Hypothalamic transcriptomes of 99 mouse strains reveal trans eQTL hotspots, splicing QTLs and novel non-coding genes.
ABSTRACT: Previous studies had shown that the integration of genome wide expression profiles, in metabolic tissues, with genetic and phenotypic variance, provided valuable insight into the underlying molecular mechanisms. We used RNA-Seq to characterize hypothalamic transcriptome in 99 inbred strains of mice from the Hybrid Mouse Diversity Panel (HMDP), a reference resource population for cardiovascular and metabolic traits. We report numerous novel transcripts supported by proteomic analyses, as well as novel non coding RNAs. High resolution genetic mapping of transcript levels in HMDP, reveals both local and trans expression Quantitative Trait Loci (eQTLs) demonstrating 2 trans eQTL 'hotspots' associated with expression of hundreds of genes. We also report thousands of alternative splicing events regulated by genetic variants. Finally, comparison with about 150 metabolic and cardiovascular traits revealed many highly significant associations. Our data provide a rich resource for understanding the many physiologic functions mediated by the hypothalamus and their genetic regulation.
Project description:Previous studies had shown that integration of genome wide expression profiles, in metabolic tissues, with genetic and phenotypic variance, provided valuable insight into the underlying molecular mechanisms. We used RNA-Seq to characterize hypothalamic transcriptome in 99 inbred strains of mice from the Hybrid Mouse Diversity Panel (HMDP), a reference resource population for cardiovascular and metabolic traits. We report numerous novel transcripts supported by proteomic analyses, as well as novel non coding RNAs. High resolution genetic mapping of transcript levels in HMDP, reveals both local and trans expression Quantitative Trait Loci (eQTLs) demonstrating 2 trans eQTL "hotspots" associated with expression of hundreds of genes. We also report thousands of alternative splicing events regulated by genetic variants. Finally, comparison with about 150 metabolic and cardiovascular traits revealed many highly significant associations. Our data provides a rich resource for understanding the many physiologic functions mediated by the hypothalamus and their genetic regulation. 282 samples, 3 biological replicates per strain
Project description:Identifying methylation quantitative trait loci (meQTLs) and integrating them with disease-associated variants from genome-wide association studies (GWAS) may illuminate functional mechanisms underlying genetic variant-disease associations. Here, we perform GWAS of >415 thousand CpG methylation sites in whole blood from 4170 individuals and map 4.7 million cis- and 630 thousand trans-meQTL variants targeting >120 thousand CpGs. Independent replication is performed in 1347 participants from two studies. By linking cis-meQTL variants with GWAS results for cardiovascular disease (CVD) traits, we identify 92 putatively causal CpGs for CVD traits by Mendelian randomization analysis. Further integrating gene expression data reveals evidence of cis CpG-transcript pairs causally linked to CVD. In addition, we identify 22 trans-meQTL hotspots each targeting more than 30 CpGs and find that trans-meQTL hotspots appear to act in cis on expression of nearby transcriptional regulatory genes. Our findings provide a powerful meQTL resource and shed light on DNA methylation involvement in human diseases.
Project description:The pig is a well-known animal model used to investigate genetic and mechanistic aspects of human disease biology. They are particularly useful in the context of obesity and metabolic diseases because other widely used models (e.g. mice) do not completely recapitulate key pathophysiological features associated with these diseases in humans. Therefore, we established a F2 pig resource population (n = 564) designed to elucidate the genetics underlying obesity and metabolic phenotypes. Segregation of obesity traits was ensured by using breeds highly divergent with respect to obesity traits in the parental generation. Several obesity and metabolic phenotypes were recorded (n = 35) from birth to slaughter (242 ± 48 days), including body composition determined at about two months of age (63 ± 10 days) via dual-energy x-ray absorptiometry (DXA) scanning. All pigs were genotyped using Illumina Porcine 60k SNP Beadchip and a combined linkage disequilibrium-linkage analysis was used to identify genome-wide significant associations for collected phenotypes. We identified 229 QTLs which associated with adiposity- and metabolic phenotypes at genome-wide significant levels. Subsequently comparative analyses were performed to identify the extent of overlap between previously identified QTLs in both humans and pigs. The combined analysis of a large number of obesity phenotypes has provided insight in the genetic architecture of the molecular mechanisms underlying these traits indicating that QTLs underlying similar phenotypes are clustered in the genome. Our analyses have further confirmed that genetic heterogeneity is an inherent characteristic of obesity traits most likely caused by segregation or fixation of different variants of the individual components belonging to cellular pathways in different populations. Several important genes previously associated to obesity in human studies, along with novel genes were identified. Altogether, this study provides novel insight that may further the current understanding of the molecular mechanisms underlying human obesity.
