Microbiota-dependent activation of the myeloid calcineurin-NFAT pathway inhibits B7H3- and B7H4-dependent anti-tumor immunity in colorectal cancer.

Bacterial sensing by intestinal tumor cells contributes to tumor growth through cell-intrinsic activation of the calcineurin-NFAT axis, but the role of this pathway in other intestinal cells remains unclear. Here, we found that myeloid-specific deletion of calcineurin in mice activated protective CD8+ T cell responses and inhibited colorectal cancer (CRC) growth. Microbial sensing by myeloid cells promoted calcineurin- and NFAT-dependent interleukin 6 (IL-6) release, expression of the co-inhibitory molecules B7H3 and B7H4 by tumor cells, and inhibition of CD8+ T cell-dependent anti-tumor immunity. Accordingly, targeting members of this pathway activated protective CD8+ T cell responses and inhibited primary and metastatic CRC growth. B7H3 and B7H4 were expressed by the majority of human primary CRCs and metastases, which was associated with low numbers of tumor-infiltrating CD8+ T cells and poor survival. Therefore, a microbiota-, calcineurin-, and B7H3/B7H4-dependent pathway controls anti-tumor immunity, revealing additional targets for immune checkpoint inhibition in microsatellite-stable CRC.

Apical bulkheads accumulate as adaptive response to impaired bile flow in liver disease.

Hepatocytes form bile canaliculi that dynamically respond to the signalling activity of bile acids and bile flow. Little is known about their responses to intraluminal pressure. During embryonic development, hepatocytes assemble apical bulkheads that increase the canalicular resistance to intraluminal pressure. Here, we investigate whether they also protect bile canaliculi against elevated pressure upon impaired bile flow in adult liver. Apical bulkheads accumulate upon bile flow obstruction in mouse models and patients with primary sclerosing cholangitis (PSC). Their loss under these conditions leads to abnormally dilated canaliculi, resembling liver cell rosettes described in other hepatic diseases. 3D reconstruction reveals that these structures are sections of cysts and tubes formed by hepatocytes. Mathematical modelling establishes that they positively correlate with canalicular pressure and occur in early PSC stages. Using primary hepatocytes and 3D organoids, we demonstrate that excessive canalicular pressure causes the loss of apical bulkheads and formation of rosettes. Our results suggest that apical bulkheads are a protective mechanism of hepatocytes against impaired bile flow, highlighting the role of canalicular pressure in liver diseases.

Genetic variation in TERT modifies the risk of hepatocellular carcinoma in alcohol-related cirrhosis: results from a genome-wide case-control study.

OBJECTIVE: Hepatocellular carcinoma (HCC) often develops in patients with alcohol-related cirrhosis at an annual risk of up to 2.5%. Some host genetic risk factors have been identified but do not account for the majority of the variance in occurrence. This study aimed to identify novel susceptibility loci for the development of HCC in people with alcohol related cirrhosis. DESIGN: Patients with alcohol-related cirrhosis and HCC (cases: n=1214) and controls without HCC (n=1866), recruited from Germany, Austria, Switzerland, Italy and the UK, were included in a two-stage genome-wide association study using a case-control design. A validation cohort of 1520 people misusing alcohol but with no evidence of liver disease was included to control for possible association effects with alcohol misuse. Genotyping was performed using the InfiniumGlobal Screening Array (V.24v2, Illumina) and the OmniExpress Array (V.24v1-0a, Illumina). RESULTS: Associations with variants rs738409 in PNPLA3 and rs58542926 in TM6SF2 previously associated with an increased risk of HCC in patients with alcohol-related cirrhosis were confirmed at genome-wide significance. A novel locus rs2242652(A) in TERT (telomerase reverse transcriptase) was also associated with a decreased risk of HCC, in the combined meta-analysis, at genome-wide significance (p=6.41×10-9, OR=0.61 (95% CI 0.52 to 0.70). This protective association remained significant after correction for sex, age, body mass index and type 2 diabetes (p=7.94×10-5, OR=0.63 (95% CI 0.50 to 0.79). Carriage of rs2242652(A) in TERT was associated with an increased leucocyte telomere length (p=2.12×10-44). CONCLUSION: This study identifies rs2242652 in TERT as a novel protective factor for HCC in patients with alcohol-related cirrhosis.

