Genetic and Epigenetic Regulation in Nonalcoholic Fatty Liver Disease (NAFLD)

Genetics and epigenetics play a key role in the development of several diseases, including nonalcoholic fatty liver disease (NAFLD). Family studies demonstrate that first degree relatives of patients with NAFLD are at a much higher risk of the disease than the general population. The development of the Genome Wide Association Study (GWAS) technology has allowed the identification of numerous genetic polymorphisms involved in the evolution of diseases (e.g., PNPLA3, MBOAT7). On the other hand, epigenetic changes interact with inherited risk factors to determine an individual’s susceptibility to NAFLD. Modifications of the histones amino-terminal ends are key factors in the maintenance of chromatin structure and gene expression (cAMP-responsive element binding protein H (CREBH) or SIRT1). Activation of SIRT1 showed potential against the physiological mechanisms related to NAFLD. Abnormal DNA methylation represents a starting point for cancer development in NAFLD patients. Besides, the evaluation of circulating miRNA profiles represents a promising approach to assess and non-invasively monitor liver disease severity. To date, there is no approved pharmacologic therapy for NAFLD and the current treatment remains weight loss with lifestyle modification and exercise. In this review, the status of research into relevant genetic and epigenetic modifiers of NAFLD progression will be discusse

Non-invasive diagnosis of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) has become one of the top concerns for the practising hepato-gastroenterologist due to the obesity epidemic and its potential to progress to an advanced liver disease that significantly impacts on overall and liver-related mortality. Due to the rapidly advancing epidemics of obesity and diabetes, a large segment of the population is at risk for NAFLD. Particularly worrisome is the emergence of NAFLD or non-alcoholic steatohepatitis (NASH) with a significant fibrotic disease in developing countries, even in patients of normal or underweight. A critical issue in patients with NAFLD is the differentiation of NASH from simple steatosis (SS). It is then of particular importance to identify NASH patients as they are at greatest risk of developing cardiovascular diseases and complications such as cirrhosis, liver failure or hepatocellular carcinoma. There is a need, in NAFLD management, to develop non-invasive methods to detect NASH and to predict advanced fibrosis stages. Therefore, we evaluated the following items: (i) A tool-based on optical analysis of liver magnetic resonance images as biomarkers for NASH and fibrosis detection by investigating patients with biopsy-proven NAFLD who underwent magnetic resonance protocols using 1.5T General Electric or Philips devices. Two imaging biomarkers (NASHMRI and FibroMRI) were developed, standardized and validated using the area under the receiver operating characteristic curve (AUROC) analysis. The results indicated NASHMRI diagnostic accuracy for steatohepatitis detection was 0.83 (95%CI: 0.73-0.93), and FibroMRI diagnostic accuracy for significant fibrosis determination was 0.85 (95%CI: 0.77-0.94). These findings were independent of the magnetic resonance system used. We conclude that the optical analysis of magnetic resonance images has high potential to define non-invasive imaging biomarkers for the detection of steatohepatitis (NASHMRI) and the prediction of significant fibrosis (FibroMRI). (ii) Genetic and epigenetic biomarkers, such as human patatin-like phospholipase domain containing 3 (PNPLA3), transmembrane 6 superfamily 2 (TM6SF2) and fibroblast growth factor 21 (FGF21) variants as well as a panel of most abundant liver microRNAs. After univariate and multivariate analysis, we confirmed that GG genotype of PNPLA3 exerted a clear role in NASH development, and we identified the impact of a novel risk variant located in FGF21 gene in significant fibrosis stages. Besides, we found overexpression of FGF21 levels in both liver and serum, directly related to NASH condition. Finally, two microRNAs (miR-200b-3p and miR-224-5p) were screened and validated in human liver tissue and plasma of biopsy proven NAFLD patients, and were found raised in NASH, conferring them potential as non-invasive biomarkers.Premio Extraordinario de Doctorado U

Imaging biomarkers for steatohepatitis and fibrosis detection in non-alcoholic fatty liver disease