Project description:OBJECTIVE:Lipocalin-2 (LCN2) is a secreted protein involved in innate immunity and has also been associated with several cardiometabolic traits in both mouse and human studies. However, the causal relationship of LCN2 to these traits is unclear, and most studies have examined only males. METHODS:Using adeno-associated viral vectors we expressed LCN2 in either adipose or liver in a tissue specific manner on the background of a whole-body Lcn2 knockout or wildtype mice. Metabolic phenotypes including body weight, body composition, plasma and liver lipids, glucose homeostasis, insulin resistance, mitochondrial phenotyping, and metabolic cage studies were monitored. RESULTS:We studied the genetics of LCN2 expression and associated clinical traits in both males and females in a panel of 100 inbred strains of mice (HMDP). The natural variation in Lcn2 expression across the HMDP exhibits high heritability, and genetic mapping suggests that it is regulated in part by Lipin1 gene variation. The correlation analyses revealed striking tissue dependent sex differences in obesity, insulin resistance, hepatic steatosis, and dyslipidemia. To understand the causal relationships, we examined the effects of expression of LCN2 selectively in liver or adipose. On a Lcn2-null background, LCN2 expression in white adipose promoted metabolic disturbances in females but not males. It acted in an autocrine/paracrine manner, resulting in mitochondrial dysfunction and an upregulation of inflammatory and fibrotic genes. On the other hand, on a null background, expression of LCN2 in liver had no discernible impact on the traits examined despite increasing the levels of circulating LCN2 more than adipose LCN2 expression. The mechanisms underlying the sex-specific action of LCN2 are unclear, but our results indicate that adipose LCN2 negatively regulates its receptor, LRP2 (or megalin), and its repressor, ER?, in a female-specific manner and that the effects of LCN2 on metabolic traits are mediated in part by LRP2. CONCLUSIONS:Following up on our population-based studies, we demonstrate that LCN2 acts in a highly sex- and tissue-specific manner in mice. Our results have important implications for human studies, emphasizing the importance of sex and the tissue source of LCN2.
Project description:We have developed an association-based approach using classical inbred strains of mice in which we correct for population structure, which is very extensive in mice, using an efficient mixed-model algorithm. Our approach includes inbred parental strains as well as recombinant inbred strains in order to capture loci with effect sizes typical of complex traits in mice (in the range of 5% of total trait variance). Over the last few years, we have typed the hybrid mouse diversity panel (HMDP) strains for a variety of clinical traits as well as intermediate phenotypes and have shown that the HMDP has sufficient power to map genes for highly complex traits with resolution that is in most cases less than a megabase. In this essay, we review our experience with the HMDP, describe various ongoing projects, and discuss how the HMDP may fit into the larger picture of common diseases and different approaches.
Project description:Genetic variations in blood cell parameters can impact clinical traits. We report here the mapping of blood cell traits in a panel of 100 inbred strains of mice of the Hybrid Mouse Diversity Panel (HMDP) using genome-wide association (GWA). We replicated a locus previously identified in using linkage analysis in several genetic crosses for mean corpuscular volume (MCV) and a number of other red blood cell traits on distal chromosome 7. Our peak for SNP association to MCV occurred in a linkage disequilibrium (LD) block spanning from 109.38 to 111.75 Mb that includes Hbb-b1, the likely causal gene. Altogether, we identified five loci controlling red blood cell traits (on chromosomes 1, 7, 11, 12, and 16), and four of these correspond to loci for red blood cell traits reported in a recent human GWA study. For white blood cells, including granulocytes, monocytes, and lymphocytes, a total of six significant loci were identified on chromosomes 1, 6, 8, 11, 12, and 15. An average of ten candidate genes were found at each locus and those were prioritized by examining functional variants in the HMDP such as missense and expression variants. These results provide intermediate phenotypes and candidate loci for genetic studies of atherosclerosis and cancer as well as inflammatory and immune disorders in mice.
