Genetic variants that are associated with common human diseases do not lead directly to disease, but instead act on intermediate, molecular phenotypes that in turn induce changes in higher-order disease traits. Therefore, identifying the molecular phenotypes that vary in response to changes in DNA and that also associate with changes in disease traits has the potential to provide the functional information required to not only identify and validate the susceptibility genes that are directly affected by changes in DNA, but also to understand the molecular networks in which such genes operate and how changes in these networks lead to changes in disease traits. Toward that end, we profiled more than 39,000 transcripts and we genotyped 782,476 unique single nucleotide polymorphisms (SNPs) in more than 400 human liver samples to characterize the genetic architecture of gene expression in the human liver, a metabolically active tissue that is important in a number of common human diseases, including obesity, diabetes, and atherosclerosis. This genome-wide association study of gene expression resulted in the detection of more than 6,000 associations between SNP genotypes and liver gene expression traits, where many of the corresponding genes identified have already been implicated in a number of human diseases. The utility of these data for elucidating the causes of common human diseases is demonstrated by integrating them with genotypic and expression data from other human and mouse populations. This provides much-needed functional support for the candidate susceptibility genes being identified at a growing number of genetic loci that have been identified as key drivers of disease from genome-wide association studies of disease. By using an integrative genomics approach, we highlight how the gene RPS26 and not ERBB3 is supported by our data as the most likely susceptibility gene for a novel type 1 diabetes locus recently identified in a large-scale, genome-wide association study. We also identify SORT1 and CELSR2 as candidate susceptibility genes for a locus recently associated with coronary artery disease and plasma low-density lipoprotein cholesterol levels in the process.

We have identified a region in the 5' flanking sequence of the glutathione S-transferase (RX:glutathione Rtransferase, EC 2.5.1.18) Ya subunit gene that contains a unique xenobiotic-responsive element (XRE). The regulatory region spans nucleotides -722 to -682 of the 5' flanking sequence and is responsible for part of the basal level as well as inducible expression of the Ya subunit gene by planar aromatic compounds such as P-naphthoflavone ((3-NF) and 3-methylcholanthrene. The DNA sequence of this region (J3-NF-responsive element) is distinct from the DNA sequence ofthe XRE found in the cytochrome P-450 IAl gene. In addition to the region containing the fi-NF-responsive element, two other regulatory regions of the Ya subunit gene have been identified. One region spans nucleotides -867 to -857 and has a DNA sequence with identity to the hepatocyte nuclear factor 1 recognition motif found in several liver-specific genes. The second region spans nucleotides -908 to -899 and contains a DNA sequence with identity to the XRE found in the cytochrome P450 IA1 gene. The XRE sequence also contributes to part of the responsiveness of the Ya subunit gene to planar aromatic compounds. Our data suggest that regulation of gene expression by planar aromatic compounds can be mediated by a DNA sequence that is distinct from the XRE sequence.The glutathione S-transferases (RX:glutathione R-transferase, EC 2.5.1.18) represent a family of proteins that catalyze the conjugation of glutathione to a variety of electrophiles. In addition, they bind with high affinity a number of exogenous hydrophobic compounds, such as heme, bilirubin, dexamethasone, and polycyclic aromatic hydrocarbons (1-7). The cytosolic forms are heterodimers or homodimers and are composed of at least seven subunits (8)(9)(10)(11)(12).Previous work from our laboratory has included the construction and characterization of cDNA clones complementary to the rat liver Ya, Yb1, Yb2, One region, which is required for basal level expression, has a DNA sequence with identity to the hepatocyte nuclear factor 1 (HNF1) recognition sequence (20, 21). The second region has a DNA sequence with identity to the xenobioticresponsive element (XRE) core sequence found in the planar aromatic hydrocarbon-responsive cytochrome P450 IA1 gene (22, 23). MATERIALS AND METHODSTissue Culture and Cell Lines. Human hepatoma cells, HepG2, were maintained in Eagle's minimum essential medium (Hazleton Laboratories, Lenexa, KS) with nonessential amino acids, sodium pyruvate, 1o (vol/vol) fetal bovine serum, penicillin (10 units/ml), and streptomycin (10 units/ ml) at 370C in humidified 7% C02/93% air. The class II variant mouse cell line, obtained from James Whitlock (Stanford University), was maintained in minimal essential medium (a-MEM) with L-glutamine (Hazleton Laboratories), 10%6 fetal bovine serum, penicillin (10 units/ml), and streptomycin (10 units/ml) at 370C in humidified 7% C02/93% air.Construction of Deletion Mutants of the 5' Flanking Sequence of the Ya Subunit Gene. The initia...

Liver cytochrome P450s (P450s) play critical roles in drug metabolism, toxicology, and metabolic processes. Despite rapid progress in the understanding of these enzymes, a systematic investigation of the full spectrum of functionality of individual P450s, the interrelationship or networks connecting them, and the genetic control of each gene/enzyme is lacking. To this end, we genotyped, expression-profiled, and measured P450 activities of 466 human liver samples and applied a systems biology approach via the integration of genetics, gene expression, and enzyme activity measurements. We found that most P450s were positively correlated among themselves and were highly correlated with known regulators as well as thousands of other genes enriched for pathways relevant to the metabolism of drugs, fatty acids, amino acids, and steroids. Genome-wide association analyses between genetic polymorphisms and P450 expression or enzyme activities revealed sets of SNPs associated with P450 traits, and suggested the existence of both cis-regulation of P450 expression (especially for CYP2D6) and more complex trans-regulation of P450 activity. Several novel SNPs associated with CYP2D6 expression and enzyme activity were validated in an independent human cohort. By constructing a weighted coexpression network and a Bayesian regulatory network, we defined the human liver transcriptional network structure, uncovered subnetworks representative of the P450 regulatory system, and identified novel candidate regulatory genes, namely, EHHADH, SLC10A1, and AKR1D1. The P450 subnetworks were then validated using gene signatures responsive to ligands of known P450 regulators in mouse and rat. This systematic survey provides a comprehensive view of the functionality, genetic control, and interactions of P450s.

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