In the last decade, the utility of whole-exome sequencing in uncovering genetic aetiologies of a variety of liver diseases has been demonstrated. These new diagnoses have guided the management, treatment, and prognostication of previously undiagnosed patients, largely thanks to improved insight into the underlying pathogenesis of their conditions. Despite its clear benefits, the uptake of genetic testing by hepatologists has been limited, in part due to limited prior genetic training and/or opportunities for continuing education. Herein, we show that Hepatology Genome Rounds, an interdisciplinary forum highlighting hepatology cases of clinical interest and educational value, are an important venue for integrating genotypic and phenotypic information to enable accurate diagnosis and appropriate management, dissemination of genomic knowledge within the field of hepatology, and ongoing education to providers and trainees in genomic medicine. We describe our single-centre experience and discuss practical considerations for clinicians interested in launching such a series. We foresee that this format will be adopted at other institutions and by additional specialties, with the aim of further incorporating genomic information into clinical medicine.

Alcohol-related liver disease is a major cause of liver disease-associated mortality, with inpatient care being a major contributor to its clinical and economic burden. Alcohol-related hepatitis (AH) is an acute inflammatory form of alcohol-related liver disease. Severe AH is associated with high short-term mortality, with infection being a common cause of death. The presence of AH is associated with increased numbers of circulating and hepatic neutrophils. We review the literature on the role of neutrophils in AH. In particular, we explain how neutrophils are recruited to the inflamed liver and how their antimicrobial functions (chemotaxis, phagocytosis, oxidative burst, NETosis) may be altered in AH. We highlight evidence for the existence of 'high-density' and 'low-density' neutrophil subsets. We also describe the potentially beneficial roles of neutrophils in the resolution of injury in AH through their effects on macrophage polarisation and hepatic regeneration. Finally, we discuss how manipulation of neutrophil recruitment/function may be used as a therapeutic strategy in AH. For example, correction of gut dysbiosis in AH could help to prevent excess neutrophil activation, or treatments could aim to enhance miR-223 function in AH. The development of markers that can reliably distinguish neutrophil subsets and of animal models that accurately reproduce human disease will be crucial for facilitating translational research in this important field.

Autoimmune hepatitis (AIH) is a rare chronic liver disease of unknown aetiology; the risk of hepatocellular carcinoma (HCC) remains unclear and risk factors are not well-defined. We aimed to investigate the risk of HCC across a multicentre AIH cohort and to identify predictive factors. We performed a retrospective, observational, multicentric study of patients included in the International Autoimmune Hepatitis Group Retrospective Registry. The assessed clinical outcomes were HCC development, liver transplantation, and death. Fine and Gray regression analysis stratified by the centre was applied to determine the effects of individual covariates; the cumulative incidence of HCC was estimated using the competing risk method with death as a competing risk. 1428 patients diagnosed with AIH from 1980 to 2020 from 22 eligible centres across Europe and Canada were included, with a median follow-up of 11.1 years (interquartile range 5.2-15.9). 293 (20.5%) patients had cirrhosis at diagnosis. During follow up, 24 patients developed HCC (1.7%) with incidence rate of 1.44 cases/1000-patient years; cumulative incidence of HCC increased over time (0.6% at 5 years, 0.9% at 10 years, 2.7% at 20 years, and 6.6% at 30 years of follow-up). Patients that developed cirrhosis during follow-up had a significantly higher incidence of HCC. The cumulative incidence of HCC was respectively 2.6%, 4.6%, 5.6% and 6.6% at 5, 10, 15, and 20 years after the development of cirrhosis. Obesity (HR 2.94, p=0.04), cirrhosis (HR 3.17, p=0.01), and AIH/PSC variant (HR 5.18, p=0.007) at baseline resulted independent risk factors for HCC development. HCC incidence in AIH is low even after cirrhosis development and is associated to risk factors as obesity, cirrhosis, and AIH/PSC variant syndrome. The risk of developing hepatocellular carcinoma (HCC) in subjects with autoimmune hepatitis (AIH) seems to be lower compared to other aetiologies of chronic liver disease. Yet, solid data for this specific patient group remains elusive, given that most of the existing evidence comes from small, single-centre studies. In our study, we found that HCC incidence in AIH patients is low even after the onset of cirrhosis. Additionally, factors such as advanced age, obesity, cirrhosis, alcohol consumption, and the presence of the AIH/PSC variant syndrome at the time of AIH diagnosis are linked to a higher risk of HCC. Based on these findings, there seems to be merit in adopting a specialized HCC monitoring program for AIH patients, taking into account their individual risk factors.