Project description:Common forms of atherosclerosis involve multiple genetic and environmental factors. While human genome-wide association studies have identified numerous loci contributing to coronary artery disease and its risk factors, these studies are unable to control environmental factors or examine detailed molecular traits in relevant tissues. We now report a study of natural variations contributing to atherosclerosis and related traits in over 100 inbred strains of mice from the Hybrid Mouse Diversity Panel (HMDP). The mice were made hyperlipidemic by transgenic expression of human apolipoprotein E-Leiden (APOE-Leiden) and human cholesteryl ester transfer protein (CETP). The mice were examined for lesion size and morphology as well as plasma lipid, insulin and glucose levels, and blood cell profiles. A subset of mice was studied for plasma levels of metabolites and cytokines. We also measured global transcript levels in aorta and liver. Finally, the uptake of acetylated LDL by macrophages from HMDP mice was quantitatively examined. Loci contributing to the traits were mapped using association analysis, and relationships among traits were examined using correlation and statistical modeling. A number of conclusions emerged. First, relationships among atherosclerosis and the risk factors in mice resemble those found in humans. Second, a number of trait-loci were identified, including some overlapping with previous human and mouse studies. Third, gene expression data enabled enrichment analysis of pathways contributing to atherosclerosis and prioritization of candidate genes at associated loci in both mice and humans. Fourth, the data provided a number of mechanistic inferences; for example, we detected no association between macrophage uptake of acetylated LDL and atherosclerosis. Fifth, broad sense heritability for atherosclerosis was much larger than narrow sense heritability, indicating an important role for gene-by-gene interactions. Sixth, stepwise linear regression showed that the combined variations in plasma metabolites, including LDL/VLDL-cholesterol, trimethylamine N-oxide (TMAO), arginine, glucose and insulin, account for approximately 30 to 40% of the variation in atherosclerotic lesion area. Overall, our data provide a rich resource for studies of complex interactions underlying atherosclerosis.
Project description:A powerful way to identify genes for complex traits it to combine genetic and genomic methods. Many trait quantitative trait loci (QTLs) for complex traits are sex specific, but the reason for this is not well understood.RNA was prepared from bone marrow derived macrophages of 93 female and 114 male F(2) mice derived from a strain intercross between apoE-deficient mice on the AKR and DBA/2 genetic backgrounds, and was subjected to transcriptome profiling using microarrays. A high density genome scan was performed using a mouse SNP chip, and expression QTLs (eQTLs) were located for expressed transcripts. Using suggestive and significant LOD score cutoffs of 3.0 and 4.3, respectively, thousands of eQTLs in the female and male cohorts were identified. At the suggestive LOD threshold the majority of the eQTLs were trans eQTLs, mapping unlinked to the position of the gene. Cis eQTLs, which mapped to the location of the gene, had much higher LOD scores than trans eQTLs, indicating their more direct effect on gene expression. The majority of cis eQTLs were common to both males and females, but only approximately 1% of the trans eQTLs were shared by both sexes. At the significant LOD threshold, the majority of eQTLs were cis eQTLs, which were mostly sex-shared, while the trans eQTLs were overwhelmingly sex-specific. Pooling the male and female data, 31% of expressed transcripts were expressed at different levels in males vs. females after correction for multiple testing.These studies demonstrate a large sex effect on gene expression and trans regulation, under conditions where male and female derived cells were cultured ex vivo and thus without the influence of endogenous sex steroids. These data suggest that eQTL data from male and female mice should be analyzed separately, as many effects, such as trans regulation are sex specific.
