Pathway enrichment analysis helps researchers gain mechanistic insight into gene lists generated from genome-scale (omics) experiments. This method identifies biological pathways that are enriched in a gene list more than would be expected by chance. We explain the procedures of pathway enrichment analysis and present a practical step-by-step guide to help interpret gene lists resulting from RNA-seq and genome-sequencing experiments. The protocol comprises three major steps: definition of a gene list from omics data, determination of statistically enriched pathways, and visualization and interpretation of the results. We describe how to use this protocol with published examples of differentially expressed genes and mutated cancer genes; however, the principles can be applied to diverse types of omics data. The protocol describes innovative visualization techniques, provides comprehensive background and troubleshooting guidelines, and uses freely available and frequently updated software, including g:Profiler, Gene Set Enrichment Analysis (GSEA), Cytoscape and EnrichmentMap. The complete protocol can be performed in ~4.5 h and is designed for use by biologists with no prior bioinformatics training.

Drug metabolizing enzymes (DMEs) play central roles in the metabolism, elimination and detoxification of xenobiotics and drugs introduced into the human body. Most of the tissues and organs in our body are well equipped with diverse and various DMEs including phase I, phase II metabolizing enzymes and phase III transporters, which are present in abundance either at the basal unstimulated level, and/or are inducible at elevated level after exposure to xenobiotics. Recently, many important advances have been made in the mechanisms that regulate the expression of these drug metabolism genes. Various nuclear receptors including the aryl hydrocarbon receptor (AhR), orphan nuclear receptors, and nuclear factor-erythoroid 2 p45-related factor 2 (Nrf2) have been shown to be the key mediators of drug-induced changes in phase I, phase II metabolizing enzymes as well as phase III transporters involved in efflux mechanisms. For instance, the expression of CYP1 genes can be induced by AhR, which dimerizes with the AhR nuclear translocator (Arnt), in response to many polycyclic aromatic hydrocarbon (PAHs). Similarly, the steroid family of orphan nuclear receptors, the constitutive androstane receptor (CAR) and pregnane X receptor (PXR), both heterodimerize with the retinoid X receptor (RXR), are shown to transcriptionally activate the promoters of CYP2B and CYP3A gene expression by xenobiotics such as phenobarbital-like compounds (CAR) and dexamethasone and rifampin-type of agents (PXR). The peroxisome proliferator activated receptor (PPAR), which is one of the first characterized members of the nuclear hormone receptor, also dimerizes with RXR and has been shown to be activated by lipid lowering agent fibrate-type of compounds leading to transcriptional activation of the promoters on CYP4A gene. CYP7A was recognized as the first target gene of the liver X receptor (LXR), in which the elimination of cholesterol depends on CYP7A. Farnesoid X receptor (FXR) was identified as a bile acid receptor, and its activation results in the inhibition of hepatic acid biosynthesis and increased transport of bile acids from intestinal lumen to the liver, and CYP7A is one of its target genes. The transcriptional activation by these receptors upon binding to the promoters located at the 5-flanking region of these CYP genes generally leads to the induction of their mRNA gene expression. The physiological and the pharmacological implications of common partner of RXR for CAR, PXR, PPAR, LXR and FXR receptors largely remain unknown and are under intense investigations. For the phase II DMEs, phase II gene inducers such as the phenolic compounds butylated hydroxyanisol (BHA), tert-butylhydroquinone (tBHQ), green tea polyphenol (GTP), (-)-epigallocatechin-3-gallate (EGCG) and the isothiocyanates (PEITC, sulforaphane) generally appear to be electrophiles. They generally possess electrophilic-mediated stress response, resulting in the activation of bZIP transcription factors Nrf2 which dimerizes with Mafs and binds to the antioxidant/electrophile resp...

The contribution of rare and low-frequency variants to human traits is largely unexplored. Here we describe insights from sequencing whole genomes (low read depth, 7×) or exomes (high read depth, 80×) of nearly 10,000 individuals from population-based and disease collections. In extensively phenotyped cohorts we characterize over 24 million novel sequence variants, generate a highly accurate imputation reference panel and identify novel alleles associated with levels of triglycerides (APOB), adiponectin (ADIPOQ) and low-density lipoprotein cholesterol (LDLR and RGAG1) from single-marker and rare variant aggregation tests. We describe population structure and functional annotation of rare and low-frequency variants, use the data to estimate the benefits of sequencing for association studies, and summarize lessons from disease-specific collections. Finally, we make available an extensive resource, including individual-level genetic and phenotypic data and web-based tools to facilitate the exploration of association results