There is a need, in NAFLD management, to develop non-invasive methods to detect steatohepatitis (NASH) and to predict advanced fibrosis stages. We evaluated a tool based on optical analysis of liver magnetic resonance images (MRI) as biomarkers for NASH and fibrosis detection by investigating patients with biopsy-proven NAFLD who underwent magnetic resonance (MR) protocols using 1.5T General Electric (GE) or Philips devices. Two imaging biomarkers (NASHMRI and FibroMRI) were developed, standardised and validated using area under the receiver operating characteristic curve (AUROC) analysis. The results indicated NASHMRI diagnostic accuracy for steatohepatitis detection was 0.83 (95% CI: 0.73–0.93) and FibroMRI diagnostic accuracy for significant fibrosis determination was 0.85 (95% CI: 0.77–0.94). These findings were independent of the MR system used. We conclude that optical analysis of MRI has high potential to define non-invasive imaging biomarkers for the detection of steatohepatitis (NASHMRI) and the prediction of significant fibrosis (FibroMRI) in NAFLD patients.Comisión Europea, 7º Programa Marco FP7/2007–2013 HEALTH-F2-2009-241762 Project Fatty Liver Inhibition of Progression (FLIP)Junta de Andalucía, Consejería de Salud PI-0488-2012/201

Definite and indeterminate nonalcoholic steatohepatitis share similar clinical features and prognosis: A longitudinal study of 1893 biopsy-proven nonalcoholic fatty liver disease subjects

Background and Aim: Histological score systems may not fully capture the essential nonalcoholic steatohepatitis (NASH) features, which is one of the leading causes of screening failure in clinical trials. We assessed the NASH distribution and its components across the fibrosis stages and their impact on the prognosis and their relationship with the concept of metabolic-associated fatty liver disease (MAFLD). Methods: Spanish multicenter study including 1893 biopsy-proven nonalcoholic fatty liver disease (NAFLD) patients from HEPAmet registry. NASH was diagnosed by NAS score ≥4 (including steatosis, ballooning and lobular inflammation) and fibrosis by Kleiner score. The presence of MAFLD was determined. Progression to cirrhosis, first episode of decompensated cirrhosis and death were collected during the follow-up (4.7 ± 3.8 years). Results: Fibrosis was F0 34.3% (649/1893), F1 27% (511/1893), F2 16.5% (312/1893), F3 15% (284/1893) and F4 7.2% (137/1893). NASH diagnosis 51.9% (982/1893), and its individual components (severe steatosis, ballooning and lobular inflammation), increased from F0 (33.6%) to F2 (68.6%), and decreased significantly in F4 patients (51.8%) (P = .0001). M ore t han 7 0% o f n on-NASH p atients s howed s ome i nflammatory activity (ballooning or lobular inflammation), showing a similar MAFLD rate than NASH (96.2% [945/982] vs. 95.2% [535/562]) and significantly higher than nonalcoholic fatty liver (NAFL) subjects (89.1% [311/349]) (P < .0001). Progression to cirrhosis was similar between NASH (9.5% [51/539]) and indeterminate NASH (7.9% [25/316]), and higher than steatosis (5% [14/263]) (logRank 8.417; P = .015). Death and decompensated cirrhosis were similar between these. Conclusions: The prevalence of steatohepatitis decreased in advanced liver disease. However, most of these patients showed some inflammatory activity histologically and had metabolic disturbances. These findings should be considered in clinical trials whose main aim is to prevent cirrhosis progression and complications, liver transplant and death

Development and Validation of a Clinical-Genetic Risk Score to Predict Hepatic Encephalopathy in Patients With Liver Cirrhosis

