A functional genomic framework to elucidate novel causal non-alcoholic fatty liver disease genes.

Background & Aims: Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver pathology in western countries, with serious public health consequences. Efforts to identify causal genes for NAFLD have been hampered by the relative paucity of human data from gold-standard magnetic resonance quantification of hepatic fat. To overcome insufficient sample size, genome-wide association studies using NAFLD surrogate phenotypes have been used, but only a small number of loci have been identified to date. In this study, we combined GWAS of NAFLD composite surrogate phenotypes with genetic colocalization studies followed by functional in vitro screens to identify bona fide causal genes for NAFLD. Approach & Results: We used the UK Biobank to explore the associations of our novel NAFLD score, and genetic colocalization to prioritize putative causal genes for in vitro validation. We created a functional genomic framework to study NAFLD genes in vitro using CRISPRi. Our data identify VKORC1, TNKS, LYPLAL1 and GPAM as regulators of lipid accumulation in hepatocytes and suggest the involvement of VKORC1 in the lipid storage related to the development of NAFLD. Conclusions: Complementary genetic and genomic approaches are useful for the identification of NAFLD genes. Our data supports VKORC1 as a bona fide NAFLD gene. We have established a functional genomic framework to study at scale putative novel NAFLD genes from human genetic association studies.

A single-cell CRISPRi platform for characterizing candidate genes relevant to metabolic disorders in human adipocytes.

CROP-Seq combines gene silencing using CRISPR interference with single-cell RNA sequencing. Here, we applied CROP-Seq to study adipogenesis and adipocyte biology. Human preadipocyte SGBS cell line expressing KRAB-dCas9 was transduced with a sgRNA library. Following selection, individual cells were captured using microfluidics at different timepoints during adipogenesis. Bioinformatic analysis of transcriptomic data was used to determine the knockdown effects, the dysregulated pathways, and to predict cellular phenotypes. Single-cell transcriptomes recapitulated adipogenesis states. For all targets, over 400 differentially expressed genes were identified at least at one timepoint. As a validation of our approach, the knockdown of PPARG and CEBPB (which encode key proadipogenic transcription factors) resulted in the inhibition of adipogenesis. Gene set enrichment analysis generated hypotheses regarding the molecular function of novel genes. MAFF knockdown led to downregulation of transcriptional response to proinflammatory cytokine TNF-α in preadipocytes and to decreased CXCL-16 and IL-6 secretion. TIPARP knockdown resulted in increased expression of adipogenesis markers. In summary, this powerful, hypothesis-free tool can identify novel regulators of adipogenesis, preadipocyte, and adipocyte function associated with metabolic disease.NEW & NOTEWORTHY Genomics efforts led to the identification of many genomic loci that are associated with metabolic traits, many of which are tied to adipose tissue function. However, determination of the causal genes, and their mechanism of action in metabolism, is a time-consuming process. Here, we use an approach to determine the transcriptional outcome of candidate gene knockdown for multiple genes at the same time in a human cell model of adipogenesis.

Integration of genetic colocalizations with physiological and pharmacological perturbations identifies cardiometabolic disease genes.

BACKGROUND: Identification of causal genes for polygenic human diseases has been extremely challenging, and our understanding of how physiological and pharmacological stimuli modulate genetic risk at disease-associated loci is limited. Specifically, insulin resistance (IR), a common feature of cardiometabolic disease, including type 2 diabetes, obesity, and dyslipidemia, lacks well-powered genome-wide association studies (GWAS), and therefore, few associated loci and causal genes have been identified. METHODS: Here, we perform and integrate linkage disequilibrium (LD)-adjusted colocalization analyses across nine cardiometabolic traits (fasting insulin, fasting glucose, insulin sensitivity, insulin sensitivity index, type 2 diabetes, triglycerides, high-density lipoprotein, body mass index, and waist-hip ratio) combined with expression and splicing quantitative trait loci (eQTLs and sQTLs) from five metabolically relevant human tissues (subcutaneous and visceral adipose, skeletal muscle, liver, and pancreas). To elucidate the upstream regulators and functional mechanisms for these genes, we integrate their transcriptional responses to 21 relevant physiological and pharmacological perturbations in human adipocytes, hepatocytes, and skeletal muscle cells and map their protein-protein interactions. RESULTS: We identify 470 colocalized loci and prioritize 207 loci with a single colocalized gene. Patterns of shared colocalizations across traits and tissues highlight different potential roles for colocalized genes in cardiometabolic disease and distinguish several genes involved in pancreatic ß-cell function from others with a more direct role in skeletal muscle, liver, and adipose tissues. At the loci with a single colocalized gene, 42 of these genes were regulated by insulin and 35 by glucose in perturbation experiments, including 17 regulated by both. Other metabolic perturbations regulated the expression of 30 more genes not regulated by glucose or insulin, pointing to other potential upstream regulators of candidate causal genes. CONCLUSIONS: Our use of transcriptional responses under metabolic perturbations to contextualize genetic associations from our custom colocalization approach provides a list of likely causal genes and their upstream regulators in the context of IR-associated cardiometabolic risk.