Epigenome-wide association study of body mass index, and the adverse outcomes of adiposity.

Approximately 1.5 billion people worldwide are overweight or affected by obesity, and are at risk of developing type 2 diabetes, cardiovascular disease and related metabolic and inflammatory disturbances. Although the mechanisms linking adiposity to associated clinical conditions are poorly understood, recent studies suggest that adiposity may influence DNA methylation, a key regulator of gene expression and molecular phenotype. Here we use epigenome-wide association to show that body mass index (BMI; a key measure of adiposity) is associated with widespread changes in DNA methylation (187 genetic loci with P < 1 × 10-7, range P = 9.2 × 10-8 to 6.0 × 10-46; n = 10,261 samples). Genetic association analyses demonstrate that the alterations in DNA methylation are predominantly the consequence of adiposity, rather than the cause. We find that methylation loci are enriched for functional genomic features in multiple tissues (P < 0.05), and show that sentinel methylation markers identify gene expression signatures at 38 loci (P < 9.0 × 10-6, range P = 5.5 × 10-6 to 6.1 × 10-35, n = 1,785 samples). The methylation loci identify genes involved in lipid and lipoprotein metabolism, substrate transport and inflammatory pathways. Finally, we show that the disturbances in DNA methylation predict future development of type 2 diabetes (relative risk per 1 standard deviation increase in methylation risk score: 2.3 (2.07-2.56); P = 1.1 × 10-54). Our results provide new insights into the biologic pathways influenced by adiposity, and may enable development of new strategies for prediction and prevention of type 2 diabetes and other adverse clinical consequences of obesity.

SETDB1 is required for intestinal epithelial differentiation and the prevention of intestinal inflammation.

OBJECTIVE: The intestinal epithelium is a rapidly renewing tissue which plays central roles in nutrient uptake, barrier function and the prevention of intestinal inflammation. Control of epithelial differentiation is essential to these processes and is dependent on cell type-specific activity of transcription factors which bind to accessible chromatin. Here, we studied the role of SET Domain Bifurcated Histone Lysine Methyltransferase 1, also known as ESET (SETDB1), a histone H3K9 methyltransferase, in intestinal epithelial homeostasis and IBD. DESIGN: We investigated mice with constitutive and inducible intestinal epithelial deletion of Setdb1, studied the expression of SETDB1 in patients with IBD and mouse models of IBD, and investigated the abundance of SETDB1 variants in healthy individuals and patients with IBD. RESULTS: Deletion of intestinal epithelial Setdb1 in mice was associated with defects in intestinal epithelial differentiation, barrier disruption, inflammation and mortality. Mechanistic studies showed that loss of SETDB1 leads to de-silencing of endogenous retroviruses, DNA damage and intestinal epithelial cell death. Predicted loss-of-function variants in human SETDB1 were considerably less frequently observed than expected, consistent with a critical role of SETDB1 in human biology. While the vast majority of patients with IBD showed unimpaired mucosal SETDB1 expression, comparison of IBD and non-IBD exomes revealed over-representation of individual rare missense variants in SETDB1 in IBD, some of which are predicted to be associated with loss of function and may contribute to the pathogenesis of intestinal inflammation. CONCLUSION: SETDB1 plays an essential role in intestinal epithelial homeostasis. Future work is required to investigate whether rare variants in SETDB1 contribute to the pathogenesis of IBD.

Loss of hepatic Mboat7 leads to liver fibrosis.