[Introduction] We aimed to define the impact of the genetic background on overt hepatic encephalopathy (HE) in patients with liver cirrhosis by developing a combined clinical-genetic risk score.[Methods] Patients suffering from liver cirrhosis from the outpatient clinics of 4 hospitals (n = 600) were included and followed up for at least 5 years until HE bouts, liver transplant, or death. Patients were genotyped for 60 candidate single nucleotide polymorphisms together with the microsatellite in the promoter region of the gene GLS.[Results] Single nucleotide polymorphisms rs601338 (FUT2), rs5743836 (TRL9), rs2562582 (SLC1A3), rs313853 (SLC1A5), and GLS microsatellite did predict independently the incidence and severity of overt HE and were included as genetic score. Competing risk analysis revealed that bilirubin (subhazard ratio [sHR] 1.30 [1.15–1.48], P < 0.001), albumin (sHR 0.90 [0.86–0.93], P < 0.001), genetic score (sHR 1.90 [1.57–2.30], P < 0.001), and previous episodes of overt HE (sHR 2.60 [1.57–4.29], P < 0.001) were independently associated to HE bouts during the follow-up with an internal (C-index 0.83) and external validation (C-index 0.74). Patients in the low-risk group had 5% and 12% risk of HE at 1 (log-rank 92.1; P < 0.001) and 5 (log-rank 124.1; P < 0.001) years, respectively, whereas 36% and 48% in the high-risk group.[Discussion] The genetic background influenced overt HE risk and severity. The clinical-genetic HE Risk score, which combined genetic background together with albumin, bilirubin, and previous episodes of overt HE, could be a useful tool to predict overt HE in patients with cirrhosis.Peer reviewe

Bacterial antigen translocation and age as BMI-independent contributing factors on systemic inflammation in NAFLD patients

Background & Aims Low‐grade systemic inflammation is a crucial landmark in NAFLD favouring disease progression and comorbidities. We evaluated the input of circulating bacterial antigens on systemic markers of inflammation in NAFLD patients. Patients & Methods Multicenter cross‐sectional study including consecutive patients with biopsy‐proven NAFLD. Demographic, metabolic and fibrosis‐related variables were collected. Circulating bacterial antigens were quantified in blood. Toll‐like receptor SNPs were genotyped. Serum cytokine levels were evaluated. Peripheral blood mononuclear cell response to bacterial antigens was evaluated in vitro. Results Three hundred and fifteen patients from five Spanish hospitals were distributed by BMI. At least, one bacterial antigenic type was found in 66 patients with BMI 30 (77.3%) (P = .014). HOMA‐IR was significantly higher in the presence of circulating antigens among patients with BMI < 30. NASH and significant fibrosis in non‐obese patients were more frequent in the presence of at least two circulating antigenic types. Allelic frequencies of TLR variants were similar to controls and did not affect clinical or laboratory parameters. Pro‐inflammatory cytokines were significantly increased in patients with bacterial antigens, regardless of BMI. TLR gene and protein expression levels were significantly increased in PBMCs from patients with bacterial antigens. Antigen concentrations independently influenced TNF‐α and IL‐6, in both BMI subgroups of patients. Age independently influenced TNF‐α and IL‐6 in non‐obese patients, and TNF‐α in obese patients. Conclusion Serum circulating bacterial antigens as well as age were BMI‐independent factors related to increased systemic inflammation in NAFLD and provides insight on the multifaceted sources of inflammation in these patients.This work has been funded by grants PI16/0967 and PI17/0535 from Instituto de Salud Carlos III, Madrid, Spain, and PROMETEO 2016/001 from Generalitat Valenciana, Valencia, Spain. IGH was recipient of a Young Investigator Grant by CIBERehd, Instituto de Salud Carlos III, Madrid, Spain

Hepatitis C Virus Clearance by Direct-Acting Antivirals Agents Improves Endothelial Dysfunction and Subclinical Atherosclerosis: HEPCAR Study

