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.
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.
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