Transient elastography for assessing and monitoring of liver steatosis and fibrosis

Summary Transient elastography (TE) is a novel method to assess and monitor non-alcoholic fatty liver disease (NAFLD) non-invasively by simultaneously quantifying steatosis using the controlled attenuation parameter (CAP) and liver stiffness using the liver stiffness measurement (LSM). We carried out four studies using TE as the central method to explore its value for research and clinical practice. We found a positive correlation between CAP and ultrasonography in the detection of steatosis in a cohort of 174 patients with chronic liver diseases. Furthermore, patatin-like phospholipase domain containing 3 (PNPLA3) p.148M, a known genetic risk factor for NAFLD, was associated with CAP, but not with LSM. No association was observed for CAP and LSM and transmembrane 6 superfamily member 2 (TM6SF2) p.167K, which is another known genetic risk factor for NAFLD. However, for both variants an association with serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities was demonstrated. Overall, carriers of the PNPLA3 risk allele [M] had an increased risk for the development of hepatic phenotypes in general and more specifically an increased odds of 2.4 for steatosis. This is the first time steatosis assessed by CAP was used in a genetic study. To investigate the short-term modulation of liver fat, we carried out the first intervention study using TE. In 60 patients with hepatic steatosis, fourteen days of a hypocaloric high-fiber, high-protein diet resulted in significant reductions in liver fat by 14% and in body weight by 4.8%. There is evidence that PNPLA3 modulated this dietary-related response. Furthermore, liver stiffness, body composition, serum liver enzymes and metabolic markers improved significantly. The UKS-study availed of CAP and LSM during occupational health check-ups and suggests that the prevalence of NAFLD is underdiagnosed in 104 hospital employees. The study also observed that TE is superior to serum parameters for diagnosing hepatic steatosis and fibrosis. By use of body impedance analysis (BIA) and a tape measure, we were able to demonstrate that body fat mass and waist circumference are the strongest predictors for steatosis. This is the first study that availed of CAP and LSM during occupational health check-ups. The recommendations for the use of TE to assess fibrosis are weaker in the German guideline as opposed to the European guideline on NAFLD. Neither of the guidelines however, provides a statement on assessing steatosis using CAP. Consequently, we analyzed almost 6,700 TE measurements obtained over a period of 3.5 years in patients and the general population. The results reflect the feasibility of TE and subsequently, we introduced the term “EGK des Hepatologen”, translating to “ECG of the hepatologist”.Zusammenfassung Die transiente Elastographie (TE) ist eine neuartige Methode zur nichtinvasiven Beurteilung und Überwachung der nichtalkoholischen Fettlebererkrankung (NAFLD), bei der gleichzeitig die Leberverfettung mittels Controlled Attenuation Parameter (CAP) und die Lebersteifigkeit mittels Lebersteifigkeitsmessung (LSM) quantifiziert wird. Die Bedeutung der Methode für Forschung und klinische Praxis wurde in vier Studien untersucht. Bei 174 Patienten mit chronischen Lebererkrankungen konnte bei der Detektion der Steatose eine positive Korrelation zwischen CAP und der Messung mittels Ultraschall gezeigt werden. Für Patatin-ähnliche Phospholipase-Domäne 3 (PNPLA3) p.148M, ein bekannter genetischer Risikofaktor für NAFLD, konnte ein Zusammenhang mit CAP, nicht aber mit LSM, gezeigt werden. Für Transmembran-6-Superfamilienmitglied 2 (TM6SF2) p.167K, einem weiteren NAFLD-Risikofaktor, konnten weder für CAP, noch für LSM ein Zusammenhang nachgewiesen werden. Eine Assoziation mit Serum-Alanin-Aminotransferase (ALT) und Aspartat-Aminotransferase (AST) Aktivitäten wurde hingegen für beide genetische Varianten gezeigt. Träger des Risikoallels [M] hatten ein erhöhtes Risiko für die Entwicklung eines hepatischen Phänotyps und eine erhöhte Wahrscheinlichkeit für die Entwicklung einer Steatose um das 2,4-Fache. In dieser Studie wurde die CAP zum ersten Mal zur Detektion der Steatose in einer genetischen Studie eingesetzt. Die kurzfristige Modulation von Leberfett wurde in der ersten Interventionsstudie mit TE durchgeführt. Bei 60 Patienten mit Steatose führten vierzehn Tage einer hypokalorischen, ballaststoffreichen und proteinreichen Diät zu einer signifikanten Reduktion des Leberfetts um 14% und des Körpergewichts um 4,8%. Es gibt Hinweise darauf, dass PNPLA3 die ernährungsbedingte Reduktion beeinflusst. Weiterhin verbesserten sich Lebersteifigkeit, Körperzusammen-setzung, Serumleberenzyme und metabolische Marker signifikant. Die UKS-Studie konnte mithilfe von CAP und LSM im Rahmen der betrieblichen Gesundheitsförderung zeigen, dass die Prävalenz von NAFLD bei 104 Krankenhausangestellten unterdiagnostiziert ist und dass TE den Serumparametern zur Diagnose von Steatose und Fibrose überlegen ist. Dabei wurde weiterhin gezeigt, dass gemessen mittels Körperimpedanzanalyse (BIA) und einem einfachen Maßband, Körperfettmasse und Taillenumfang die stärksten Prädiktoren für die Steatose sind. Dies ist die erste Studie, bei der bei arbeitsmedizinischen Vorsorgeuntersuchungen auf CAP und LSM angewendet wurde. Die Empfehlungen zur Verwendung von TE zur Beurteilung der Fibrose sind in der deutschen Leitlinie gegenüber der europäischen Leitlinie zu NAFLD schwächer ausgeprägt.Keine der Leitlinien macht eine Aussage zur Beurteilung der Leberverfettung mittels CAP. Eine Auswertung von fast 6.700 TE-Messungen bei Patienten und der Allgemeinbevölkerung über einen Zeitraum von 3,5 Jahren macht die Durchführbarkeit der transienten Elastographie deutlich und lässt sich durch den dafür eingeführten Begriff "EGK des Hepatologen" trefflich beschreiben