© 2020 The Author(s).[INTRODUCTION]: Hepatitis C virus (HCV) infection has been related to increased cardiovascular (CV) risk. The aim of this study was to analyze the impact of sustained virological response (SVR) on endothelial dysfunction and subclinical atherosclerosis in patients with hepatitis C virus treated with direct-acting antiviral agents. [METHODS]: A total of 114 patients were prospectively recruited and underwent CV risk assessment including (i) endothelial dysfunction determined through laser Doppler flowmetry and (ii) subclinical atherosclerosis, elucidated by the ankle-brachial index (ABI). Atherogenic lipid profile (total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides); markers of oxidative stress (oxidized low-density lipoprotein antibodies [OLAbs]), soluble markers of adhesion (vascular cell adhesion molecule [VCAM], e-selectin, and soluble markers of angiogenesis; and vascular endothelial growth factor, endothelial [EMPs] and platelet [PMPs] apoptotic microparticles, and cell-free DNA [cfDNA]) were measured. All determinations were performed at baseline, 12 weeks (SVR time), and 1 year after treatment. [RESULTS]: In patients with endothelial dysfunction, area of hyperemia improved after virus clearance (P = 0.013) and was related to significant decrease in VCAM, e-selectin (P < 0.001), and cfDNA (P = 0.017) and to increased OLAb levels (P = 0.001). In patients with subclinical atherosclerosis at baseline, a significantly improved ABI was seen after HCV clearance (P < 0.001). Levels of both EMPs and PMPs also decreased after SVR and at follow-up (P = 0.006 and P = 0.002, respectively). [DISCUSSION]: HCV clearance improved not only liver function but also endothelial dysfunction and subclinical atherosclerosis promoted by decrease in levels of VCAM, e-selectin, cfDNA, and PMPs and EMPs.Postdoctoral fellowship from the Spanish Government (Juan de la Cierva fellowship FJC1-2014-21675). Instituto de Salud Carlos III Project GLD17/00203

Liver injury in non-alcoholic fatty liver disease is associated with urea cycle enzyme dysregulation

The main aim was to evaluate changes in urea cycle enzymes in NAFLD patients and in two preclinical animal models mimicking this entity. Seventeen liver specimens from NAFLD patients were included for immunohistochemistry and gene expression analyses. Three-hundred-and-eighty-two biopsy-proven NAFLD patients were genotyped for rs1047891, a functional variant located in carbamoyl phosphate synthetase-1 (CPS1) gene. Two preclinical models were employed to analyse CPS1 by immunohistochemistry, a choline deficient high-fat diet model (CDA-HFD) and a high fat diet LDLr knockout model (LDLr −/−). A significant downregulation in mRNA was observed in CPS1 and ornithine transcarbamylase (OTC1) in simple steatosis and NASH-fibrosis patients versus controls. Further, age, obesity (BMI > 30 kg/m), diabetes mellitus and ALT werefound to be risk factors whereas A-allele from CPS1 was a protective factor from liver fibrosis. CPS1 hepatic expression was diminished in parallel with the increase of fibrosis, and its levels reverted up to normality after changing diet in CDA-HFD mice. In conclusion, liver fibrosis and steatosis were associated with a reduction in both gene and protein expression patterns of mitochondrial urea cycle enzymes. A-allele from a variant on CPS1 may protect from fibrosis development. CPS1 expression is restored in a preclinical model when the main trigger of the liver damage disappears.The research leading to these results has received funding from the Consejería de Salud de la Junta de Andalucía under grant agreement PC-0148-2016-0148 and PE-0451-2018 and Instituto de Salud Carlos III under grant agreements CD21/00095, PI16/01842, PI19/01404, PI19/00589, IFI18/00041, FI20/00201, CD18/00126 and EHD18PI04/2021. Rocío Gallego-Durán has received the Andrew K Burroughs Fellowship from European Association for the Study of the Liver (EASL), Aprendizaje de Nuevas Tecnologías fellowship from Asociación Española para el Estudio del Hígado (AEEH) and CIBERehd Grant to support researcher’s mobility

