpd蛋白的半衰期，以及b)确定Dpd蛋白的可能的不稳定元件(S)；3)针对DPD缺乏症和其他嘧啶分解代谢途径缺陷的诊断方法，包括a)表型测试和b)基因测试；4)其他可能导致5-FU严重毒性的因素的作用，包括a)5-FU作用位点-胸苷合成酶基因表达的变化，b)合成代谢酶基因表达的变化，例如尿苷和胸苷磷酸化酶、激酶和旋转磷酸核糖转移酶，以及c)其他分解代谢酶基因表达的变化，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毒性癌症患者(迄今已超过350人)采集血液，2)进行药代动力学研究，检测5-FU毒性癌症患者(每名患者8小时；迄今超过25名患者)的嘧啶分解代谢，3)实验室工作人员从已进行药代动力学研究的5-FU毒性癌症患者身上分离血浆，4)培养和维持5-FU毒性癌症患者、DPD缺乏、DPYS缺陷和BUP缺乏的受试者/癌症患者的成纤维细胞。研究人群和结果：这项研究项目的成功进展将转化为未来对接受氟嘧啶药物治疗的患者的改善护理。

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.