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1.  The genetic architecture of NAFLD among inbred strains of mice 
eLife  null;4:e05607.
To identify genetic and environmental factors contributing to the pathogenesis of non-alcoholic fatty liver disease, we examined liver steatosis and related clinical and molecular traits in more than 100 unique inbred mouse strains, which were fed a diet rich in fat and carbohydrates. A >30-fold variation in hepatic TG accumulation was observed among the strains. Genome-wide association studies revealed three loci associated with hepatic TG accumulation. Utilizing transcriptomic data from the liver and adipose tissue, we identified several high-confidence candidate genes for hepatic steatosis, including Gde1, a glycerophosphodiester phosphodiesterase not previously implicated in triglyceride metabolism. We confirmed the role of Gde1 by in vivo hepatic over-expression and shRNA knockdown studies. We hypothesize that Gde1 expression increases TG production by contributing to the production of glycerol-3-phosphate. Our multi-level data, including transcript levels, metabolite levels, and gut microbiota composition, provide a framework for understanding genetic and environmental interactions underlying hepatic steatosis.
eLife digest
Non-alcoholic fatty liver disease is a major health problem worldwide and is caused by an abnormal build-up of fat molecules in liver cells that disrupts how the cells work. Although many people with the disease show only mild or no symptoms, if the disease progresses the consequences—such as organ damage and an increased risk of liver cancer—can be severe.
Although non-alcoholic fatty liver disease has been linked with obesity and diabetes, how it develops is poorly understood. The most widely supported explanation suggests that the disease begins with an imbalance in the process that normally maintains the correct amount of fat molecules called triglycerides inside cells. As a result, triglycerides accumulate in the liver cells in a process known as steatosis, which is then thought to make the liver vulnerable to further problems. However, this theory has been questioned by genetic experiments that suggest triglyceride build-up actually protects cells from other kinds of damage.
Hui et al. studied mice that had been fed a diet that was high in fat and sugar. The extent of liver steatosis varied considerably between the mice, with some mice accumulating 30 times more triglyceride in their liver than others. The underlying variation in the genes of the mice was then examined to investigate whether this can explain the differences in liver condition. This revealed at least three DNA stretches that appear to be linked to triglyceride accumulation in the liver, including several genes that appear to be active during steatosis. One of these genes, known as Gde1, had not previously been shown to have a role in controlling how cells make and use triglycerides.
To confirm the role of Gde1, Hui et al. artificially turned the gene on in some mice and prevented it from turning on in others. Turning on Gde1 significantly increased the amount of triglyceride in the liver and keeping it turned off decreased triglyceride levels. Hui et al. suggest that this is because Gde1 helps to make a precursor molecule that is needed to build triglycerides. Certain gut bacteria also appear to be linked to steatosis.
This study used a population-based approach in mice to examine genetic factors in the development of fatty liver disease. The challenge now is to find out how the genes work and to understand their interactions with each other and with the environment.
PMCID: PMC4493743  PMID: 26067236
hepatic steatosis; genome-wide association; transcriptome; microbiome; metabolome; mouse
2.  Genetic regulation of human adipose microRNA expression and its consequences for metabolic traits 
Human Molecular Genetics  2013;22(15):3023-3037.
The genetics of messenger RNA (mRNA) expression has been extensively studied in humans and other organisms, but little is known about genetic factors contributing to microRNA (miRNA) expression. We examined natural variation of miRNA expression in adipose tissue in a population of 200 men who have been carefully characterized for metabolic syndrome (MetSyn) phenotypes as part of the Metabolic Syndrome in Men (METSIM) study. We genotyped the subjects using high-density single-nucleotide polymorphism microarrays and quantified the mRNA abundance using genome-wide expression arrays and miRNA abundance using next-generation sequencing. We reliably quantified 356 miRNA species that were expressed in human adipose tissue, a limited number of which made up most of the expressed miRNAs. We mapped the miRNA abundance as an expression quantitative trait and determined cis regulation of expression for nine of the miRNAs and of the processing of one miRNA (miR-28). The degree of genetic variation of miRNA expression was substantially less than that of mRNAs. For the majority of the miRNAs, genetic regulation of expression was independent of the expression of mRNA from which the miRNA is transcribed. We also showed that for 108 miRNAs, mapped reads displayed widespread variation from the canonical sequence. We found a total of 24 miRNAs to be significantly associated with MetSyn traits. We suggest a regulatory role for miR-204-5p which was predicted to inhibit acetyl coenzyme A carboxylase β, a key fatty acid oxidation enzyme that has been shown to play a role in regulating body fat and insulin resistance in adipose tissue.