Interactions of physical activity, muscular fitness, adiposity, and genetic risk for NAFLD.

Genetic predisposition and unhealthy lifestyle are risk factors for nonalcoholic fatty liver disease (NAFLD). We investigated whether the genetic risk of NAFLD is modified by physical activity, muscular fitness, and/or adiposity. In up to 242,524 UK Biobank participants without excessive alcohol intake or known liver disease, we examined cross-sectional interactions and joint associations of physical activity, muscular fitness, body mass index (BMI), and a genetic risk score (GRS) with alanine aminotransferase (ALT) levels and the proxy definition for suspected NAFLD of ALT levels > 30 U/L in women and >40 U/L in men. Genetic predisposition to NAFLD was quantified using a GRS consisting of 68 loci known to be associated with chronically elevated ALT. Physical activity was assessed using accelerometry, and muscular fitness was estimated by measuring handgrip strength. We found that increased physical activity and grip strength modestly attenuate genetic predisposition to elevation in ALT levels, whereas higher BMI markedly amplifies it (all p values < 0.001). Among those with normal weight and high level of physical activity, the odds of suspected NAFLD were 1.6-fold higher in those with high versus low genetic risk (reference group). In those with high genetic risk, the odds of suspected NAFLD were 12-fold higher in obese participants with low physical activity versus those with normal weight and high physical activity (odds ratio for NAFLD = 19.2 and 1.6, respectively, vs. reference group). Conclusion: In individuals with high genetic predisposition for NAFLD, maintaining a normal body weight and increased physical activity may reduce the risk of NAFLD.

An integrated approach to identify environmental modulators of genetic risk factors for complex traits.

Complex traits and diseases can be influenced by both genetics and environment. However, given the large number of environmental stimuli and power challenges for gene-by-environment testing, it remains a critical challenge to identify and prioritize specific disease-relevant environmental exposures. We propose a framework for leveraging signals from transcriptional responses to environmental perturbations to identify disease-relevant perturbations that can modulate genetic risk for complex traits and inform the functions of genetic variants associated with complex traits. We perturbed human skeletal-muscle-, fat-, and liver-relevant cell lines with 21 perturbations affecting insulin resistance, glucose homeostasis, and metabolic regulation in humans and identified thousands of environmentally responsive genes. By combining these data with GWASs from 31 distinct polygenic traits, we show that the heritability of multiple traits is enriched in regions surrounding genes responsive to specific perturbations and, further, that environmentally responsive genes are enriched for associations with specific diseases and phenotypes from the GWAS Catalog. Overall, we demonstrate the advantages of large-scale characterization of transcriptional changes in diversely stimulated and pathologically relevant cells to identify disease-relevant perturbations.

Signaling defects associated with insulin resistance in nondiabetic and diabetic individuals and modification by sex.

Insulin resistance is present in one-quarter of the general population, predisposing these people to a wide range of diseases. Our aim was to identify cell-intrinsic determinants of insulin resistance in this population using induced pluripotent stem cell-derived (iPSC-derived) myoblasts (iMyos). We found that these cells exhibited a large network of altered protein phosphorylation in vitro. Integrating these data with data from type 2 diabetic iMyos revealed critical sites of conserved altered phosphorylation in IRS-1, AKT, mTOR, and TBC1D1 in addition to changes in protein phosphorylation involved in Rho/Rac signaling, chromatin organization, and RNA processing. There were also striking differences in the phosphoproteome in cells from men versus women. These sex-specific and insulin-resistance defects were linked to functional differences in downstream actions. Thus, there are cell-autonomous signaling alterations associated with insulin resistance within the general population and important differences between men and women, many of which also occur in diabetes, that contribute to differences in physiology and disease.

Isthmin-1 is an adipokine that promotes glucose uptake and improves glucose tolerance and hepatic steatosis.