miR-873-5p targets mitochondrial GNMT-Complex II interface contributing to non-alcoholic fatty liver disease

Objective: Non-alcoholic fatly liver disease (NAFLD) is a complex pathology in which several dysfunctions, including alterations in metabolic pathways, mitochondrial functionality and unbalanced lipid import/export, lead to lipid accumulation and progression to inflammation and fibrosis. The enzyme glycine N-methyltransferase (GNMT), the most important enzyme implicated in S-adenosylmethionine catabolism in the liver, is downregulated during NAFLD progression. We have studied the mechanism involved in GNMT downregulation by its repressor microRNA miR-873-5p and the metabolic pathways affected in NAFLD as well as the benefit of recovery GNMT expression. Methods: miR-873-5p and GNMT expression were evaluated in liver biopsies of NAFLD/NASH patients. Different in vitro and in vivo NAFLD murine models were used to assess miR-873-5p/GNMT involvement in fatty liver progression through targeting of the miR-873-5p as NAFLD therapy. Results: We describe a new function of GNMT as an essential regulator of Complex II activity in the electron transport chain in the mitochondria. In NAFLD, GNMT expression is controlled by miR-873-5p in the hepatocytes, leading to disruptions in mitochondria! functionality in a preclinical murine non-alcoholic steatohepatitis (NASH) model. Upregulation of miR-873-5p is shown in the liver of NAFLD/NASH patients, correlating with hepatic GNMT depletion. Importantly, NASH therapies based on anti-miR-873-5p resolve lipid accumulation, inflammation and fibrosis by enhancing fatty acid beta-oxidation in the mitochondria. Therefore, miR-873-5p inhibitor emerges as a potential tool for NASH treatment. Conclusion: GNMT participates in the regulation of metabolic pathways and mitochondria! functionality through the regulation of Complex II activity in the electron transport chain. In NAFLD, GNMT is repressed by miR-873-5p and its targeting arises as a valuable therapeutic option for treatment. (C) 2019 The Authors. Published by Elsevier GmbH.This work was supported by grants from NIH (US Department of Health and Human services)-R01AT001576 (to S.C.L., J.M.M., and M.L.M.-C.), Ministerio de Economia, Industria y Competitividad: SAF2017-87301-R (to M.L.M.-C.), SAF2015-64352-R (to P.A.), Gobierno Vasco-Departamento de Salud 2013111114 (to M.L.M.-C.), Gobierno Vasco-Departamento de Educacion IT-336-10 (to PA), BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia BIO15/CA/016/BD (M.L.M.-C.), ELKARTEK 2016, Departamento de Industria del Gobierno Vasco (to M.L.M.-C), Asociacion Espanola contra el Cancer (to T.C.D., P.F.-T. and M.L.M.-C.), Mitotherapeutix (to M.L.M.-C.), Daniel Alagille award from EASL (to T.C.D), Fundacion Cientifica de la Asociacion Espanola Contra el Cancer (AECC Scientific Foundation) Rare Tumor Calls 2017 (to M.L.M.-C.), La Caixa Foundation Program (to M.L.M.-C.), Ayudas Fundacion BBVA a Equipos de Investigacion Cientifica 2019 (to M.L.M.-C.). Ciberehd_ISCIII_MINECO is funded by the Instituto de Salud Carlos III. We thank this work produced with the support of a 2017 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation (to M.V.R.). This work was supported by Fonds National de la Recherche Luxembourg and the Deutsche Forschungsgemeinschaft (C12/BM/3975937, FL/997/7-1, Inter "HepmiRSTAT", to I.B. and F.L.). We thank MINECO for the Severo Ochoa Excellence Accreditation (SEV2016-0644)

