Genetic Predictors of Human Liver CYP Expression & Activity

描述（由申请人提供）：本研究的长期目标是了解四个候选基因的遗传变异如何影响肝细胞色素P450 （CYP）活性和mRNA表达。这是必要的，因为尽管在鉴定顺式CYP核苷酸多样性方面付出了相当大的努力，但宿主对药物治疗的反应和毒性仍然存在无法解释的变异性，这受到肝脏CYP基因介导的药物清除的显著影响。我们认为CYPs的表达实际上是多种多态性相互作用调控基因表达和酶活性的综合作用的复杂性状。我们提出，人类肝脏GWAS的最新结果与基于系统生物学的网络分析相结合，通过识别负责肝脏CYP变异性的新的候选基因，在药物遗传学研究领域创造了前所未有的机会。具体来说，最近与我们的合作者进行的一项研究利用网络和途径分析工具阐明了主要的CYP网络和上游调节因子，其遗传变异会干扰下游调节CYP的表达。本提案的主要目标是扩展这些新的初步发现，以确定与CYP表达/活性改变相关的候选基因的功能变异。我们的主要方法将是人类肝脏的历史表型到基因型分析。在这种方法中，我们的最初目标是使用肝脏基因表达和活性作为表型测量，并对表型样品进行深度重测序，特别是在表达/活性的表型分布的尾部，并确定导致表型变异性的变异。对大量多样的表型样本的需求通常是实施这种方法的限制因素。然而，我们有700多个肝脏样本的组织。最近，数百个这样的肝脏参与了一项基因组广泛关联研究（GWAS #1），其中包括九种CYP活性的表型分析。这个应用是独一无二的，因为我们也有广泛的肝脏表型，通过独立的GWAS #2和3肝脏资源进行验证。我们使用多种实验方法从cdna的深度重测序开始鉴定候选基因遗传变异（方法1）。接下来的方法2和方法3确定了导致mRNA加工和转录改变的多态性，并最终改变了候选基因的mRNA表达。对于每种方法，我们检查了GWAS#1肝脏中候选基因mRNA表达、CYP活性和表达的多态性的关联，并测试了GWAS# 2、3肝脏中的复制。方法4进行必要的机制研究，以证明任何变体如何改变候选基因mRNA的表达。总的来说，这些目标将填补现有的知识空白，通过确定新的遗传因素可变CYP介导的药物代谢。

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand how genetic variation in four candidate genes influences hepatic cytochrome P450 (CYP) activity and mRNA expression. This is necessary because, despite considerable effort to identify cis CYP nucleotide diversity, there is still unexplained variability in host response to and toxicity from drug therapies that is significantly influenced by hepatic CYP gene mediated drug clearance. We propose that expression of CYPs is really a complex trait resulting from the combined effect of multiple polymorphisms interacting to regulate gene expression and enzyme activity. We propose that recent results from human liver GWAS integrated with systems biology based network analysis has created unprecedented opportunities in the pharmacogenetic research area by identifying novel candidate genes responsible for hepatic CYP variability. Specifically a recent study with our collaborators, exploited network and pathway analysis tools to illuminate the primary CYP network and the upstream regulators whose genetic variation perturbs expression of the downstream regulated CYPs. A major goal of this proposal is to extend these novel preliminary findings to identify the functional variation in candidate genes associated with altered CYP expression/activity. Our primary approach will be our historical phenotype to genotype analysis in human livers. In this approach, our initial goal is to use liver gene expression and activity as phenotypic measures and to deep resequence the phenotyped samples, particularly the tails of the phenotypic distribution in expression/activity and identify variants that account for phenotypic variability. The need for large and diverse sets of phenotyped samples is usually a limiting factor in implementing this approach. However, we have tissue from over 700 liver samples. Hundreds of these livers were recently part of a collaborative genome wide association study (GWAS #1) that included phenotyping for nine CYP activities. This application is unique in because we also have extensively phenotyped livers for validation available through independent GWAS #2 and 3 liver resources. We use a variety of experimental approaches to identify candidate gene genetic variation starting with deep resequencing of the cDNAs (Approach 1). This is followed in Approaches 2 and 3 that identify polymorphisms leading to altered mRNA processing and transcription, and ultimately altered mRNA expression of the candidate gene. For each approach we examine the association of polymorphisms to candidate gene mRNA expression and CYP activity and expression in liver GWAS#1 livers and test for replication in GWAS #2,3 livers. Approach 4 carries out required mechanistic studies to demonstrate how any variants change candidate gene mRNA expression. In total, these aims will fill an existing knowledge gap by identifying novel genetic contributors to variable CYP mediated drug metabolism. PUBLIC HEALTH RELEVANCE: CYPs oxidatively metabolize the majority of the orally effective drugs in use today. Nevertheless, despite many years of cis CYP pharmacogenetics, knowledge is still incomplete on the genetic factors influencing CYP mediated metabolism. This study is, for the first time, pursuing novel candidate gene regulators of CYPs identified through integration of pharmacogenetics with liver system biology. Successful completion of the studies proposed herein will improve public health by advancing "personalized medicine" through understanding of how polymorphisms in the candidate genes affect CYP expression and activity, and will ultimately improve genetically tailored patient dosing.