PMCID: PMC3699064  PMID: 23562819
3.  Genetic regulation of mouse liver metabolite levels 
Molecular Systems Biology  2014;10(5):730.
We profiled and analyzed 283 metabolites representing eight major classes of molecules including Lipids, Carbohydrates, Amino Acids, Peptides, Xenobiotics, Vitamins and Cofactors, Energy Metabolism, and Nucleotides in mouse liver of 104 inbred and recombinant inbred strains. We find that metabolites exhibit a wide range of variation, as has been previously observed with metabolites in blood serum. Using genome‐wide association analysis, we mapped 40% of the quantified metabolites to at least one locus in the genome and for 75% of the loci mapped we identified at least one candidate gene by local expression QTL analysis of the transcripts. Moreover, we validated 2 of 3 of the significant loci examined by adenoviral overexpression of the genes in mice. In our GWAS results, we find that at significant loci the peak markers explained on average between 20 and 40% of variation in the metabolites. Moreover, 39% of loci found to be regulating liver metabolites in mice were also found in human GWAS results for serum metabolites, providing support for similarity in genetic regulation of metabolites between mice and human. We also integrated the metabolomic data with transcriptomic and clinical phenotypic data to evaluate the extent of co‐variation across various biological scales.
PMCID: PMC4188043  PMID: 24860088
genome‐wide association; local eQTL; metabolome; QTL; transcriptome
4.  Intergenerational genomic DNA methylation patterns in mouse hybrid strains 
Genome Biology  2014;15(5):R68.
DNA methylation is a contributing factor to both rare and common human diseases, and plays a major role in development and gene silencing. While the variation of DNA methylation among individuals has been partially characterized, the degree to which methylation patterns are preserved across generations is still poorly understood. To determine the extent of methylation differences between two generations of mice we examined DNA methylation patterns in the livers of eight parental and F1 mice from C57BL/6J and DBA/2J mouse strains using bisulfite sequencing.
We find a large proportion of reproducible methylation differences between C57BL/6J and DBA/2J chromosomes in CpGs, which are highly heritable between parent and F1 mice. We also find sex differences in methylation levels in 396 genes, and 11% of these are differentially expressed between females and males. Using a recently developed approach to identify allelically methylated regions independently of genotypic differences, we identify 112 novel putative imprinted genes and microRNAs, and validate imprinting at the RNA level in 10 of these genes.
The majority of DNA methylation differences among individuals are associated with genetic differences, and a much smaller proportion of these epigenetic differences are due to sex, imprinting or stochastic intergenerational effects. Epigenetic differences can be a determining factor in heritable traits and should be considered in association studies for molecular and clinical traits, as we observed that methylation differences in the mouse model are highly heritable and can have functional consequences on molecular traits such as gene expression.
PMCID: PMC4076608  PMID: 24887417
5.  Unraveling inflammatory responses using systems genetics and gene-environment interactions in macrophages 
Cell  2012;151(3):658-670.
Many common diseases have an important inflammatory component mediated in part by macrophages. Here we used a systems genetics strategy to examine the role of common genetic variation in macrophage responses to inflammatory stimuli.
We examined genome-wide transcript levels in macrophages from 92 strains of the Hybrid Mouse Diversity Panel. We exposed macrophages to control media, bacterial lipopolysaccharide, or oxidized phospholipids. We performed association mapping under each condition and identified several thousand expression quantitative trait loci (eQTL), gene-by-environment interactions and several eQTL “hotspots” that specifically control LPS responses. We validated an eQTL hotspot in chromosome 8 using siRNA knock-down of candidate genes and identified the gene 2310061C15Rik, as a novel regulator of inflammatory responses in macrophages.
We have created a public database where the data presented here can be used as a resource for understanding many common inflammatory traits which are modeled in the mouse, and for the dissection of regulatory relationships between genes.
PMCID: PMC3513387  PMID: 23101632
6.  Network for Activation of Human Endothelial Cells by Oxidized Phospholipids: A Critical Role of Heme Oxygenase 1 
Circulation research  2011;109(5):e27-e41.
Ox-PAPC accumulates in atherosclerotic lesions, is pro-atherogenic, and influences the expression of over 1000 genes in endothelial cells.
To elucidate the major pathways involved in Ox-PAPC action, we conducted a systems analysis of endothelial cell gene expression after exposure to Ox-PAPC.