miR-873-5p targets mitochondrialGNMT-Complex II interface contributing tonon-alcoholic fatty liver disease

Objective:Non-alcoholic fatty liver disease (NAFLD) is a complex pathology in which several dysfunctions, including alterations in metabolicpathways, mitochondrial functionality and unbalanced lipid import/export, lead to lipid accumulation and progression to inflammation andfibrosis.The enzyme glycine N-methyltransferase (GNMT), the most important enzyme implicated in S-adenosylmethionine catabolism in the liver, isdownregulated during NAFLD progression. We have studied the mechanism involved in GNMT downregulation by its repressor microRNA miR-873-5p and the metabolic pathways affected in NAFLD as well as the benefit of recovery GNMT expression.Methods:miR-873-5p and GNMT expression were evaluated in liver biopsies of NAFLD/NASH patients. Differentin vitroandin vivoNAFLD murinemodels were used to assess miR-873-5p/GNMT involvement in fatty liver progression through targeting of the miR-873-5p as NAFLD therapy.Results:We describe a new function of GNMT as an essential regulator of Complex II activity in the electron transport chain in the mitochondria.In NAFLD, GNMT expression is controlled by miR-873-5p in the hepatocytes, leading to disruptions in mitochondrial functionality in a preclinicalmurine non-alcoholic steatohepatitis (NASH) model. Upregulation of miR-873-5p is shown in the liver of NAFLD/NASH patients, correlating withhepatic GNMT depletion. Importantly, NASH therapies based on anti-miR-873-5p resolve lipid accumulation, inflammation andfibrosis byenhancing fatty acidb-oxidation in the mitochondria. Therefore, miR-873-5p inhibitor emerges as a potential tool for NASH treatment.Conclusion:GNMT participates in the regulation of metabolic pathways and mitochondrial functionality through the regulation of Complex II activityin the electron transport chain. In NAFLD, GNMT is repressed by miR-873-5p and its targeting arises as a valuable therapeutic option for treatment

Low Accuracy of FIB-4 and NAFLD Fibrosis Scores for Screening for Liver Fibrosis in the Population