Project description:Analyses of QTLs for expression levels (eQTLs) of the genes reveal genetic relationship between expression variation and the regulator, thus unlocking the information for identifying the regulatory network. Oligo-nucleotide expression microarrays hybridized with RNA can simultaneously provide data for molecular markers and transcript abundance. In this study, we used Affymetrix GeneChip Rice Genome Array to analyze eQTLs in rice shoots at 72 h after germination from 110 recombinant inbred lines (RILs) derived from a cross between Zhenshan 97 and Minghui 63. Totally 1,632 single feature polymorphisms (SFPs) plus 23 PCR markers were identified and placed into 601 recombinant bins, spanning 1,459 cM in length, which were used as markers to genotype the RILs. We obtained 16,372 expression traits (e-traits) each with at least one eQTL, resulting in 26,051 eQTLs in total, including both cis- and trans-eQTLs. We also identified 171 eQTL hotspots among rice genome, each of which controls transcript variations of many e-traits. Gene Ontology analysis revealed enrichment of certain functional categories of genes in some of the eQTL hotspots. In particular, eQTLs for e-traits involving DNA metabolic process was significantly enriched in several eQTL hotspots on chromosomes 3, 5 and 10. Several transcription factors colocalizing with cis-eQTLs showed significant correlations with hundreds of e-traits, indicating possible co-regulation. We also detected correlations between the QTLs for shoot dry weight and eQTLs, revealing possible candidate genes for the trait. These results provided the clues for identification and characterization of regulatory network in the whole genome at the transcriptional level. Overall design: To dissect the genetic variation between the two rice indica varieties Minghui 63 and Zhenshan 97, a total of 110 RILs from Minghui 63 and Zhenshan 97 and parents were sampled. And the Affymetrix Genechip rice Genome Array was used to investigate their dynamic transcript levels. Two independent biological replicates were sampled from each RIL, and three replicates for each parent.resulting in a dataset of 226 microarrays.
Project description:We report a multi-omic study of sex differences and gene-by-sex interactions across a panel of 100 inbred strains of mice (the Hybrid Mouse Diversity Panel, HMDP), with a focus on metabolic and cardiovascular traits. For all traits examined, including obesity, insulin resistance, fatty liver, atherosclerosis, and gut microbiota composition, sex differences were influenced by genetic background. Loci identified by genome-wide association studies (GWAS) of the traits were frequently influenced by sex. Lyplal1, a gene implicated in human obesity, was shown to underlie a sex-specific locus for diet induced obesity. Many of the sex-dependent traits showed interdependencies as judged by correlation and shared gene expression patterns, indicating higher order regulation. Global gene expression analyses of tissues across the HMDP indicated that sex differences in mitochondrial functions in adipose contributed to many of the traits. Consistent with this, we observed that females tended to be more resistant to the adverse effects of a high fat diet, with smaller adipocytes and increased “browning” of white adipose tissue as compared to males. Sex-specific differences in mitochondrial activity were confirmed by examining respiration of isolated mitochondria. Gonadectomy experiments revealed thousands of genes influenced by sex hormones. In liver, a tissue exhibiting particularly strong differences in gene expression between tissues, sex hormones appeared to be the primary driver of the differences, whereas in adipose organizational effects of sex appeared to be more important. Overall design: Sixteen male and sixteen female C57BL/6J were purchased from The Jackson Laboratory (Bar Harbor). Mice were either maintained on a chow diet (Ralston Purina Company) or placed on an HF/HS diet (Research Diets D12266B) at 8 weeks of age until 16 weeks of age. At 6 weeks of age the mice were gonadectomized under isoflurane anesthesia. Scrotal regions of male mice were bilaterally incised, testes removed, and the incisions closed with wound clips. Ovaries of female mice were removed through an incision just below the rib cage. There were four mice per group. The muscle layer was sutured, and the incision closed with wound clips. In sham-operated control mice, incisions were made and closed as described above. The gonads were briefly manipulated, but remained intact. Gonadal fat and liver samples were taken for RNASeq expression profiling.