Margarete Mehrabian

Mapping the Genetic Architecture of Gene Expression in Human Liver

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.

Identification of 5-Lipoxygenase as a Major Gene Contributing to Atherosclerosis Susceptibility in Mice

We previously reported the identification of a locus on mouse chromosome 6 that confers almost total resistance to atherogenesis, even on a hypercholesterolemic (LDL receptor–null) background. 5-Lipoxygenase (5-LO) is the rate-limiting enzyme in leukotriene synthesis and was among the chromosome 6 locus candidate genes that we examined. The levels of 5-LO mRNA were reduced about 5-fold in a congenic strain, designated CON6, containing the resistant chromosome 6 region derived from the CAST/Ei strain (CAST), as compared with the background C57BL/6J (B6) strain. 5-LO protein levels were similarly reduced in the CON6 mice. Sequencing of the 5-LO cDNA revealed several differences between CON6 and the B6 strain. To test the whether 5-LO is responsible for the resistant phenotype, we bred a 5-LO knockout allele onto an LDL receptor–null (LDLR−/−) background. On this background, the mice bred poorly and only heterozygous 5-LO knockout mice were obtained. These mice showed a dramatic decrease (>26-fold;P <0.0005) in aortic lesion development, similar to the CON6 mice. Immunohistochemistry revealed that 5-LO was abundantly expressed in atherosclerotic lesions of apoE− /− and LDLR−/− deficient mice, appearing to colocalize with a subset of macrophages but not with all macrophage-staining regions. When bone marrow from 5-LO+/− mice was transplanted into LDLR−/−, there was a significant reduction in atherogenesis, suggesting that macrophage 5-LO is responsible, at least in part, for the effect on atherosclerosis. These results indicate that 5-LO contributes importantly to the atherogenic process and they provide strong presumptive evidence that reduced 5-LO expression is partly responsible for the resistance to atherosclerosis in CON6 mice.

The nature and identification of quantitative trait loci: a community’s view

This white paper by eighty members of the Complex Trait Consortium presents a communitys view on the approaches and statistical analyses that are needed for the identification of genetic loci that determine quantitative traits. Quantitative trait loci (QTLs) can be identified in several ways, but is there a definitive test of whether a candidate locus actually corresponds to a specific QTL?

Genetic Control of Obesity and Gut Microbiota Composition in Response to High-Fat, High-Sucrose Diet in Mice

Obesity is a highly heritable disease driven by complex interactions between genetic and environmental factors. Human genome-wide association studies (GWAS) have identified a number of loci contributing to obesity; however, a major limitation of these studies is the inability to assess environmental interactions common to obesity. Using a systems genetics approach, we measured obesity traits, global gene expression, and gut microbiota composition in response to a high-fat/high-sucrose (HF/HS) diet of more than 100 inbred strains of mice. Here we show that HF/HS feeding promotes robust, strain-specific changes in obesity that are not accounted for by food intake and provide evidence for a genetically determined set point for obesity. GWAS analysis identified 11 genome-wide significant loci associated with obesity traits, several of which overlap with loci identified in human studies. We also show strong relationships between genotype and gut microbiota plasticity during HF/HS feeding and identify gut microbial phylotypes associated with obesity.

Role of Group II Secretory Phospholipase A2 in Atherosclerosis 1. Increased Atherogenesis and Altered Lipoproteins in Transgenic Mice Expressing Group IIa Phospholipase A2

Some observations have suggested that the extracellular group IIa phospholipase A2 (sPLA2), previously implicated in chronic inflammatory conditions such as arthritis, may contribute to atherosclerosis. We have examined this hypothesis by studying transgenic mice expressing the human enzyme. Compared with nontransgenic littermates, the transgenic mice exhibited dramatically increased atherosclerotic lesions when maintained on a high-fat, high-cholesterol diet. Surprisingly, the transgenic mice also exhibited significant atherosclerotic lesions when maintained on a low-fat chow diet. Immunohistochemical staining indicated that sPLA2 was present in the atherosclerotic lesions of the transgenic mice. On both chow and atherogenic diets, the transgenic mice exhibited decreased levels of HDLs and slightly increased levels of LDLs compared with nontransgenic littermates. These data indicate that group IIa sPLA2 may promote atherogenesis, in part, through its effects on lipoprotein levels. These data also provide a possible mechanism for the observation that there is an increased incidence of coronary artery disease in many chronic inflammatory diseases.

Gut Microbial Metabolite TMAO Enhances Platelet Hyperreactivity and Thrombosis Risk

Normal platelet function is critical to blood hemostasis and maintenance of a closed circulatory system. Heightened platelet reactivity, however, is associated with cardiometabolic diseases and enhanced potential for thrombotic events. We now show gut microbes, through generation of trimethylamine N-oxide (TMAO), directly contribute to platelet hyperreactivity and enhanced thrombosis potential. Plasma TMAO levels in subjects (n > 4,000) independently predicted incident (3 years) thrombosis (heart attack, stroke) risk. Direct exposure of platelets to TMAO enhanced sub-maximal stimulus-dependent platelet activation from multiple agonists through augmented Ca(2+) release from intracellular stores. Animal model studies employing dietary choline or TMAO, germ-free mice, and microbial transplantation collectively confirm a role for gut microbiota and TMAO in modulating platelet hyperresponsiveness and thrombosis potential and identify microbial taxa associated with plasma TMAO and thrombosis potential. Collectively, the present results reveal a previously unrecognized mechanistic link between specific dietary nutrients, gut microbes, platelet function, and thrombosis risk.