Background & AimsFibrosis-4 (FIB-4) and the nonalcoholic fatty liver disease fibrosis score (NFS) are the 2 most popular noninvasive blood-based serum tests proposed for widespread fibrosis screening. We therefore aimed to describe the accuracy of FIB-4 and NFS to detect elevated liver stiffness as an indicator of hepatic fibrosis in low-prevalence populations.MethodsThis study included a total of 5129 patients with concomitant measurement of FIB-4, NFS, and liver stiffness measurement (LSM) by Fibroscan (Echosens, France) from 5 independent population-based cohorts from Spain, Hong Kong, Denmark, England, and France; 3979 participants from the general population and 1150 from at-risk cohorts due to alcohol, diabetes, or obesity. We correlated LSM with FIB-4 and NFS, and calculated pre- and post-test predictive values of FIB-4 and NFS to detect elevated LSM at 8 kPa and 12 kPa cutoffs. The mean age was 53 ± 12 years, the mean body mass index was 27 ± 5 kg/m2, and 2439 (57%) were women. One in 10 patients (552; 11%) had liver stiffness ≥8 kPa, but 239 of those (43%) had a normal FIB-4, and 171 (31%) had normal NFS. The proportion of false-negatives was higher in at-risk patients than the general population. FIB-4 was false-negative in 11% of diabetic subjects, compared with 2.5% false-negatives with NFS. Waist circumference outperformed FIB-4 and NFS for detecting LSM ≥8 kPa in the general population. Almost one-third (28%–29%) of elevated FIB-4/NFS were false-positive in both the general population and at-risk cohorts.ConclusionsFIB-4 and NFS are suboptimal for screening purposes due to a high risk of overdiagnosis and a non-negligible percentage of false-negatives, especially in patients with risk factors for chronic liver disease. Waist circumference emerged as a potential first step to identify patients at risk for liver fibrosis in the general population

LiverScreen project: study protocol for screening for liver fibrosis in the general population in European countries

Background: The development of liver cirrhosis is usually an asymptomatic process until late stages when complications occur. The potential reversibility of the disease is dependent on early diagnosis of liver fibrosis and timely targeted treatment. Recently, the use of non-invasive tools has been suggested for screening of liver fibrosis, especially in subjects with risk factors for chronic liver disease. Nevertheless, large population-based studies with cost-effectiveness analyses are still lacking to support the widespread use of such tools. The aim of this study is to investigate whether non-invasive liver stiffness measurement in the general population is useful to identify subjects with asymptomatic, advanced chronic liver disease. Methods: This study aims to include 30,000 subjects from eight European countries. Subjects from the general population aged ≥ 40 years without known liver disease will be invited to participate in the study either through phone calls/letters or through their primary care center. In the first study visit, subjects will undergo bloodwork as well as hepatic fat quantification and liver stiffness measurement (LSM) by vibration-controlled transient elastography. If LSM is ≥ 8 kPa and/or if ALT levels are ≥1.5 x upper limit of normal, subjects will be referred to hospital for further evaluation and consideration of liver biopsy. The primary outcome is the percentage of subjects with LSM ≥ 8kPa. In addition, a health economic evaluation will be performed to assess the cost-effectiveness and budget impact of such an intervention. The project is funded by the European Commission H2020 program. Discussion: This study comes at an especially important time, as the burden of chronic liver diseases is expected to increase in the coming years. There is consequently an urgent need to change our current approach, from diagnosing the disease late when the impact of interventions may be limited to diagnosing the disease earlier, when the patient is asymptomatic and free of complications, and the disease potentially reversible. Ultimately, the LiverScreen study will serve as a basis from which diagnostic pathways can be developed and adapted to the specific socio-economic and healthcare conditions in each country

Liver Fibrosis and Metabolic Alterations in Adults With alpha-1-antitrypsin Deficiency Caused by the Pi*ZZ Mutation

