Bi-directional gene set enrichment and canonical correlation analysis identify key diet-sensitive pathways and biomarkers of metabolic syndrome

peer-reviewedBackground Currently, a number of bioinformatics methods are available to generate appropriate lists of genes from a microarray experiment. While these lists represent an accurate primary analysis of the data, fewer options exist to contextualise those lists. The development and validation of such methods is crucial to the wider application of microarray technology in the clinical setting. Two key challenges in clinical bioinformatics involve appropriate statistical modelling of dynamic transcriptomic changes, and extraction of clinically relevant meaning from very large datasets. Results Here, we apply an approach to gene set enrichment analysis that allows for detection of bi-directional enrichment within a gene set. Furthermore, we apply canonical correlation analysis and Fisher's exact test, using plasma marker data with known clinical relevance to aid identification of the most important gene and pathway changes in our transcriptomic dataset. After a 28-day dietary intervention with high-CLA beef, a range of plasma markers indicated a marked improvement in the metabolic health of genetically obese mice. Tissue transcriptomic profiles indicated that the effects were most dramatic in liver (1270 genes significantly changed; p < 0.05), followed by muscle (601 genes) and adipose (16 genes). Results from modified GSEA showed that the high-CLA beef diet affected diverse biological processes across the three tissues, and that the majority of pathway changes reached significance only with the bi-directional test. Combining the liver tissue microarray results with plasma marker data revealed 110 CLA-sensitive genes showing strong canonical correlation with one or more plasma markers of metabolic health, and 9 significantly overrepresented pathways among this set; each of these pathways was also significantly changed by the high-CLA diet. Closer inspection of two of these pathways - selenoamino acid metabolism and steroid biosynthesis - illustrated clear diet-sensitive changes in constituent genes, as well as strong correlations between gene expression and plasma markers of metabolic syndrome independent of the dietary effect. Conclusion Bi-directional gene set enrichment analysis more accurately reflects dynamic regulatory behaviour in biochemical pathways, and as such highlighted biologically relevant changes that were not detected using a traditional approach. In such cases where transcriptomic response to treatment is exceptionally large, canonical correlation analysis in conjunction with Fisher's exact test highlights the subset of pathways showing strongest correlation with the clinical markers of interest. In this case, we have identified selenoamino acid metabolism and steroid biosynthesis as key pathways mediating the observed relationship between metabolic health and high-CLA beef. These results indicate that this type of analysis has the potential to generate novel transcriptome-based biomarkers of disease.Department of Agriculture and Food, Ireland - Food Institutional Research Measure (project no. 5254); IRCSET postgraduate scholarship scheme (MJM); Science Foundation Ireland Principal Investigator Programme (HMR) Programme

Systems biology of human adipocytes: from genes and molecular networks to heterogeneity and metabolic phenotypes

Obesity and its co-morbidities are among the leading health problems facing the developed world. Multiple genetic and environmental factors are known to have a significant impact on obesity development, however, heterogeneity of adipose tissue also contributes to obesity and its complications. Regional variation in adipose tissue has been associated with disease risks. For example, accumulation of visceral white adipose tissue (VAT), when compared to accumulation of subcutaneous white adipose tissue (SAT), is associated with an increased risk of diabetes and metabolic syndrome. Accumulation of brown adipose tissue (BAT), when compared to accumulation of white adipose tissue (WAT), on the other hand, is associated with lower BMI and higher insulin sensitivity. The goal of our project is to identify molecular targets that can be used as diagnostics, prognostics, or for reprogramming adipose tissue to a healthier phenotype (e.g. reprogramming VAT to SAT or WAT to BAT). To this end, we used three approaches. First, we used metabolic modeling to compare brown and white adipocyte metabolic profiles to predict and experimentally validate flux differences in the metabolic networks. Through this, we predicted and discovered a difference in urea secretion between these two classes of adipocytes. Second, we conducted transcriptome analysis of preadipocytes derived from SAT and VAT to identify several differentially expressed genes. Among them, we focused on Membrane Metallo-Endopeptidase (MME/Neprilysin) and showed experimentally that MME regulated the inflammatory response and insulin signaling in white preadipocytes by differentially affecting the insulin receptor (IR) subunits by increasing IRα but not IRβ. Finally, we used single-cell transcriptomics in differentiating human white preadipocytes derived from a single adipose depot to identify two subpopulations populations and a novel gene cluster of zinc finger proteins involved in white preadipocyte differentiation. The results presented here identify several key targets underlying the molecular and metabolic heterogeneity of adipose tissue