Methods and Results
We used the variable responses of primary endothelial cells from 149 individuals exposed to Ox-PAPC to construct a network consisting of 11 groups of genes or modules. Modules were enriched for a broad range of GO pathways, some of which have not been previously identified as major Ox-PAPC targets. Further validating our method of network construction, modules were consistent with relationships established by cell biology studies of Ox-PAPC effects on endothelial cells. This network provides novel hypotheses about molecular interactions, as well as candidate molecular regulators of inflammation and atherosclerosis. We validate several hypotheses based on network connections and genomic association. Our network analysis predicted that the hub gene CHAC1 was regulated by the ATF4 arm of the unfolded protein response pathway and here we showed that ATF4 directly activates an element in the CHAC1 promoter. We show that variation in basal levels of HMOX1 contribute to the response to Ox-PAPC, consistent with its position as a hub in our network. We also identified GPR39 as a regulator of HMOX1 levels and showed that it modulates the promoter activity of HMOX1. We further showed that OKL38/OSGN1, the hub gene in the blue module, is a key regulator of both inflammatory and anti-inflammatory molecules.
Our systems genetics approach has provided a broad view of the pathways involved in the response of endothelial cells to Ox-PAPC and also identified novel regulatory mechanisms.
PMCID: PMC3163234  PMID: 21737788
Endothelial Cells; Oxidized Phospholipids; Gene Expression; Heme Oxygenase; Network; Systems Genetics; Genome-Wide Association Studies
7.  NF-E2–Related Factor 2 Promotes Atherosclerosis by Effects on Plasma Lipoproteins and Cholesterol Transport That Overshadow Antioxidant Protection 
To test the hypothesis that NF-E2–related factor 2 (Nrf2) expression plays an antiatherogenic role by its vascular antioxidant and anti-inflammatory properties.
Methods and Results
Nrf2 is an important transcription factor that regulates the expression of phase 2 detoxifying enzymes and antioxidant genes. Its expression in vascular cells appears to be an important factor in the protection against vascular oxidative stress and inflammation. We developed Nrf2 heterozygous (HET) and homozygous knockout (KO) mice on an apolipoprotein (apo) E–null background by sequential breeding, resulting in Nrf2−/−, apoE−/− (KO), Nrf2−/+, apoE−/− (HET) and Nrf2+/+, and apoE−/− wild-type littermates. KO mice exhibited decreased levels of antioxidant genes with evidence of increased reactive oxygen species generation compared with wild-type controls. Surprisingly, KO males exhibited 47% and 53% reductions in the degree of aortic atherosclerosis compared with HET or wild-type littermates, respectively. Decreased atherosclerosis in KO mice correlated with lower plasma total cholesterol in a sex-dependent manner. KO mice also had a decreased hepatic cholesterol content and a lower expression of lipogenic genes, suggesting that hepatic lipogenesis could be reduced. In addition, KO mice exhibited atherosclerotic plaques characterized by a lesser macrophage component and decreased foam cell formation in an in vitro lipid-loading assay. This was associated with a lower rate of cholesterol influx, mediated in part by decreased expression of the scavenger receptor CD36.
Nrf2 expression unexpectedly promotes atherosclerotic lesion formation in a sex-dependent manner, most likely by a combination of systemic metabolic and local vascular effects.
PMCID: PMC3037185  PMID: 20947826
atherosclerosis; cytokines; lipoproteins; reactive oxygen species; foam cell formation; lipogenesis; Nrf2
8.  Copy number variation influences gene expression and metabolic traits in mice 
Human Molecular Genetics  2009;18(21):4118-4129.
Copy number variants (CNVs) are genomic segments which are duplicated or deleted among different individuals. CNVs have been implicated in both Mendelian and complex traits, including immune and behavioral disorders, but the study of the mechanisms by which CNVs influence gene expression and clinical phenotypes in humans is complicated by the limited access to tissues and by population heterogeneity. We now report studies of the effect of 19 CNVs on gene expression and metabolic traits in a mouse intercross between strains C57BL/6J and C3H/HeJ. We found that 83% of genes predicted to occur within CNVs were differentially expressed. The expression of most CNV genes was correlated with copy number, but we also observed evidence that gene expression was altered in genes flanking CNVs, suggesting that CNVs may contain regulatory elements for these genes. Several CNVs mapped to hotspots, genomic regions influencing expression of tens or hundreds of genes. Several metabolic traits including cholesterol, triglycerides, glucose and body weight mapped to three CNVs in the genome, in mouse chromosomes 1, 4 and 17. Predicted CNV genes, such as Itlna, Defcr-1, Trim12 and Trim34 were highly correlated with these traits. Our results suggest that CNVs have a significant impact on gene expression and that CNVs may be playing a role in the mechanisms underlying metabolic traits in mice.
PMCID: PMC2758141  PMID: 19648292

Results 1-8 (8)