Oxidative stress has been implicated in the etiology of neurodegenerative disease, cancer and aging. Indeed, the reactive oxygen and nitrogen species generated by inflammatory cells that created oxidative stress is thought to be one of the major factor by which chronic inflammation contributes to neoplastic transformation as well as many other diseases. We have recently reported that mice lacking nuclear factor-erythroid 2-related factor 2 (Nrf2) are more susceptibility to dextran sulfate sodium (DSS)-induced colitis and colorectal carcinogenesis. Nrf2 is a basic leucine zipper redoxsensitive transcriptional factor that plays a center role in ARE (antioxidant response element)-mediated induction of phase II detoxifying and antioxidant enzymes. We found that increased susceptibility of Nrf2 deficient mice to DSS-induced colitis and colorectal cancer was associated with decreased expression of antioxidant/phase II detoxifying enzymes in parallel with upregulation of pro-inflammatory cytokines/biomarkers. These findings suggest that Nrf2 may play an important role in defense against oxidative stress possibly by activation of cellular antioxidant machinery as well as suppression of pro-inflammatory signaling pathways. In addition, in vivo and in vitro data generated from our laboratory suggest that many dietary compounds can differentially regulate Nrf2-mediated antioxidant/anti-inflammatory signaling pathways as the first line defense or induce apoptosis once the cells have been damaged. In this review, we will summarize our thoughts on the potential cross-talks between Nrf2 and NF-κB pathways. Although the mechanisms involved in the cross-talk between these signaling pathways are still illusive, targeting Nrf2-antioxidative stress signaling is an ideal strategy to prevent or treat oxidative-stress related diseases.

Osteoporosis is a common disease diagnosed primarily by measurement of bone mineral density (BMD). We undertook a genome-wide association study in 142,487 individuals from the UK Biobank to identify loci associated with BMD estimated by quantitative ultrasound of the heel (“eBMD”). We identified 307 conditionally independent SNPs attaining genome-wide significance at 203 loci, explaining approximately 12% of the phenotypic variance. These included 153 novel loci, and several rare variants with large effect sizes. To investigate underlying mechanisms we undertook: 1) bioinformatic, functional genomic annotation and human osteoblast expression studies; 2) gene function prediction; 3) skeletal phenotyping of 120 knockout mice with deletions of genes adjacent to lead independent SNPs; and 4) analysis of gene expression in mouse osteoblasts, osteocytes and osteoclasts. These studies strongly implicate GPC6 as a novel determinant of BMD and also identify abnormal skeletal phenotypes in knockout mice for a further 100 prioritized genes.

In acute myeloid leukaemia, long-term survival is poor as most patients relapse despite achieving remission. Historically, the failure of therapy has been thought to be due to mutations that produce drug resistance, possibly arising as a consequence of the mutagenic properties of chemotherapy drugs. However, other lines of evidence have pointed to the pre-existence of drug-resistant cells. For example, deep sequencing of paired diagnosis and relapse acute myeloid leukaemia samples has provided direct evidence that relapse in some cases is generated from minor genetic subclones present at diagnosis that survive chemotherapy, suggesting that resistant cells are generated by evolutionary processes before treatment and are selected by therapy. Nevertheless, the mechanisms of therapy failure and capacity for leukaemic regeneration remain obscure, as sequence analysis alone does not provide insight into the cell types that are fated to drive relapse. Although leukaemia stem cells have been linked to relapse owing to their dormancy and self-renewal properties, and leukaemia stem cell gene expression signatures are highly predictive of therapy failure, experimental studies have been primarily correlative and a role for leukaemia stem cells in acute myeloid leukaemia relapse has not been directly proved. Here, through combined genetic and functional analysis of purified subpopulations and xenografts from paired diagnosis/relapse samples, we identify therapy-resistant cells already present at diagnosis and two major patterns of relapse. In some cases, relapse originated from rare leukaemia stem cells with a haematopoietic stem/progenitor cell phenotype, while in other instances relapse developed from larger subclones of immunophenotypically committed leukaemia cells that retained strong stemness transcriptional signatures. The identification of distinct patterns of relapse should lead to improved methods for disease management and monitoring in acute myeloid leukaemia. Moreover, the shared functional and transcriptional stemness properties that underlie both cellular origins of relapse emphasize the importance of developing new therapeutic approaches that target stemness to prevent relapse.

Sulforaphane, a dietary isothiocyanate, possesses potent chemopreventive effects through the induction of cellular detoxifying/antioxidant enzymes via the transcription factor nuclear factor E2-related factor 2 (Nrf2). To investigate carcinogenesis mechanisms related to the regulation of Nrf2, we examined the tumor incidence and tumor numbers per mouse in Nrf2 wild-type (+/+) and Nrf2 knockout (À/À) mice. 7,12-Dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate treatments resulted in an increase in the incidence of skin tumors and tumor numbers per mouse in both genotypes; however, both indices were markedly higher in Nrf2(À/À) mice as compared with Nrf2(+/+) mice. Western blot analysis revealed that Nrf2 as well as heme oxygenase-1, a protein regulated by Nrf2 were not expressed in skin tumors from mice of either genotype, whereas expression of heme oxygenase-1 in Nrf2(+/+) mice was much higher than that in Nrf2(À/À) mice in nontumor skin samples. Next, we examined the chemopreventive efficacy of sulforaphane in mice with both genotypes. Topical application of 100 nmol of sulforaphane once a day for 14 days prior to 7,12-dimethylbenz(a)anthracene/12-O -tetradecanoylphorbol-13-acetate applications decreased the incidence of skin tumor in the Nrf2(+/+) mice when compared with the vehicle-treated group. Importantly, there was no chemoprotective effect elicited by sulforaphane pretreatment in the Nrf2(À/À) mice group. Taken together, our results show for the first time that Nrf2(À/À) mice are more susceptible to skin tumorigenesis and that the chemopreventive effects of sulforaphane are mediated, at least in part, through Nrf2. (Cancer Res 2006; 66(16): 8293-6)

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