Consumption of dietary emulsifiers increases sensitivity to social stress in mice: a potential role for the COX molecular pathway

Chronic low-grade inflammation and exposure to stress are key contributing factors in the etiology and progression of many neuropsychiatric disorders. Dietary emulsifiers are commonly added to processed foods and are classified by the Food and Drug Administration (FDA) as generally recognized as safe (GRAS). Recently, however, it has been revealed that these additives at translationally relevant doses can cause low-grade inflammation, gut dysbiosis, and may even increase baseline anxiety-like behavior. The latter finding suggests that dietary emulsifiers impact brain areas that modulate stress responses. We used RNA-Seq to examine whether chronic consumption of either polysorbate 80 (P80) or carboxymethylcellulose (CMC) is associated with changes in gene expression in the amygdala and PVN and used Ingenuity Pathway Analysis to identify enriched molecular pathways that may underlie an anxiety-like phenotype. Emulsifier consumption resulted in alterations in gene expression of various immediate early, stress-related, and immune-related genes in brain regions that are known to be important in the generation of behavioral and neuroendocrine responses to stress-provoking stimuli. We also hypothesized that emulsifier-treated mice exhibit sensitized behavioral, hormonal, and neuronal activity responses to stress. To test this hypothesis, C57Bl6/J mice were subjected to acute defeat conditions after 12 weeks of emulsifier or water consumption. When subjected to social defeat, emulsifier-treated mice showed increases in social avoidance and circulating corticosterone as well as alterations in neuronal activity as measured by c-Fos immunofluorescence. Subsequently, given the observed increased expression of PTGS2 (COX-2) in the amygdala, we tested the hypothesis that increased inflammation through the COX pathway is a mechanism driving emulsifier-induced increases is stress sensitivity. Groups were as described above, but mice were also divided into aspirin (25mg/kg/day) and placebo intervention groups. We found that aspirin, a COX pathway inhibitor, appears to block the increase in social avoidance observed in emulsifier-treated mice. These data demonstrate that ingestion of dietary emulsifiers at concentrations analogous to those ingested by humans increases sensitivity to social stress in mice. Further, it appears that the COX pathway may be a prime mechanistic candidate by which emulsifier-induced increases in sensitivity to social stress occurs

Liver X Receptor cis-Repression and Cholesterol Efflux Restrain Innate Immunity and Coronary Artery Disease

Atherosclerotic cardiovascular disease secondary to deposition of apolipoprotein B-containing lipoproteins in the artery wall is a leading cause of mortality. Therapies that reduce serum levels of atherogenic lipoprotein-cholesterol have been successful in reducing cardiovascular mortality, but this approach requires long-term treatment and substantial residual risk remains. Here, we investigate mechanistic determinants of atherosclerosis protection by two potential therapeutic approaches for lowering of residual cardiovascular risk. Using mouse models, we show that the nuclear receptor liver X receptor exerts an anti-inflammatory activity on innate immunity and atherosclerosis through both promotion of cholesterol efflux and a direct cis-repressive activity affecting neutrophil inflammation. We then assess the causal role of the cholesterol efflux pathway in human cardiovascular events by using genetic variants that modify high density lipoprotein-cholesterol in instrumental variable analysis. We show that this pathway is associated with protection from cardiovascular disease in a precise and robust Mendelian randomization analysis on an FDR-controlled set of variants, which suggests a causal effect. Thus, agents that target the cholesterol efflux and liver X receptor cis-repression pathways may be protective in atherosclerosis

