Pharmacogenomics of Human P450 Oxidoreductase

描述（由申请人提供）：进入人体的大多数药物、外源性物质和环境污染物通过肝细胞内质网中的P450酶代谢。这种微粒体II型P450酶，包括参与肾上腺和性腺类固醇激素合成的几种酶，需要来自P450氧化还原酶（POR）的电子捐赠来介导催化。POR是一种680个氨基酸，78 kDa的蛋白质，含有两个黄素，黄素腺嘌呤二核苷酸部分（FAD）和黄素单核苷酸（FMN）。肝脏P450酶存在大量遗传变异。这种变异，加上药物转运蛋白和受体的遗传变异，解释了药物代谢中的一些但不是全部遗传变异。我们认为，药物代谢中一些无法解释的遗传变异可以用POR的遗传变异来解释。本实验室于2004年发现POR缺乏症是一种新的类固醇激素生物合成障碍。通过对患者DNA的分析，以及对大量正常个体的测序，我们已经鉴定出35种POR氨基酸序列变异。在我们最近对842名正常人的研究中，我们发现近28%的人类POR等位基因携带A503 V序列变体。体外试验表明，A503 V在基于细胞色素c和P450 c17的试验中活性降低，但在基于CYP 1A 2和CYP 2C 19的试验中活性未降低。对这些POR错义突变体与细胞色素c、类固醇生成P450 c17（CYP 17）以及药物代谢CYP 1A 2和CYP 2C 19的活性的测定表明，特定POR突变体与一种靶点P450的活性可能无法预测其与另一种P450酶的活性。因此，分析POR序列变体在药物代谢药物遗传学中的潜在作用既重要又复杂。我们假设POR的遗传变异有助于人类药物代谢的变化，轻度缺陷的POR序列变异可能与肝脏P450酶中其他无害的轻度缺陷变异协同作用，导致药物代谢的重要变化。为了开始描述POR对人类药物遗传学的贡献，我们提出了三个具体目标。目的1是确定A503 V POR变体和常见致病POR突变体A287 P和R457 H对肝脏药物代谢P450酶的催化活性。目的2是确定A503 V变体是否具有与肝脏药物代谢P450酶的常见变体的活性的选择性丧失（或获得）。目的3是表征POR基因启动子的活性，并确定其是否包含影响POR表达水平的多态性。这些研究将建立与主要药物代谢肝P450酶相关的POR变体的体外生物化学，并将允许我们设计研究以确定A503 V变体是否影响体内临床研究中的药物代谢。这些目标的完成将提供关于POR在人类药物遗传学中的潜在作用的急需信息。公共卫生相关性：不同种族背景的人如何代谢药物存在很大差异;了解这些差异对于优化临床环境中的药物剂量至关重要。组间的大部分差异是基于编码药物转运蛋白和药物代谢酶的基因的差异。这个项目探索了第三种可能性，一种叫做POR的基因，它是药物代谢酶活性所必需的。

DESCRIPTION (provided by applicant): Most drugs, xenobiotics and environmental pollutants that enter the human body are metabolized by P450 enzymes in the endoplasmic reticulum of liver cells. Such microsomal type II P450 enzymes, which include several enzymes involved in adrenal and gonadal steroid hormone synthesis, require electron donation from P450 oxidoreductase (POR) to mediate catalysis. POR is a 680 amino-acid, 78 kDa protein that contains two flavins, a flavin adenine dinucleotide moiety (FAD), and a flavin mononucleotide (FMN). There is substantial genetic variation in the hepatic P450 enzymes. This variation, plus the genetic variation in drug transporters and receptors, accounts for some, but not all, genetic variation in drug metabolism. We propose that some of this unexplained genetic variation in drug metabolism may be explained by genetic variation in POR. Our laboratory discovered POR deficiency as a new disorder of steroid hormone biosynthesis in 2004. Through analysis of patient DNAs, and sequencing of a large cohort of normal individuals, we have identified 35 POR amino acid sequence variants. In our recent study of 842 normal individuals, we discovered that nearly 28% of human POR alleles carry the A503V sequence variant. In vitro assays show that A503V has decreased activities in assays based on cytochrome c and P450c17, but not in assays based on CYP1A2 and CYP2C19. Assays of the activities of these POR missense mutants with cytochrome c, steroidogenic P450c17 (CYP17), and drug-metabolizing CYP1A2 and CYP2C19 have shown that the activity of a particular POR mutant with one target P450 may not predict its activity with another P450 enzyme. Thus the analysis of the potential role of POR sequence variants in the pharmacogenetics of drug metabolism is both important and complicated. We hypothesize that genetic variants of POR contribute to variation in human drug metabolism and that mildly defective POR sequence variants may synergize with otherwise innocuous, mildly defective variants in hepatic P450 enzymes to cause important variations in drug metabolism. To begin to characterize the contributions of POR to human pharmacogenetics, we propose three specific aims. Aim 1 is to determine the catalytic activities of the A503V POR variant and the common disease-causing POR mutants A287P and R457H with hepatic, drug-metabolizing P450 enzymes. Aim 2 is to determine whether the A503V variant has selective loss (or gain) of activity with common variants of hepatic, drug-metabolizing P450 enzymes. Aim 3 is to characterize the activity of the POR gene promoter and determine whether it contains polymorphisms that influence the level of POR expression. These studies will establish the in vitro biochemistry of POR variants in association with the principal drug-metabolizing hepatic P450 enzymes, and will permit us to design studies to determine whether the A503V variant affects drug metabolism in clinical studies in vivo. Completion of these aims will provide critically needed information about the potential role of POR in human pharmacogenetics. PUBLIC HEALTH RELEVANCE: There is substantial variation in how people of different ethnic backgrounds metabolize drugs; understanding such variations is crucial for optimizing drug dosing in clinical settings. Much of the variation between groups is based on variations in genes encoding drug transporters and drug-metabolizing enzymes. This project explores a third possibility, a gene called POR, that is needed for the activity of drug-metabolizing enzymes.