With the increasing prevalence of type 2 diabetes and fatty liver disease, there is still an unmet need to better treat hyperglycemia and hyperlipidemia. Here, we identify isthmin-1 (Ism1) as an adipokine and one that has a dual role in increasing adipose glucose uptake while suppressing hepatic lipid synthesis. Ism1 ablation results in impaired glucose tolerance, reduced adipose glucose uptake, and reduced insulin sensitivity, demonstrating an endogenous function for Ism1 in glucose regulation. Mechanistically, Ism1 activates a PI3K-AKT signaling pathway independently of the insulin and insulin-like growth factor receptors. Notably, while the glucoregulatory function is shared with insulin, Ism1 counteracts lipid accumulation in the liver by switching hepatocytes from a lipogenic to a protein synthesis state. Furthermore, therapeutic dosing of recombinant Ism1 improves diabetes in diet-induced obese mice and ameliorates hepatic steatosis in a diet-induced fatty liver mouse model. These findings uncover an unexpected, bioactive protein hormone that might have simultaneous therapeutic potential for diabetes and fatty liver disease.

Identification of rare and common regulatory variants in pluripotent cells using population-scale transcriptomics.

Induced pluripotent stem cells (iPSCs) are an established cellular system to study the impact of genetic variants in derived cell types and developmental contexts. However, in their pluripotent state, the disease impact of genetic variants is less well known. Here, we integrate data from 1,367 human iPSC lines to comprehensively map common and rare regulatory variants in human pluripotent cells. Using this population-scale resource, we report hundreds of new colocalization events for human traits specific to iPSCs, and find increased power to identify rare regulatory variants compared with somatic tissues. Finally, we demonstrate how iPSCs enable the identification of causal genes for rare diseases.

Predictive network modeling in human induced pluripotent stem cells identifies key driver genes for insulin responsiveness.

Insulin resistance (IR) precedes the development of type 2 diabetes (T2D) and increases cardiovascular disease risk. Although genome wide association studies (GWAS) have uncovered new loci associated with T2D, their contribution to explain the mechanisms leading to decreased insulin sensitivity has been very limited. Thus, new approaches are necessary to explore the genetic architecture of insulin resistance. To that end, we generated an iPSC library across the spectrum of insulin sensitivity in humans. RNA-seq based analysis of 310 induced pluripotent stem cell (iPSC) clones derived from 100 individuals allowed us to identify differentially expressed genes between insulin resistant and sensitive iPSC lines. Analysis of the co-expression architecture uncovered several insulin sensitivity-relevant gene sub-networks, and predictive network modeling identified a set of key driver genes that regulate these co-expression modules. Functional validation in human adipocytes and skeletal muscle cells (SKMCs) confirmed the relevance of the key driver candidate genes for insulin responsiveness.

Genomic integrity of human induced pluripotent stem cells across nine studies in the NHLBI NextGen program.

Human induced pluripotent stem cell (hiPSC) lines have previously been generated through the NHLBI sponsored NextGen program at nine individual study sites. Here, we examined the structural integrity of 506 hiPSC lines as determined by copy number variations (CNVs). We observed that 149 hiPSC lines acquired 258 CNVs relative to donor DNA. We identified six recurrent regions of CNVs on chromosomes 1, 2, 3, 16 and 20 that overlapped with cancer associated genes. Furthermore, the genes mapping to regions of acquired CNVs show an enrichment in cancer related biological processes (IL6 production) and signaling cascades (JNK cascade & NFκB cascade). The genomic region of instability on chr20 (chr20q11.2) includes transcriptomic signatures for cancer associated genes such as ID1, BCL2L1, TPX2, PDRG1 and HCK. Of these HCK shows statistically significant differential expression between carrier and non-carrier hiPSC lines. Overall, while a low level of genomic instability was observed in the NextGen generated hiPSC lines, the observation of structural instability in regions with known cancer associated genes substantiates the importance of systematic evaluation of genetic variations in hiPSCs before using them as disease/research models.

FAM13A affects body fat distribution and adipocyte function.

Genetic variation in the FAM13A (Family with Sequence Similarity 13 Member A) locus has been associated with several glycemic and metabolic traits in genome-wide association studies (GWAS). Here, we demonstrate that in humans, FAM13A alleles are associated with increased FAM13A expression in subcutaneous adipose tissue (SAT) and an insulin resistance-related phenotype (e.g. higher waist-to-hip ratio and fasting insulin levels, but lower body fat). In human adipocyte models, knockdown of FAM13A in preadipocytes accelerates adipocyte differentiation. In mice, Fam13a knockout (KO) have a lower visceral to subcutaneous fat (VAT/SAT) ratio after high-fat diet challenge, in comparison to their wild-type counterparts. Subcutaneous adipocytes in KO mice show a size distribution shift toward an increased number of smaller adipocytes, along with an improved adipogenic potential. Our results indicate that GWAS-associated variants within the FAM13A locus alter adipose FAM13A expression, which in turn, regulates adipocyte differentiation and contribute to changes in body fat distribution.