Lymphedema cholestasis syndrome 1 or Aagenaes syndrome is a condition characterized by neonatal cholestasis, lymphedema, and giant cell hepatitis. The genetic background of this autosomal recessive disease was unknown up to now. A total of 26 patients with Aagenaes syndrome and 17 parents were investigated with whole-genome sequencing and/or Sanger sequencing. PCR and western blot analyses were used to assess levels of mRNA and protein, respectively. CRISPR/Cas9 was used to generate the variant in HEK293T cells. Light microscopy, transmission electron microscopy and immunohistochemistry for biliary transport proteins were performed in liver biopsies. One specific variant (c.-98G>T) in the 5'-untranslated region of Unc-45 myosin chaperone A (UNC45A) was identified in all tested patients with Aagenaes syndrome. Nineteen were homozygous for the c.-98G>T variant and seven were compound heterozygous for the variant in the 5'-untranslated region and an exonic loss-of-function variant in UNC45A. Patients with Aagenaes syndrome exhibited lower expression of UNC45A mRNA and protein than controls, and this was reproduced in a CRISPR/Cas9-created cell model. Liver biopsies from the neonatal period demonstrated cholestasis, paucity of bile ducts and pronounced formation of multinucleated giant cells. Immunohistochemistry revealed mislocalization of the hepatobiliary transport proteins BSEP (bile salt export pump) and MRP2 (multidrug resistance-associated protein 2). c.-98G>T in the 5'-untranslated region of UNC45A is the causative genetic variant in Aagenaes syndrome. The genetic background of Aagenaes syndrome, a disease presenting with cholestasis and lymphedema in childhood, was unknown until now. A variant in the 5'-untranslated region of the Unc-45 myosin chaperone A (UNC45A) was identified in all tested patients with Aagenaes syndrome, providing evidence of the genetic background of the disease. Identification of the genetic background provides a tool for diagnosis of patients with Aagenaes syndrome before lymphedema is evident.

Accurate risk stratification for hepatocellular carcinoma (HCC) after achieving a sustained viral response (SVR) is necessary for optimal surveillance. We aimed to develop and validate a machine learning (ML) model to predict the risk of HCC after achieving an SVR in individual patients. In this multicenter cohort study, 1742 patients with chronic hepatitis C who achieved an SVR were enrolled. Five ML models were developed including DeepSurv, gradient boosting survival analysis, random survival forest (RSF), survival support vector machine, and a conventional Cox proportional hazard model. Model performance was evaluated using Harrel' c-index and was externally validated in an independent cohort (977 patients). During the mean observation period of 5.4 years, 122 patients developed HCC (83 in the derivation cohort and 39 in the external validation cohort). The RSF model showed the best discrimination ability using seven parameters at the achievement of an SVR with a c-index of 0.839 in the external validation cohort and a high discriminative ability when the patients were categorized into three risk groups (P <0.001). Furthermore, this RSF model enabled the generation of an individualized predictive curve for HCC occurrence for each patient with an app available online. We developed and externally validated an RSF model with good predictive performance for the risk of HCC after an SVR. The application of this novel model is available on the website. This model could provide the data to consider an effective surveillance method. Further studies are needed to make recommendations for surveillance policies tailored to the medical situation in each country. A novel prediction model for HCC occurrence in patients after hepatitis C virus eradication was developed using machine learning algorithms. This model, using seven commonly measured parameters, has been shown to have a good predictive ability for HCC development and could provide a personalized surveillance system.

