Gene set enrichment in eQTL data identifies novel annotations and pathway regulators.

Genome-wide gene expression profiling has been extensively used to generate biological hypotheses based on differential expression. Recently, many studies have used microarrays to measure gene expression levels across genetic mapping populations. These gene expression phenotypes have been used for genome-wide association analyses, an analysis referred to as expression QTL (eQTL) mapping. Here, eQTL analysis was performed in adipose tissue from 28 inbred strains of mice. We focused our analysis on "trans-eQTL bands", defined as instances in which the expression patterns of many genes were all associated to a common genetic locus. Genes comprising trans-eQTL bands were screened for enrichments in functional gene sets representing known biological pathways, and genes located at associated trans-eQTL band loci were considered candidate transcriptional modulators. We demonstrate that these patterns were enriched for previously characterized relationships between known upstream transcriptional regulators and their downstream target genes. Moreover, we used this strategy to identify both novel regulators and novel members of known pathways. Finally, based on a putative regulatory relationship identified in our analysis, we identified and validated a previously uncharacterized role for cyclin H in the regulation of oxidative phosphorylation. We believe that the specific molecular hypotheses generated in this study will reveal many additional pathway members and regulators, and that the analysis approaches described herein will be broadly applicable to other eQTL data sets.

Genomewide association analysis in diverse inbred mice: power and population structure.

The discovery of quantitative trait loci (QTL) in model organisms has relied heavily on the ability to perform controlled breeding to generate genotypic and phenotypic diversity. Recently, we and others have demonstrated the use of an existing set of diverse inbred mice (referred to here as the mouse diversity panel, MDP) as a QTL mapping population. The use of the MDP population has many advantages relative to traditional F(2) mapping populations, including increased phenotypic diversity, a higher recombination frequency, and the ability to collect genotype and phenotype data in community databases. However, these methods are complicated by population structure inherent in the MDP and the lack of an analytical framework to assess statistical power. To address these issues, we measured gene expression levels in hypothalamus across the MDP. We then mapped these phenotypes as quantitative traits with our association algorithm, resulting in a large set of expression QTL (eQTL). We utilized these eQTL, and specifically cis-eQTL, to develop a novel nonparametric method for association analysis in structured populations like the MDP. These eQTL data confirmed that the MDP is a suitable mapping population for QTL discovery and that eQTL results can serve as a gold standard for relative measures of statistical power.

Adenosine A(2A) receptors play a role in the pathogenesis of hepatic cirrhosis.

1. Adenosine is a potent endogenous regulator of inflammation and tissue repair. Adenosine, which is released from injured and hypoxic tissue or in response to toxins and medications, may induce pulmonary fibrosis in mice, presumably via interaction with a specific adenosine receptor. We therefore determined whether adenosine and its receptors contribute to the pathogenesis of hepatic fibrosis. 2. As in other tissues and cell types, adenosine is released in vitro in response to the fibrogenic stimuli ethanol (40 mg dl(-1)) and methotrexate (100 nM). 3. Adenosine A(2A) receptors are expressed on rat and human hepatic stellate cell lines and adenosine A(2A) receptor occupancy promotes collagen production by these cells. Liver sections from mice treated with the hepatotoxins carbon tetrachloride (CCl(4)) (0.05 ml in oil, 50 : 50 v : v, subcutaneously) and thioacetamide (100 mg kg(-1) in PBS, intraperitoneally) released more adenosine than those from untreated mice when cultured ex vivo. 4. Adenosine A(2A) receptor-deficient, but not wild-type or A(3) receptor-deficient, mice are protected from development of hepatic fibrosis following CCl(4) or thioacetamide exposure. 5. Similarly, caffeine (50 mg kg(-1) day(-1), po), a nonselective adenosine receptor antagonist, and ZM241385 (25 mg kg(-1) bid), a more selective antagonist of the adenosine A(2A) receptor, diminished hepatic fibrosis in wild-type mice exposed to either CCl(4) or thioacetamide. 6. These results demonstrate that hepatic adenosine A(2A) receptors play an active role in the pathogenesis of hepatic fibrosis, and suggest a novel therapeutic target in the treatment and prevention of hepatic cirrhosis.

Genetically based resistance to the antiinflammatory effects of methotrexate in the air-pouch model of acute inflammation.

OBJECTIVE: Low-dose methotrexate (MTX), a mainstay in the treatment of rheumatoid arthritis, is effective in only 60-70% of patients, a finding mirrored by poor antiinflammatory efficacy in some animal models, most notably collagen-induced arthritis. To determine whether genetic factors or the model itself is responsible for the poor response to MTX, we directly compared the responses of 4 inbred mouse strains to MTX in the air-pouch model of acute inflammation. METHODS: The exudate leukocyte count and adenosine concentration were determined in inbred mice treated with MTX (0.75 mg/kg intraperitoneally every week for 4 weeks) or vehicle 4 hours after injection of carrageenan into the air pouch using previously described methods. Quantitative trait locus mapping was performed using an in silico, or computer-based, method to identify loci potentially associated with each phenotype. RESULTS: MTX significantly reduced the exudate leukocyte count in C57BL/6J and BALB/cJ mice, but not DBA/1J (the strain used in the collagen-induced arthritis model) or DBA/2J mice. In a parallel manner, MTX increased adenosine concentration in inflammatory exudates of C57BL/6J and BALB/cJ mice, but not DBA/1J or DBA/2J mice. Antiinflammatory and adenosine responses to MTX in DBA/1J x C57BL/6J F(1) and F(2) offspring were most consistent with single genetic loci being responsible for each phenotype. In silico mapping identified partially overlapping loci containing candidate genes involved in both responses. CONCLUSION: Genetic factors contribute to the antiinflammatory efficacy of MTX, and a single locus involved in MTX-induced adenosine up-regulation is likely responsible for the observed resistance to MTX in DBA/1J mice.

An interaction between genetic factors and gender determines the magnitude of the inflammatory response in the mouse air pouch model of acute inflammation.

The widely used mouse air pouch model of acute inflammation is inducible in a variety of inbred strains, but the potential influence of genetic background and gender on inflammation severity has never been examined. We directly compared the degree of inflammation induced in the air pouch model across four commonly utilized inbred strains in both male and female mice. We then applied an in silico mapping method to identify loci potentially associated with determining inflammation severity for each gender. Air pouches were induced by subcutaneous injection 3 (3 cc) and 5 (1.5 cc) days prior to the experiment. 4h after carrageenan injection, exudates were retrieved and leukocyte concentration quantified using a hemocytometer. The in silico mapping method was applied as described below. The strain order for mean leukocyte count/mL in inflamed exudates differed between genders. In males, the order was C57BL/6J > BALB/cByJ > DBA/2J > DBA/1J, while in females the order was BALB/cByJ > DBA/2J > C57BL/6J > DBA/1J. The difference in inflammation severity between genders reached significance only in C57BL/6J mice. Independent in silico analysis based on phenotypic data from male versus female mice identified distinct sets of loci as potentially associated with the exudate count reached. We conclude that the degree of inflammation induced in the mouse air pouch model of inflammation is strain-specific and, therefore, genetically based, and the pattern of interstrain differences is altered in male relative to female mice. The loci identified by in silico mapping likely contain genes with differential roles in determining this phenotype between genders.