Induced Pluripotent Stem Cell-Derived Endothelial Cells in Insulin Resistance and Metabolic Syndrome.

Insulin resistance leads to a number of metabolic and cellular abnormalities including endothelial dysfunction that increase the risk of vascular disease. Although it has been particularly challenging to study the genetic determinants that predispose to abnormal function of the endothelium in insulin-resistant states, the possibility of deriving endothelial cells from induced pluripotent stem cells generated from individuals with detailed clinical phenotyping, including accurate measurements of insulin resistance accompanied by multilevel omic data (eg, genetic and genomic characterization), has opened new avenues to study this relationship. Unfortunately, several technical barriers have hampered these efforts. In the present review, we summarize the current status of induced pluripotent stem cell-derived endothelial cells for modeling endothelial dysfunction associated with insulin resistance and discuss the challenges to overcoming these limitations.

TCF21 and the environmental sensor aryl-hydrocarbon receptor cooperate to activate a pro-inflammatory gene expression program in coronary artery smooth muscle cells.

Both environmental factors and genetic loci have been associated with coronary artery disease (CAD), however gene-gene and gene-environment interactions that might identify molecular mechanisms of risk are not easily studied by human genetic approaches. We have previously identified the transcription factor TCF21 as the causal CAD gene at 6q23.2 and characterized its downstream transcriptional network that is enriched for CAD GWAS genes. Here we investigate the hypothesis that TCF21 interacts with a downstream target gene, the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor that mediates the cellular response to environmental contaminants, including dioxin and polycyclic aromatic hydrocarbons (e.g., tobacco smoke). Perturbation of TCF21 expression in human coronary artery smooth muscle cells (HCASMC) revealed that TCF21 promotes expression of AHR, its heterodimerization partner ARNT, and cooperates with these factors to upregulate a number of inflammatory downstream disease related genes including IL1A, MMP1, and CYP1A1. TCF21 was shown to bind in AHR, ARNT and downstream target gene loci, and co-localization was noted for AHR-ARNT and TCF21 binding sites genome-wide in regions of HCASMC open chromatin. These regions of co-localization were found to be enriched for GWAS signals associated with cardio-metabolic as well as chronic inflammatory disease phenotypes. Finally, we show that similar to TCF21, AHR gene expression is increased in atherosclerotic lesions in mice in vivo using laser capture microdissection, and AHR protein is localized in human carotid atherosclerotic lesions where it is associated with protein kinases with a critical role in innate immune response. These data suggest that TCF21 can cooperate with AHR to activate an inflammatory gene expression program that is exacerbated by environmental stimuli, and may contribute to the overall risk for CAD.

Analysis of Transcriptional Variability in a Large Human iPSC Library Reveals Genetic and Non-genetic Determinants of Heterogeneity.

Variability in induced pluripotent stem cell (iPSC) lines remains a concern for disease modeling and regenerative medicine. We have used RNA-sequencing analysis and linear mixed models to examine the sources of gene expression variability in 317 human iPSC lines from 101 individuals. We found that ∼50% of genome-wide expression variability is explained by variation across individuals and identified a set of expression quantitative trait loci that contribute to this variation. These analyses coupled with allele-specific expression show that iPSCs retain a donor-specific gene expression pattern. Network, pathway, and key driver analyses showed that Polycomb targets contribute significantly to the non-genetic variability seen within and across individuals, highlighting this chromatin regulator as a likely source of reprogramming-based variability. Our findings therefore shed light on variation between iPSC lines and illustrate the potential for our dataset and other similar large-scale analyses to identify underlying drivers relevant to iPSC applications.

Nat1 Deficiency Is Associated with Mitochondrial Dysfunction and Exercise Intolerance in Mice.

We recently identified human N-acetyltransferase 2 (NAT2) as an insulin resistance (IR) gene. Here, we examine the cellular mechanism linking NAT2 to IR and find that Nat1 (mouse ortholog of NAT2) is co-regulated with key mitochondrial genes. RNAi-mediated silencing of Nat1 led to mitochondrial dysfunction characterized by increased intracellular reactive oxygen species and mitochondrial fragmentation as well as decreased mitochondrial membrane potential, biogenesis, mass, cellular respiration, and ATP generation. These effects were consistent in 3T3-L1 adipocytes, C2C12 myoblasts, and in tissues from Nat1-deficient mice, including white adipose tissue, heart, and skeletal muscle. Nat1-deficient mice had changes in plasma metabolites and lipids consistent with a decreased ability to utilize fats for energy and a decrease in basal metabolic rate and exercise capacity without altered thermogenesis. Collectively, our results suggest that Nat1 deficiency results in mitochondrial dysfunction, which may constitute a mechanistic link between this gene and IR.