Gilbert's syndrome, also known as benign hyperbilirubinaemia, was described more than 100 years ago. It has usually been considered a physiological abnormality characterised by a mild elevation of the systemic level of unconjugated bilirubin, in the absence of any underlying liver or overt haemolytic disease. However, since the re-discovery of the potent antioxidant effects of bilirubin in the late 1980s, as well as multiple intracellular signalling pathways affected by bilirubin, an ever-increasing body of evidence suggests that individuals with Gilbert's syndrome may benefit from the mild hyperbilirubinaemia and are actually protected from the development of a wide variety of "diseases of civilisation" such as cardiovascular diseases, certain cancers, and autoimmune or neurodegenerative diseases. This review analyses the current state of medical knowledge given recent discoveries in this rapidly developing field, as well as their possible clinical significance, and provides a new perspective on this condition.

Hepatoblastoma (HB) is the most frequent childhood liver cancer. Patients with aggressive tumors have limited therapeutic options; therefore, a better understanding of HB pathogenesis is needed to improve treatment. HBs have a very low mutational burden; however, epigenetic alterations are increasingly recognized. We aimed to identify epigenetic regulators consistently dysregulated in HB and to evaluate the therapeutic efficacy of their targeting in clinically relevant models. We performed a comprehensive transcriptomic analysis of 180 epigenetic genes. Data from fetal, pediatric, adult, peritumoral (n= 72) and tumoral (n= 91) tissues were integrated. Selected epigenetic drugs were tested in HB cells. The most relevant epigenetic target identified was validated in primary HB cells, HB organoids, a patient-derived xenograft model, and a genetic mouse model. Transcriptomic, proteomic and metabolomic mechanistic analyses were performed. Altered expression of genes regulating DNA methylation and histone modifications was consistently observed in association with molecular and clinical features of poor prognosis. The histone methyltransferase G9a was markedly upregulated in tumors with epigenetic and transcriptomic traits of increased malignancy. Pharmacological targeting of G9a significantly inhibited growth of HB cells, organoids and patient-derived xenografts. Development of HB induced by oncogenic forms of β-catenin and YAP1 was ablated in mice with hepatocyte-specific deletion of G9a. We observed that HBs undergo significant transcriptional rewiring in genes involved in amino acid metabolism and ribosomal biogenesis. G9a inhibition counteracted these pro-tumorigenic adaptations. Mechanistically, G9a targeting potently repressed the expression of c-MYC and ATF4, master regulators of HB metabolic reprogramming. HBs display a profound dysregulation of the epigenetic machinery. Pharmacological targeting of key epigenetic effectors exposes metabolic vulnerabilities that can be leveraged to improve the treatment of these patients. In spite of recent advances in the management of hepatoblastoma (HB), treatment resistance and drug toxicity are still major concerns. This systematic study reveals the remarkable dysregulation in the expression of epigenetic genes in HB tissues. Through pharmacological and genetic experimental approaches, we demonstrate that the histone-lysine-methyltransferase G9a is an excellent drug target in HB, which can also be harnessed to enhance the efficacy of chemotherapy. Furthermore, our study highlights the profound pro-tumorigenic metabolic rewiring of HB cells orchestrated by G9a in coordination with the c-MYC oncogene. From a broader perspective, our findings suggest that anti-G9a therapies may also be effective in other c-MYC-dependent tumors.