OBJECTIVE: The rs641738C>T variant located near the membrane-bound O-acyltransferase domain containing 7 (MBOAT7) locus is associated with fibrosis in liver diseases, including non-alcoholic fatty liver disease (NAFLD), alcohol-related liver disease, hepatitis B and C. We aim to understand the mechanism by which the rs641738C>T variant contributes to pathogenesis of NAFLD. DESIGN: Mice with hepatocyte-specific deletion of MBOAT7 (Mboat7Δhep) were generated and livers were characterised by histology, flow cytometry, qPCR, RNA sequencing and lipidomics. We analysed the association of rs641738C>T genotype with liver inflammation and fibrosis in 846 NAFLD patients and obtained genotype-specific liver lipidomes from 280 human biopsies. RESULTS: Allelic imbalance analysis of heterozygous human liver samples pointed to lower expression of the MBOAT7 transcript on the rs641738C>T haplotype. Mboat7Δhep mice showed spontaneous steatosis characterised by increased hepatic cholesterol ester content after 10 weeks. After 6 weeks on a high fat, methionine-low, choline-deficient diet, mice developed increased hepatic fibrosis as measured by picrosirius staining (p<0.05), hydroxyproline content (p<0.05) and transcriptomics, while the inflammatory cell populations and inflammatory mediators were minimally affected. In a human biopsied NAFLD cohort, MBOAT7 rs641738C>T was associated with fibrosis (p=0.004) independent of the presence of histological inflammation. Liver lipidomes of Mboat7Δhep mice and human rs641738TT carriers with fibrosis showed increased total lysophosphatidylinositol levels. The altered lysophosphatidylinositol and phosphatidylinositol subspecies in MBOAT7Δhep livers and human rs641738TT carriers were similar. CONCLUSION: Mboat7 deficiency in mice and human points to an inflammation-independent pathway of liver fibrosis that may be mediated by lipid signalling and a potentially targetable treatment option in NAFLD.

Nonalcoholic fatty liver disease stratification by liver lipidomics.

Nonalcoholic fatty liver disease (NAFLD) is a common metabolic dysfunction leading to hepatic steatosis. However, NAFLD's global impact on the liver lipidome is poorly understood. Using high-resolution shotgun mass spectrometry, we quantified the molar abundance of 316 species from 22 major lipid classes in liver biopsies of 365 patients, including nonsteatotic patients with normal or excessive weight, patients diagnosed with NAFL (nonalcoholic fatty liver) or NASH (nonalcoholic steatohepatitis), and patients bearing common mutations of NAFLD-related protein factors. We confirmed the progressive accumulation of di- and triacylglycerols and cholesteryl esters in the liver of NAFL and NASH patients, while the bulk composition of glycerophospho- and sphingolipids remained unchanged. Further stratification by biclustering analysis identified sphingomyelin species comprising n24:2 fatty acid moieties as membrane lipid markers of NAFLD. Normalized relative abundance of sphingomyelins SM 43:3;2 and SM 43:1;2 containing n24:2 and n24:0 fatty acid moieties, respectively, showed opposite trends during NAFLD progression and distinguished NAFL and NASH lipidomes from the lipidome of nonsteatotic livers. Together with several glycerophospholipids containing a C22:6 fatty acid moiety, these lipids serve as markers of early and advanced stages of NAFL.

Genome-Wide Association Study for Alcohol-Related Cirrhosis Identifies Risk Loci in MARC1 and HNRNPUL1.

BACKGROUND AND AIMS: Little is known about genetic factors that affect development of alcohol-related cirrhosis. We performed a genome-wide association study (GWAS) of samples from the United Kingdom Biobank (UKB) to identify polymorphisms associated with risk of alcohol-related liver disease. METHODS: We performed a GWAS of 35,839 participants in the UKB with high intake of alcohol against markers of hepatic fibrosis (FIB-4, APRI, and Forns index scores) and hepatocellular injury (levels of aminotransferases). Loci identified in the discovery analysis were tested for their association with alcohol-related cirrhosis in 3 separate European cohorts (phase 1 validation cohort; n=2545). Variants associated with alcohol-related cirrhosis in the validation at a false discovery rate of less than 20% were then directly genotyped in 2 additional European validation cohorts (phase 2 validation, n=2068). RESULTS: In the GWAS of the discovery cohort, we identified 50 independent risk loci with genome-wide significance (P < 5 × 10-8). Nine of these loci were significantly associated with alcohol-related cirrhosis in the phase 1 validation cohort; 6 of these 9 loci were significantly associated with alcohol-related cirrhosis in phase 2 validation cohort, at a false discovery rate below 5%. The loci included variants in the mitochondrial amidoxime reducing component 1 gene (MARC1) and the heterogeneous nuclear ribonucleoprotein U like 1 gene (HNRNPUL1). After we adjusted for age, sex, body mass index, and type-2 diabetes in the phase 2 validation cohort, the minor A allele of MARC1:rs2642438 was associated with reduced risk of alcohol-related cirrhosis (adjusted odds ratio, 0.76; P=.0027); conversely, the minor C allele of HNRNPUL1:rs15052 was associated with an increased risk of alcohol-related cirrhosis (adjusted odds ratio, 1.30; P=.020). CONCLUSIONS: In a GWAS of samples from the UKB, we identified and validated (in 5 European cohorts) single-nucleotide polymorphisms that affect risk of alcohol-related cirrhosis in opposite directions: the minor A allele in MARC1:rs2642438 decreases risk, whereas the minor C allele in HNRNPUL1:rs15052 increases risk.