Impaired brain glymphatic flow in experimental hepatic encephalopathy

Background & Aims: Neuronal function is exquisitely sensitive to alterations in the extracellular environment. In patients with hepatic encephalopathy (HE), accumulation of metabolic waste products and noxious substances in the interstitial fluid of the brain is thought to result from liver disease and may contribute to neuronal dysfunction and cognitive impairment. This study was designed to test the hypothesis that the accumulation of these substances, such as bile acids, may result from reduced clearance from the brain. Methods: In a rat model of chronic liver disease with minimal HE (the bile duct ligation [BDL] model), we used emerging dynamic contrast-enhanced MRI and mass-spectroscopy techniques to assess the efficacy of the glymphatic system, which facilitates clearance of solutes from the brain. Immunofluorescence of aquaporin-4 (AQP4) and behavioural experiments were also performed. Results: We identified discrete brain regions (olfactory bulb, prefrontal cortex and hippocampus) of altered glymphatic clearance in BDL rats, which aligned with cognitive/behavioural deficits. Reduced AQP4 expression was observed in the olfactory bulb and prefrontal cortex in HE, which could contribute to the pathophysiological mechanisms underlying the impairment in glymphatic function in BDL rats. Conclusions: This study provides the first experimental evidence of impaired glymphatic flow in HE, potentially mediated by decreased AQP4 expression in the affected regions. Lay summary: The 'glymphatic system' is a newly discovered brain-wide pathway that facilitates clearance of various sub-stances that accumulate in the brain due to its activity. This study evaluated whether the function of this system is altered in a model of brain dysfunction that occurs in cirrhosis. For the first time, we identified that the clearance of substances from the brain in cirrhosis is reduced because this clearance system is defective. This study proposes a new mechanism of brain dysfunction in patients with cirrhosis and provides new targets for therapy

Co-expression gene network analysis reveals novel regulatory pathways involved in porto-sinusoidal vascular disease

[Background & Aims] Porto-sinusoidal vascular disease (PSVD) is a rare vascular liver disease of unknown etiology that causes portal hypertension. It usually affects young individuals and shortens live expectancy. The deregulated pathways involved in PSVD development are unknown and therefore we lack curative treatments. The purpose of this study was to integrate transcriptomic and clinical data by comprehensive network-based modeling in order to uncover altered biological processes in patients with PSVD.[Methods] We obtained liver tissue samples from 20 consecutive patients with PSVD and 21 sex- and age-matched patients with cirrhosis and 13 histologically normal livers (HNL) (initial cohort) and performed transcriptomic analysis. Microarray data were analyzed using weighted gene correlation network analysis to identify clusters of highly correlated genes differently expressed in patients with PSVD. We next evaluated the molecular pathways enriched in patients with PSVD and the core-related genes from the most significantly enriched pathways in patients with PSVD. Our main findings were validated using RNA sequencing in a different cohort of PSVD, cirrhosis and HNL (n = 8 for each group).[Results] Patients with PSVD have a distinctive genetic profile enriched mainly in canonical pathways involving hemostasis and coagulation but also lipid metabolism and oxidative phosphorylation. Serpin family (SERPINC1), the apolipoproteins (APOA, APOB, APOC), ATP synthases (ATP5G1, ATP5B), fibrinogen genes (FGB, FGA) and alpha-2-macroglobulin were identified as highly connective genes that may have an important role in PSVD pathogenesis.[Conclusion] PSVD has a unique transcriptomic profile and we have identified deregulation of pathways involved in vascular homeostasis as the main pathogenic event of disease development. [Lay summary] Porto-sinusoidal vascular disease is a rare but life-shortening disease that affects mainly young people. Knowledge of the disrupted pathways involved in its development will help to identify novel therapeutic targets and new treatments. Using a systems biology approach, we identify that pathways regulating endothelial function and tone may act as drivers of porto-sinusoidal vascular disease.This study was supported by the Instituto de Salud Carlos III FIS PI17/00398, the Ministry of Education and Science, Spain (SAF-2016-75767-R); Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR-SGR2017-517) a grant from Generalitat de Catalunya, Fondo Europeo de Desarrollo Regional (FEDER) and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), funded by Instituto de Salud Carlos III. Marta Magaz is a recipient of a Río Hortega grant from Instituto de Salud Carlos III. Pol Olivas has been funded by Contractes Clínic de Recerca ”Emili Letang-Josep Font’’ 2020, granted by Hospital Clínic de Barcelona.Peer reviewe