Comparative Analysis of Small Non-Coding RNA and Messenger RNA Expression in Somatic Cell Nuclear Transfer and In Vitro-Fertilized Bovine Embryos During Early Development Through the Maternal-to-Embryonic Transition

Cloning animals using somatic cell nuclear transfer (scNT) was first successfully demonstrated with the birth of Dolly the sheep, but the process of cloning remains highly inefficient. By improving our understanding of the errors that may occur during cloned cattle embryo development, we could obtain a greater understanding of how specific molecular events contribute to successful development. The central dogma of biology refers to the process of DNA being transcribed into messenger RNA (mRNA) and the translation of mRNA into proteins, which ultimately carry out the functions encoded by genes. The epigenetic code is defined as the array of chemical modifications, or “marks”, to DNA molecules that do not change the genome sequence but do allow for control of gene expression. During early development, genome reprogramming involves the removal of epigenetic marks from the sperm and egg and re-establishment of marks for the embryonic genome that code for proper gene expression to support embryo development. The point during this process at which the embryo’s genes are turned on is known as embryonic genome activation (EGA). Small non-coding RNAs (sncRNAs), including microRNAs (miRNAs), may also contribute to the this process. For example, miRNA molecules do not code for proteins themselves, but rather bind to mRNAs and effectively block their translation into protein. We hypothesized that aberrant expression of sncRNAs in cloned embryos may lead to anomalous abundance of mRNA molecules, thus explaining poor development of cloned embryos. First, we used RNA sequencing to examine the total population of sncRNAs in cattle embryos produced by in vitro fertilization (IVF) and found a dramatic shift in populations at the EGA. Next, we collected both sncRNA and mRNA from scNT cattle embryos, and again performed sequencing of both RNA fractions. We found that few sncRNAs were abnormally expressed in scNT embryos, with all differences appearing after EGA at the morula developmental stage. However, notable differences in the populations of sncRNAs were evident when comparing embryos by developmental stage. For populations of mRNA, we observed dramatic differences when comparing scNT and IVF cattle embryos, with the highest number of changes occurring at the EGA (8-cell stage) and after (morula stage). While changes in specific miRNA molecules (miR-34a and miR-345) were negatively correlated with some of their predicted target mRNAs, this pattern was not widespread as would be expected if these sncRNAs are functionally binding to all of the predicted mRNA targets. Collectively, our observations suggest that other mechanisms leading to altered expression of mRNA in cloned embryos may be responsible for their relatively poor development

Observational causality from -omics

Some human traits like disease are heritable, which means that they run in families. This indicates that there must be something on the DNA that affects an individual’s susceptibility to developing a trait. In the last 15 years, scientists from around the world have been very successful in mapping the locations on the DNA that are associated to traits like disease, finding thousands of loci, and hundreds of DNA locations per trait, making them truly complex traits. So, we have a very good understanding about which locations on the DNA are important for developing complex traits like disease. Unfortunately, it’s still unclear how these locations on the DNA affect an individual’s trait. In this thesis I investigate how we can best understand the DNA locations that affect trait susceptibility and in doing so, identify the causes for human traits like disease. One important technique that we have used to test for finding these causal relationships is called Mendelian randomization. Mendelian randomization identifies naturally occurring experiments that have happened in observational data. In principle, Mendelian randomization can conclude the same things from observational data as from an experimental study. So called `observational causality` has many benefits as it’s cheaper than an experiment, and is less burdensome on the subjects, as they are not subjected to any intervention. The causes that I’m interested in are so called `-omics` traits. -omics traits are molecular measurements that are usually strongly regulated by the DNA. This strong DNA regulation makes -omics traits interesting candidates to understand the mechanism behind the genetic loci of other traits. In this thesis we have investigated gene expression, protein levels and microbiome measurements as our -omics traits of interest for a wide variety of traits including celiac disease and LDL-cholesterol levels