Sex-dependent dynamics of metabolism in primary mouse hepatocytes.

The liver is one of the most sexually dimorphic organs. The hepatic metabolic pathways that are subject to sexual dimorphism include xenobiotic, amino acid and lipid metabolism. Non-alcoholic fatty liver disease and hepatocellular carcinoma are among diseases with sex-dependent prevalence, progression and outcome. Although male and female livers differ in their abilities to metabolize foreign compounds, including drugs, sex-dependent treatment and pharmacological dynamics are rarely applied in all relevant cases. Therefore, it is important to consider hepatic sexual dimorphism when developing new treatment strategies and to understand the underlying mechanisms in model systems. We isolated primary hepatocytes from male and female C57BL6/N mice and examined the sex-dependent transcriptome, proteome and extracellular metabolome parameters in the course of culturing them for 96 h. The sex-specific gene expression of the general xenobiotic pathway altered and the female-specific expression of Cyp2b13 and Cyp2b9 was significantly reduced during culture. Sex-dependent differences of several signaling pathways increased, including genes related to serotonin and melatonin degradation. Furthermore, the ratios of male and female gene expression were inversed for other pathways, such as amino acid degradation, beta-oxidation, androgen signaling and hepatic steatosis. Because the primary hepatocytes were cultivated without the influence of known regulators of sexual dimorphism, these results suggest currently unknown modulatory mechanisms of sexual dimorphism in vitro. The large sex-dependent differences in the regulation and dynamics of drug metabolism observed during cultivation can have an immense influence on the evaluation of pharmacodynamic processes when conducting initial preclinical trials to investigate potential new drugs.

Heterozygous carriage of the alpha1-antitrypsin Pi*Z variant increases the risk to develop liver cirrhosis.

OBJECTIVE: Homozygous alpha1-antitrypsin (AAT) deficiency increases the risk for developing cirrhosis, whereas the relevance of heterozygous carriage remains unclear. Hence, we evaluated the impact of the two most relevant AAT variants ('Pi*Z' and 'Pi*S'), present in up to 10% of Caucasians, on subjects with non-alcoholic fatty liver disease (NAFLD) or alcohol misuse. DESIGN: We analysed multicentric case-control cohorts consisting of 1184 people with biopsy-proven NAFLD and of 2462 people with chronic alcohol misuse, both cohorts comprising cases with cirrhosis and controls without cirrhosis. Genotyping for the Pi*Z and Pi*S variants was performed. RESULTS: The Pi*Z variant presented in 13.8% of patients with cirrhotic NAFLD but only in 2.4% of counterparts without liver fibrosis (p<0.0001). Accordingly, the Pi*Z variant increased the risk of NAFLD subjects to develop cirrhosis (adjusted OR=7.3 (95% CI 2.2 to 24.8)). Likewise, the Pi*Z variant presented in 6.2% of alcohol misusers with cirrhosis but only in 2.2% of alcohol misusers without significant liver injury (p<0.0001). Correspondingly, alcohol misusers carrying the Pi*Z variant were prone to develop cirrhosis (adjusted OR=5.8 (95% CI 2.9 to 11.7)). In contrast, the Pi*S variant was not associated with NAFLD-related cirrhosis and only borderline with alcohol-related cirrhosis (adjusted OR=1.47 (95% CI 0.99 to 2.19)). CONCLUSION: The Pi*Z variant is the hitherto strongest single nucleotide polymorphism-based risk factor for cirrhosis in NAFLD and alcohol misuse, whereas the Pi*S variant confers only a weak risk in alcohol misusers. As 2%-4% of Caucasians are Pi*Z carriers, this finding should be considered in genetic counselling of affected individuals.