Validation of candidate causal genes for obesity that affect shared metabolic pathways and networks

A principal task in dissecting the genetics of complex traits is to identify causal genes for disease phenotypes. We previously developed a method to infer causal relationships among genes through the integration of DNA variation, gene transcription and phenotypic information. Here we have validated our method through the characterization of transgenic and knockout mouse models of genes predicted to be causal for abdominal obesity. Perturbation of eight out of the nine genes, with Gas7, Me1 and Gpx3 being newly confirmed, resulted in significant changes in obesity-related traits. Liver expression signatures revealed alterations in common metabolic pathways and networks contributing to abdominal obesity and overlapped with a macrophage-enriched metabolic network module that is highly associated with metabolic traits in mice and humans. Integration of gene expression in the design and analysis of traditional F2 intercross studies allows high-confidence prediction of causal genes and identification of pathways and networks involved.

Integrating genotypic and expression data in a segregating mouse population to identify 5-lipoxygenase as a susceptibility gene for obesity and bone traits:

Forward genetic approaches to identify genes involved in complex traits such as common human diseases have met with limited success. Fine mapping of linkage regions and validation of positional candidates are time-consuming and not always successful. Here we detail a hybrid procedure to map loci involved in complex traits that leverages the strengths of forward and reverse genetic approaches. By integrating genotypic and expression data in a segregating mouse population, we show how clusters of expression quantitative trait loci linking to regions of the genome accurately reflect the underlying perturbation to the transcriptional network induced by DNA variations in genes that control the complex traits. By matching patterns of gene expression in a segregating population with expression responses induced by single-gene perturbation experiments, we show how genes controlling clusters of expression and clinical quantitative trait loci can be mapped directly. We demonstrate the utility of this approach by identifying 5-lipoxygenase as underlying previously identified quantitative trait loci in an F2 cross between strains C57BL/6J and DBA/2J and showing that it has pleiotropic effects on body fat, lipid levels and bone density.Forward genetic approaches to identify genes involved in complex traits such as common human diseases have met with limited success. Fine mapping of linkage regions and validation of positional candidates are time-consuming and not always successful. Here we detail a hybrid procedure to map loci involved in complex traits that leverages the strengths of forward and reverse genetic approaches. By integrating genotypic and expression data in a segregating mouse population, we show how clusters of expression quantitative trait loci linking to regions of the genome accurately reflect the underlying perturbation to the transcriptional network induced by DNA variations in genes that control the complex traits. By matching patterns of gene expression in a segregating population with expression responses induced by single-gene perturbation experiments, we show how genes controlling clusters of expression and clinical quantitative trait loci can be mapped directly. We demonstrate the utility of this approach by identifying 5-lipoxygenase as underlying previously identified quantitative trait loci in an F2 cross between strains C57BL/6J and DBA/2J and showing that it has pleiotropic effects on body fat, lipid levels and bone density.

Minimally modified low density lipoprotein is biologically active in vivo in mice.

Minimally modified low density lipoprotein (MM-LDL), derived by mild iron oxidation or prolonged storage at 4 degrees C, has been shown to induce certain inflammatory responses in vascular cells in tissue culture. These include induction of monocyte (but not neutrophil) adherence to endothelial cells (EC), induction of EC production of colony stimulating factors (CSF), and induction of EC and smooth muscle cell production of monocyte chemotactic protein (MCP-1). To test for biologic activity in vivo, microgram quantities of MM-LDL were injected into mice, sera were assayed for CSF activity, and tissues were subjected to Northern analysis. After injection of MM-LDL, CSF activity increased approximately 7-26-fold but remained near control levels after injection of native LDL. Essentially all of the induced CSF activity was due to macrophage CSF as judged by antibody inhibition. Injection of MM-LDL into a mouse strain (C3H/HeJ) that is resistant to bacterial LPS gave similar results, indicating that the induction of CSF was not due to contaminating LPS and suggesting that there are differences in the pathways by which LPS and MM-LDL trigger cytokine production. In addition, after injection of MM-LDL, mRNA for JE, the mouse homologue of MCP-1, was markedly induced in various tissues, but was not induced after injection of native LDL. We conclude, therefore, that MM-LDL is biologically active in vivo and may contribute to the early stages of atherosclerosis by acting as an inflammatory agent.

Genetic loci controlling body fat, lipoprotein metabolism, and insulin levels in a multifactorial mouse model.

We analyzed the inheritance of body fat, leptin levels, plasma lipoprotein levels, insulin levels, and related traits in an intercross between inbred mouse strains CAST/Ei and C57BL/6J. CAST/Ei mice are unusually lean, with only approximately 8% of body weight as fat, whereas C57BL/6J mice have approximately 18% body fat. Quantitative trait locus analysis using > 200 F2 mice revealed highly significant loci (lod scores > 4.3) on chromosomes 2 (three separate loci) and 9 that contribute to mouse fat-pad mass for mice on a high-fat diet. Some loci also influenced plasma lipoprotein levels and insulin levels either on chow or high-fat diets. Two loci for body fat and lipoprotein levels (on central and distal chromosome 2) coincided with a locus having strong effects on hepatic lipase activity, an activity associated with visceral obesity and lipoprotein levels in humans. A locus contributing to plasma leptin levels (lod score 5.3) but not obesity was identified on chromosome 4, near the leptin receptor gene. These data identify candidate regions and candidate genes for studies of human obesity and diabetes, and suggest obesity is highly complex in terms of the number of genetic factors involved. Finally, they support the existence of specific genetic interactions between body fat, insulin metabolism, and lipoprotein metabolism.