ABSTRACT: To identify the genes and pathways that underlie cardiovascular and metabolic phenotypes we performed an integrated analysis of a mouse C57BL/6J x A/J F2 (B6AF2) cross by relating genome-wide gene expression data from adipose, kidney, and liver tissues to physiological endpoints measured in the population. We have identified a large number of trait QTLs including loci driving variation in cardiac function on chromosomes 2 and 6 and a hotspot for adiposity, energy metabolism, and glucose traits on chromosome 8. Integration of adipose gene expression data identified a core set of genes that drive the chromosome 8 adiposity QTL. This chromosome 8 trans eQTL signature contains genes associated with mitochondrial function and oxidative phosphorylation and maps to a subnetwork with conserved function in humans that was previously implicated in human obesity. In addition, human eSNPs corresponding to orthologous genes from the signature show enrichment for association to type II diabetes in the DIAGRAM cohort, supporting the idea that the chromosome 8 locus perturbs a molecular network that in humans senses variations in DNA and in turn affects metabolic disease risk. We functionally validate predictions from this approach by demonstrating metabolic phenotypes in knockout mice for three genes from the trans eQTL signature, Akr1b8, Emr1, and Rgs2. In addition we show that the transcriptional signatures for knockout of two of these genes, Akr1b8 and Rgs2, map to the F2 network modules associated with the chromosome 8 trans eQTL signature and that these modules are in turn very significantly correlated with adiposity in the F2 population. Overall this study demonstrates how integrating gene expression data with QTL analysis in a network-based framework can aid in the elucidation of the molecular drivers of disease that can be translated from mice to humans. An F2 population was derived from a C57BL/6J x A/J mouse cross (B6AF2) and tissues were collected in 360 male and female progeny for microarray analysis. RNA extraction, probe preparation, and array hybridizations were all carried out at the Rosetta Inpharmatics Gene Expression Laboratory (Seattle, WA). Mouse tissues (gonadal adipose, kidney medulla, kidney cortex, hypothalamus) were pulverized prior to homogenization in a solution of GITC/BME (1:50 ratio) using a Covaris S2 cryo-prep (Covaris, Inc, Woburn, MA), followed by addition of a TRIzol water solution (4:1 ratio). 100% Chloroform was added to the TRIzol/GITC lysate (1:5 ratio) to facilitate separation of the organic and aqueous components using the phaselock (Eppendorf) system. The aqueous supernatant was further purified using a Promega SV-96 total RNA kit (Promega, Madison, WI), incorporating a DNase treatment. Total RNA samples were assayed for quality using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA) and for yield using Ribogreen (Ambion, Austin, TX) metrics prior to amplification. All samples, with the exception of kidney medulla, were amplified and labeled using a custom automated version of a 5 µg RT/IVT protocol and hybridizations to custom Agilent microarrays were performed as described [58]. The custom ink-jet microarrays used in this study were manufactured by Agilent Technologies and consisted of 4,732 control probes and 39,558 non-control oligonucleotides derived from mouse Unigene clusters, combined with RefSeq sequences and RIKEN full-length cDNA clones. For each individual animal tissue sample, labeled complementary RNA (cRNA) was hybridized against a pool of labeled cRNAs constructed from equal aliquots of RNA for that specific tissue from at least 200 individuals.