Genome-wide association analysis of diverticular disease points towards neuromuscular, connective tissue and epithelial pathomechanisms.

OBJECTIVE: Diverticular disease is a common complex disorder characterised by mucosal outpouchings of the colonic wall that manifests through complications such as diverticulitis, perforation and bleeding. We report the to date largest genome-wide association study (GWAS) to identify genetic risk factors for diverticular disease. DESIGN: Discovery GWAS analysis was performed on UK Biobank imputed genotypes using 31 964 cases and 419 135 controls of European descent. Associations were replicated in a European sample of 3893 cases and 2829 diverticula-free controls and evaluated for risk contribution to diverticulitis and uncomplicated diverticulosis. Transcripts at top 20 replicating loci were analysed by real-time quatitative PCR in preparations of the mucosal, submucosal and muscular layer of colon. The localisation of expressed protein at selected loci was investigated by immunohistochemistry. RESULTS: We discovered 48 risk loci, of which 12 are novel, with genome-wide significance and consistent OR in the replication sample. Nominal replication (p<0.05) was observed for 27 loci, and additional 8 in meta-analysis with a population-based cohort. The most significant novel risk variant rs9960286 is located near CTAGE1 with a p value of 2.3×10-10 and 0.002 (ORallelic=1.14 (95% CI 1.05 to 1.24)) in the replication analysis. Four loci showed stronger effects for diverticulitis, PHGR1 (OR 1.32, 95% CI 1.12 to 1.56), FAM155A-2 (OR 1.21, 95% CI 1.04 to 1.42), CALCB (OR 1.17, 95% CI 1.03 to 1.33) and S100A10 (OR 1.17, 95% CI 1.03 to 1.33). CONCLUSION: In silico analyses point to diverticulosis primarily as a disorder of intestinal neuromuscular function and of impaired connective fibre support, while an additional diverticulitis risk might be conferred by epithelial dysfunction.

Aberrant DNA methylation of ADAMTS16 in colorectal and other epithelial cancers.

BACKGROUND: ADAMs (a disintegrin and metalloproteinase) have long been associated with tumor progression. Recent findings indicate that members of the closely related ADAMTS (ADAMs with thrombospondin motifs) family are also critically involved in carcinogenesis. Gene silencing through DNA methylation at CpG loci around e.g. transcription start or enhancer sites is a major mechanism in cancer development. Here, we aimed at identifying genes of the ADAM and ADAMTS family showing altered DNA methylation in the development or colorectal cancer (CRC) and other epithelial tumors. METHODS: We investigated potential changes of DNA methylation affecting ADAM and ADAMTS genes in 117 CRC, 40 lung cancer (LC) and 15 oral squamous-cell carcinoma (SCC) samples. Tumor tissue was analyzed in comparison to adjacent non-malignant tissue of the same patients. The methylation status of 1145 CpGs in 51 ADAM and ADAMTS genes was measured with the HumanMethylation450 BeadChip Array. ADAMTS16 protein expression was analyzed in CRC samples by immunohistochemistry. RESULTS: In CRC, we identified 72 CpGs in 18 genes which were significantly affected by hyper- or hypomethylation in the tumor tissue compared to the adjacent non-malignant tissue. While notable/frequent alterations in methylation patterns within ADAM genes were not observed, conspicuous changes were found in ADAMTS16 and ADAMTS2. To figure out whether these differences would be CRC specific, additional LC and SCC tissue samples were analyzed. Overall, 78 differentially methylated CpGs were found in LC and 29 in SCC. Strikingly, 8 CpGs located in the ADAMTS16 gene were commonly differentially methylated in all three cancer entities. Six CpGs in the promoter region were hypermethylated, whereas 2 CpGs in the gene body were hypomethylated indicative of gene silencing. In line with these findings, ADAMTS16 protein was strongly expressed in globlet cells and colonocytes in control tissue but not in CRC samples. Functional in vitro studies using the colorectal carcinoma cell line HT29 revealed that ADAMTS16 expression restrained tumor cell proliferation. CONCLUSIONS: We identified ADAMTS16 as novel gene with cancer-specific promoter hypermethylation in CRC, LC and SCC patients implicating ADAMTS16 as potential biomarker for these tumors. Moreover, our results provide evidence that ADAMTS16 may have tumor suppressor properties.