In patients with compensated alcohol-related cirrhosis, reliable prognostic biomarkers are lacking. Keratin-18 and hepatocyte-derived large extracellular vesicle (lEV) concentrations reflect disease activity, but their ability to predict liver-related events is unknown. We measured plasma keratin-18 and hepatocyte lEV concentrations in 500 patients with Child-Pugh class A alcohol-related cirrhosis. The ability of these hepatocyte-derived biomarkers, alone or combined with model for end-stage liver disease (MELD) and FibroTest scores, to predict liver-related events at 2 years was analyzed, taking into account the alcohol consumption at inclusion and during follow-up. Keratin-18 and hepatocyte lEV concentrations increased with alcohol consumption. In patients without active alcohol consumption at enrollment (n= 419), keratin-18 concentration predicted liver-related events at 2 years, independently of FibroTest and MELD. Patients with both keratin-18 concentrations >285 U/L and FibroTest >0.74 had a 24% cumulative incidence of liver-related events at 2 years, vs. 5% to 14% in other groups of patients. Similar results were obtained when combining keratin-18 concentrations >285 U/L with MELD >10. In patients with active alcohol consumption at enrollment (n= 81), hepatocyte lEVs predicted liver-related events at 2 years, independently of FibroTest and MELD. Patients with both hepatocyte lEV concentrations >50 U/L and FibroTest >0.74 had a 62% cumulative incidence of liver-related events at 2 years, vs. 8% to 13% in other groups of patients. Combining hepatocyte lEV concentrations >50 U/L with MELD >10 had a lower discriminative ability. Similar results were obtained when using decompensation of cirrhosis, defined according to Baveno VII criteria, as an endpoint. In patients with Child-Pugh class A alcohol-related cirrhosis, combining hepatocyte-derived biomarkers with FibroTest or MELD scores identifies patients at high risk of liver-related events, and could be used for risk stratification and patient selection in clinical trials. In patients with compensated alcohol-related cirrhosis, reliable predictors of outcome are lacking. In patients with Child-Pugh class A alcohol-related cirrhosis, combining hepatocyte-derived biomarkers (keratin-18 and hepatocyte-large extracellular vesicles) with FibroTest or MELD scores identifies those at high risk of liver-related events at 2 years. The identified patients at high risk of liver-related events are the target-of-choice population for intensive surveillance (e.g., referral to tertiary care centers; intensive control of risk factors) and inclusion in clinical trials.

Hepatocellular carcinoma (HCC) is among the most prevalent and fatal cancers worldwide. The tumor microenvironment (TME) contributes to the poor response of patients with HCC to current therapies, while tumor vascular endothelial cells (ECs) are fundamental TME components significantly contributing to tumor progression. However, the specific functions and mechanisms of tumor vascular ECs in HCC remains unclear. We screened and validated the specific diacylglycerol kinase gamma (DGKG) hyper-expression in HCC tumor vascular ECs. Single-cell RNA sequencing (scRNA-seq), cytometry by time-of-flight (CyTOF), and in vitro and in vivo studies were performed to investigate the functions and mechanisms of endothelial DGKG. Multiplexed immunohistochemistry (mIHC) staining and flow cytometry were used to evaluate changes in the TME. Functionally, endothelial DGKG promotes tumor angiogenesis and immunosuppressive regulatory T (Treg) cell differentiation in HCC. Of significance, we found that hypoxia-inducible factor-1α (HIF-1α) activates DGKG transcription by directly binding to its promoter region under hypoxia. Upregulated DGKG promotes HCC progression by recruiting ubiquitin specific peptidase 16 (USP16) to facilitate zinc finger E-box-binding homeobox 2 (ZEB2) deubiquitination and cytokine transforming growth factor-beta 1 (TGF-β1) positive feedback loop resultant activation, thus inducing tumor angiogenesis and Treg differentiation. Importantly, targeting endothelial DGKG potentiated the efficiency of dual blockade of PD-1 and VEGFR-2. Hypoxia-induced EC-specific DGKG hyper-expression promotes tumor angiogenesis and immune evasion via the ZEB2/TGF-β1 axis, suggesting EC-specific DGKG as a potential therapeutic target for HCC.