Identification and validation of N-acetyltransferase 2 as an insulin sensitivity gene.

Decreased insulin sensitivity, also referred to as insulin resistance (IR), is a fundamental abnormality in patients with type 2 diabetes and a risk factor for cardiovascular disease. While IR predisposition is heritable, the genetic basis remains largely unknown. The GENEticS of Insulin Sensitivity consortium conducted a genome-wide association study (GWAS) for direct measures of insulin sensitivity, such as euglycemic clamp or insulin suppression test, in 2,764 European individuals, with replication in an additional 2,860 individuals. The presence of a nonsynonymous variant of N-acetyltransferase 2 (NAT2) [rs1208 (803A>G, K268R)] was strongly associated with decreased insulin sensitivity that was independent of BMI. The rs1208 "A" allele was nominally associated with IR-related traits, including increased fasting glucose, hemoglobin A1C, total and LDL cholesterol, triglycerides, and coronary artery disease. NAT2 acetylates arylamine and hydrazine drugs and carcinogens, but predicted acetylator NAT2 phenotypes were not associated with insulin sensitivity. In a murine adipocyte cell line, silencing of NAT2 ortholog Nat1 decreased insulin-mediated glucose uptake, increased basal and isoproterenol-stimulated lipolysis, and decreased adipocyte differentiation, while Nat1 overexpression produced opposite effects. Nat1-deficient mice had elevations in fasting blood glucose, insulin, and triglycerides and decreased insulin sensitivity, as measured by glucose and insulin tolerance tests, with intermediate effects in Nat1 heterozygote mice. Our results support a role for NAT2 in insulin sensitivity.

Regulation of human bone marrow stromal cell proliferation and differentiation capacity by glucocorticoid receptor and AP-1 crosstalk.

Although marrow adipocytes and osteoblasts derive from a common bone marrow stromal cells (BMSCs), the mechanisms that underlie osteoporosis-associated bone loss and marrow adipogenesis during prolonged steroid treatment are unclear. We show in human BMSCs (hBMSCs) that glucocorticoid receptor (GR) signaling in response to high concentrations of glucocorticoid (GC) supports adipogenesis but inhibits osteogenesis by reducing c-Jun expression and hBMSC proliferation. Conversely, significantly lower concentrations of GC, which permit hBMSC proliferation, are necessary for normal bone mineralization. In contrast, platelet-derived growth factor (PDGF) signaling increases both JNK/c-Jun activity and hBMSC expansion, favoring osteogenic differentiation instead of adipogenesis. Indeed, PDGF antagonizes the proadipogenic qualities of GC/GR signaling. Thus our results reveal a novel c-Jun-centered regulatory network of signaling pathways in differentiating hBMSCs that controls the proliferation-dependent balance between osteogenesis and adipogenesis.

ERK2 protein regulates the proliferation of human mesenchymal stem cells without affecting their mobilization and differentiation potential.

Human Mesenchymal Stem Cells (hMSC), derived mainly from adult bone marrow, are valuable models for the study of processes involved in stem cell self-renewal and differentiation. As the Extracellular signal-Regulated Kinase (ERK) signalling pathway is a major contributor to cellular growth, differentiation and survival, we have studied the functions of this kinase in hMSC activity. Ablation of ERK2 gene expression (but not ERK1) by RNA interference significantly reduced proliferation of hMSC. This reduction was due to a defect in Cyclin D1 expression and subsequent arrest in the G0/G1 phase of the cell cycle. hMSC growth is enhanced through culture medium supplementation with growth factors (GFs) such as Platelet-Derived Growth Factor (PDGF), basic Fibroblast Growth Factor (bFGF) or Epidermal Growth Factor (EGF). However, these supplements could not rescue the defect observed after ERK2 knockdown, suggesting a common signalling pathway used by these GFs for proliferation. In contrast, ERK1/2 may be dissociated from chemotactic signalling induced by the same GFs. Additionally, hMSCs were capable of differentiating into adipocytes even in the absence of either ERK1 or ERK2 proteins. Our data show that hMSCs do not require cell division to enter the adipogenic differentiation process, indicating that clonal amplification of these cells is not a critical step. However, cell-cell contact seems to be an essential requirement to be able to differentiate into mature adipocytes.