Comparison of Gene Expression Patterns Between Mouse Models of Nonalcoholic Fatty Liver Disease and Liver Tissues From Patients.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder in industrialized countries. Mouse models of NAFLD have been used in studies of pathogenesis and treatment, and have certain features of the human disease. We performed a systematic transcriptome-wide analysis of liver tissues from patients at different stages of NAFLD progression (ranging from healthy obese individuals to those with steatosis), as well as rodent models of NAFLD, to identify those that most closely resemble human disease progression in terms of gene expression patterns. METHODS: We performed a systematic evaluation of genome-wide messenger RNA expression using liver tissues collected from mice fed a standard chow diet (controls) and 9 mouse models of NAFLD: mice on a high-fat diet (with or without fructose), mice on a Western-type diet, mice on a methionine- and choline-deficient diet, mice on a high-fat diet given streptozotocin, and mice with disruption of Pten in hepatocytes. We compared gene expression patterns with those of liver tissues from 25 patients with nonalcoholic steatohepatitis (NASH), 27 patients with NAFLD, 15 healthy obese individuals, and 39 healthy nonobese individuals (controls). Liver samples were obtained from patients undergoing liver biopsy for suspected NAFLD or NASH, or during liver or bariatric surgeries. Data sets were analyzed using the limma R-package. Overlap of functional profiles was analyzed by gene set enrichment analysis profiles. RESULTS: We found differences between human and mouse transcriptomes to be significantly larger than differences between disease stages or models. Of the 65 genes with significantly altered expression in patients with NASH and 177 genes with significantly altered expression in patients with NAFLD, compared with controls, only 1-18 of these genes also differed significantly in expression between mouse models of NAFLD and control mice. However, expression of genes that regulate pathways associated with the development of NAFLD were altered in some mouse models (such as pathways associated with lipid metabolism). On a pathway level, gene expression patterns in livers of mice on the high-fat diet were associated more closely with human fatty liver disease than other models. CONCLUSIONS: In comparing gene expression profiles between liver tissues from different mouse models of NAFLD and patients with different stages of NAFLD, we found very little overlap. Our data set is available for studies of pathways that contribute to the development of NASH and NAFLD and selection of the most applicable mouse models (http://www.nash-profiler.com).

Obesity accelerates epigenetic aging of human liver.

Because of the dearth of biomarkers of aging, it has been difficult to test the hypothesis that obesity increases tissue age. Here we use a novel epigenetic biomarker of aging (referred to as an "epigenetic clock") to study the relationship between high body mass index (BMI) and the DNA methylation ages of human blood, liver, muscle, and adipose tissue. A significant correlation between BMI and epigenetic age acceleration could only be observed for liver (r = 0.42, P = 6.8 × 10(-4) in dataset 1 and r = 0.42, P = 1.2 × 10(-4) in dataset 2). On average, epigenetic age increased by 3.3 y for each 10 BMI units. The detected age acceleration in liver is not associated with the Nonalcoholic Fatty Liver Disease Activity Score or any of its component traits after adjustment for BMI. The 279 genes that are underexpressed in older liver samples are highly enriched (1.2 × 10(-9)) with nuclear mitochondrial genes that play a role in oxidative phosphorylation and electron transport. The epigenetic age acceleration, which is not reversible in the short term after rapid weight loss induced by bariatric surgery, may play a role in liver-related comorbidities of obesity, such as insulin resistance and liver cancer.

A genome-wide association scan identifies the hepatic cholesterol transporter ABCG8 as a susceptibility factor for human gallstone disease.