BACKGROUND & AIMS: Alpha-1 antitrypsin deficiency (AATD) is among the most common genetic disorders. Severe AATD is caused by a homozygous mutation in the SERPINA1 gene that encodes the Glu342Lys substitution (called the Pi*Z mutation, Pi*ZZ genotype). Pi*ZZ carriers may develop lung and liver diseases. Mutation- associated lung disorders have been well studied, but less is known about the effects in liver. We assessed the liver disease burden and associated features in adults with this form of AATD. METHODS: We collected data from 554 Pi*ZZ adults (403 in an exploratory cohort, 151 in a confirmatory cohort), in 9 European countries, with AATD who were homozygous for the Pi*Z mutation, and 234 adults without the Pi*Z mutation (controls), all without pre-existing liver disease. We collected data on demographic parameters, comorbidities, lung- and liver-related health, and blood samples for laboratory analysis. Liver fibrosis was assessed non-invasively via the serum tests Aspartate Aminotransferase to Platelet Ratio Index and HepaScore and via transient elastography. Liver steatosis was determined via transient elastography-based controlled attenuation parameter. We performed histologic analyses of livers from transgenic mice that overexpress the AATD-associated Pi*Z variant. RESULTS: Serum levels of liver enzymes were significantly higher in Pi*ZZ carriers vs controls. Based on non-invasive tests for liver fibrosis, significant fibrosis was suspected in 20%–36% of Pi*ZZ carriers, whereas signs of advanced fibrosis were 9- to 20-fold more common in Pi*ZZ carriers compared to non-carriers. Male sex; age older than 50 years; increased levels of alanine aminotransferase, aspartate aminotransferase, or g-glutamyl transferase; and low numbers of platelets were associated with higher liver fibrosis burden. We did not find evidence for a relationship between lung function and liver fibrosis. Controlled attenuation parameter 280 dB/m, suggesting severe steatosis, was detected in 39% of Pi*ZZ carriers vs 31% of controls. Carriers of Pi*ZZ had lower serum concentrations of triglyceride and low- and very-lowdensity lipoprotein cholesterol than controls, suggesting impaired hepatic secretion of lipid. Livers from Pi*Zoverexpressing mice had steatosis and down-regulation of genes involved in lipid secretion. CONCLUSIONS: In studies of AATD adults with the Pi*ZZ mutation, and of Pi*Z-overexpressing mice, we found evidence of liver steatosisinfo:eu-repo/semantics/publishedVersio

Altered profiles of circulating cytokines in chronic liver diseases (NAFLD/HCC): Impact of the PNPLA3I148M risk allele

peer reviewedBackground: Individuals carrying the risk variant p.I148M of patatin-like phospholipase domain-containing protein 3 (PNPLA3) have a higher susceptibility to fatty liver diseases and associated complications, including HCC, a cancer closely linked to chronic inflammation. Here, we assessed circulating cytokine profiles for patients with chronic liver diseases genotyped for PNPLA3. Methods: Serum concentrations of 22 cytokines were measured by multiplex sandwich-ELISA. The cohort comprised 123 individuals: 67 patients with NAFLD without cirrhosis (57 steatosis, 10 NASH), 24 patients with NAFLD with cirrhosis, 21 patients with HCC (15 cirrhosis), and 11 healthy controls. Receiver operator characteristic analyses were performed to assess the suitability of the cytokine profiles for the prediction of steatosis, cirrhosis, and HCC. Results: HGF, IL-6, and IL-8 levels were increased in patients, with ∼2-fold higher levels in patients with cirrhosis versus healthy, while platelet derived growth factor-BB (PDGF-BB) and regulated on activation, normal T cell expressed and secreted (RANTES) showed lower concentrations compared to controls. Migration inhibitory factor and monocyte chemoattractant protein-1 (MCP-1) were found at higher levels in NAFLD samples (maximum: NAFLD-cirrhosis) versus healthy controls and HCC samples. In receiver operator characteristic analyses, migration inhibitory factor, IL-8, IL-6, and monocyte chemoattractant protein-1 yielded high sensitivity scores for predicting noncirrhotic NAFLD (vs. healthy). The top combination to predict cirrhosis was HGF plus PDGF-BB. Migration inhibitory factor performed best to discriminate HCC from NAFLD; the addition of monokine induced gamma (MIG), RANTES, IL-4, macrophage colony-stimulating factor (M-CSF), or IL-17A as second parameters further increased the AUC values (> 0.9). No significant impact of the PNPLA3 I148M allele on cytokine levels was observed in this cohort. Conclusions: Cytokines have biomarker potential in patients with fatty liver, possibly suited for early HCC detection in patients with fatty liver. Patients carrying the PNPLA3 risk allele did not present significantly different levels of circulating cytokines