With an overall prevalence of 10-20%, gallstone disease (cholelithiasis) represents one of the most frequent and economically relevant health problems of industrialized countries. We performed an association scan of >500,000 SNPs in 280 individuals with gallstones and 360 controls. A follow-up study of the 235 most significant SNPs in 1,105 affected individuals and 873 controls replicated the disease association of SNP A-1791411 in ABCG8 (allelic P value P(CCA) = 4.1 x 10(-9)), which was subsequently attributed to coding variant rs11887534 (D19H). Additional replication was achieved in 728 German (P = 2.8 x 10(-7)) and 167 Chilean subjects (P = 0.02). The overall odds ratio for D19H carriership was 2.2 (95% confidence interval: 1.8-2.6, P = 1.4 x 10(-14)) in the full German sample. Association was stronger in subjects with cholesterol gallstones (odds ratio = 3.3), suggesting that His19 might be associated with a more efficient transport of cholesterol into the bile.

Genome-wide search for novel human uORFs and N-terminal protein extensions using ribosomal footprinting.

So far, the annotation of translation initiation sites (TISs) has been based mostly upon bioinformatics rather than experimental evidence. We adapted ribosomal footprinting to puromycin-treated cells to generate a transcriptome-wide map of TISs in a human monocytic cell line. A neural network was trained on the ribosomal footprints observed at previously annotated AUG translation initiation codons (TICs), and used for the ab initio prediction of TISs in 5062 transcripts with sufficient sequence coverage. Functional interpretation suggested 2994 novel upstream open reading frames (uORFs) in the 5' UTR, 1406 uORFs overlapping with the coding sequence, and 546 N-terminal protein extensions. The TIS detection method was validated on the basis of previously published alternative TISs and uORFs. Among primates, TICs in newly annotated TISs were significantly more conserved than control codons, both for AUGs and near-cognate codons. The transcriptome-wide map of novel candidate TISs derived as part of the study will shed further light on the way in which human proteome diversity is influenced by alternative translation initiation and regulation.

Metabolomic tissue signature in human non-alcoholic fatty liver disease identifies protective candidate metabolites.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder in industrialized countries, yet its pathophysiology is incompletely understood. Small-molecule metabolite screens may offer new insights into disease mechanisms and reveal new treatment targets. METHODS: Discovery (N = 33) and replication (N = 66) of liver biopsies spanning the range from normal liver histology to non-alcoholic steatohepatitis (NASH) were ascertained ensuring rapid freezing under 30 s in patients. 252 metabolites were assessed using GC/MS. Replicated metabolites were evaluated in a murine high-fat diet model of NAFLD. RESULTS: In a two-stage metabolic screening, hydroquinone (HQ, p(combined) = 3.0 × 10(-4)) and nicotinic acid (NA, p(combined) = 3.9 × 10(-9)) were inversely correlated with histological NAFLD severity. A murine high-fat diet model of NAFLD demonstrated a protective effect of these two substances against NAFLD: Supplementation with 1% HQ reduced only liver steatosis, whereas 0.6% NA reduced both liver fat content and serum transaminase levels and induced a complex regulatory network of genes linked to NALFD pathogenesis in a global expression pathway analysis. Human nutritional intake of NA equivalent was also consistent with a protective effect of NA against NASH progression. CONCLUSION: This first small-molecular screen of human liver tissue identified two replicated protective metabolites. Either the use of NA or targeting its regulatory pathways might be explored to treat or prevent human NAFLD.

Genetic and functional identification of the likely causative variant for cholesterol gallstone disease at the ABCG5/8 lithogenic locus.

UNLABELLED: The sterolin locus (ABCG5/ABCG8) confers susceptibility for cholesterol gallstone disease in humans. Both the responsible variant and the molecular mechanism causing an increased incidence of gallstones in these patients have as yet not been identified. Genetic mapping utilized patient samples from Germany (2,808 cases, 2,089 controls), Chile (680 cases, 442 controls), Denmark (366 cases, 766 controls), India (247 cases, 224 controls), and China (280 cases, 244 controls). Analysis of allelic imbalance in complementary DNA (cDNA) samples from human liver (n = 22) was performed using pyrosequencing. Transiently transfected HEK293 cells were used for [(3) H]-cholesterol export assays, analysis of protein expression, and localization of allelic constructs. Through fine mapping in German and Chilean samples, an ∼250 kB disease-associated interval could be defined for this locus. Lack of allelic imbalance or allelic splicing of the ABCG5 and ABCG8 transcripts in human liver limited the search to coding single nucleotide polymorphisms. Subsequent mutation detection and genotyping yielded two disease-associated variants: ABCG5-R50C (P = 4.94 × 10(-9) ) and ABCG8-D19H (P = 1.74 × 10(-10) ) in high pairwise linkage disequilibrium (r(2) = 0.95). [(3) H]-cholesterol export assays of allelic constructs harboring these genetic candidate variants demonstrated increased transport activity (3.2-fold, P = 0.003) only for the ABCG8-19H variant, which was also superior in nested logistic regression models in German (P = 0.018), Chilean (P = 0.030), and Chinese (P = 0.040) patient samples. CONCLUSION: This variant thus provides a molecular basis for biliary cholesterol hypersecretion as the mechanism for cholesterol gallstone formation, thereby drawing a link between "postgenomic" and "pregenomic" pathophysiological knowledge about this common complex disorder. (HEPATOLOGY 2012).

Cell atlas of the regenerating human liver after portal vein embolization.

The liver has the remarkable capacity to regenerate. In the clinic, regeneration is induced by portal vein embolization, which redirects portal blood flow, resulting in liver hypertrophy in locations with increased blood supply, and atrophy of embolized segments. Here, we apply single-cell and single-nucleus transcriptomics on healthy, hypertrophied, and atrophied patient-derived liver samples to explore cell states in the regenerating liver. Our data unveils pervasive upregulation of genes associated with developmental processes, cellular adhesion, and inflammation in post-portal vein embolization liver, disrupted portal-central hepatocyte zonation, and altered cell subtype composition of endothelial and immune cells. Interlineage crosstalk analysis reveals mesenchymal cells as an interaction hub between immune and endothelial cells, and highlights the importance of extracellular matrix proteins in liver regeneration. Moreover, we establish tissue-scale iterative indirect immunofluorescence imaging for high-dimensional spatial analysis of perivascular microenvironments, uncovering changes to tissue architecture in regenerating liver lobules. Altogether, our data is a rich resource revealing cellular and histological changes in human liver regeneration.

Distinct DNA methylation patterns in cirrhotic liver and hepatocellular carcinoma.

Abberrant DNA methylation is one of the hallmarks of cancerogenesis. Our study aims to delineate differential DNA methylation in cirrhosis and hepatic cancerogenesis. Patterns of methylation of 27,578 individual CpG loci in 12 hepatocellular carcinomas (HCCs), 15 cirrhotic controls and 12 normal liver samples were investigated using an array-based technology. A supervised principal component analysis (PCA) revealed 167 hypomethylated loci and 100 hypermethylated loci in cirrhosis and HCC as compared to normal controls. Thus, these loci show a "cirrhotic" methylation pattern that is maintained in HCC. In pairwise supervised PCAs between normal liver, cirrhosis and HCC, eight loci were significantly changed in all analyses differentiating the three groups (p < 0.0001). Of these, five loci showed highest methylation levels in HCC and lowest in control tissue (LOC55908, CELSR1, CRMP1, GNRH2, ALOX12 and ANGPTL7), whereas two loci showed the opposite direction of change (SPRR3 and TNFSF15). Genes hypermethylated between normal liver to cirrhosis, which maintain this methylation pattern during the development of HCC, are depleted for CpG islands, high CpG content promoters and polycomb repressive complex 2 (PRC2) targets in embryonic stem cells. In contrast, genes selectively hypermethylated in HCC as compared to nonmalignant samples showed an enrichment of CpG islands, high CpG content promoters and PRC2 target genes (p < 0.0001). Cirrhosis and HCC show distinct patterns of differential methylation with regards to promoter structure, PRC2 targets and CpG islands.