Combined effects of PNPLA3, TM6SF2 and HSD17B13 variants on severity of biopsy-proven non-alcoholic fatty liver disease.

OBJECTIVE: Several single-nucleotide polymorphisms have been identified to be disadvantageous or protective in regard to disease severity in patients with non-alcoholic fatty liver disease (NAFLD). However, it is unclear, whether including genetic risk factor(s) either alone or combined into risk stratification algorithms for NAFLD actually provides incremental benefit over clinical risk factors. DESIGN: Patients with biopsy-proven NAFLD were genotyped for the PNPLA3-rs738409(minor allele:G), TM6SF2-rs58542926(minor allele:T) and HSD17B13- rs72613567 (minor allele:TA) variants. The NAFLD activity score (NAS) and fibrosis stage (F0-F4) were used to grade and stage all liver biopsy samples. Patients from seven centers throughout Central Europe were considered for the study. RESULTS: 703 patients were included: NAS ≥ 5:173(24.6%); Fibrosis: F3-4:81(11.5%). PNPLA3 G/G genotype was associated with a NAS ≥ 5(aOR 2.23, p = 0.007) and advanced fibrosis (aOR-3.48, p < 0.001).TM6SF2 T/- was associated with advanced fibrosis (aOR 1.99, p = 0.023). HSD17B13 TA/- was associated with a lower probability of NAS ≥ 5(TA/T: aOR 0.65, p = 0.041, TA/TA: aOR 0.40, p = 0.033). Regarding the predictive capability for NAS ≥ 5, well-known risk factors (age, sex, BMI, diabetes, and ALT; baseline model) had an AUC of 0.758, Addition of PNPLA3(AUC 0.766), HSB17B13(AUC 0.766), and their combination(AUC 0.775), but not of TM6SF2(AUC 0.762), resulted in a higher diagnostic accuracy of the model. Addition of genetic markers for the prediction of advanced fibrosis (baseline model: age, sex, BMI, diabetes: AUC 0.777) resulted in a higher AUC if PNPLA3(AUC 0.789), and TM6SF2(AUC 0.786) but not if HSD17B13(0.777) were added. CONCLUSION: In biopsy-proven NAFLD, PNPLA3 G/-, TM6SF2 T/- and HSD17B13 TA/- carriage are associated with severity of NAFLD. Incorporating these genetic risk factors into risk stratification models might improve their predictive accuracy for severity of NAFLD and/or advanced fibrosis on liver biopsy.

Prevalence of anti-Hepatitis E antibodies and impact on disease severity in non-alcoholic fatty liver disease.

AIM: In most immune-competent individuals, hepatitis E (HEV) infections appear silent. It is unclear whether past HEV infections deteriorate disease severity in patients with non-alcoholic fatty liver disease (NAFLD). METHODS: Patients with biopsy-proven NAFLD and data on anti-HEV immunoglobulin M (HEV-IgM) and anti-HEV IgG antibodies (HEV-IgG) were included. The NAFLD activity score (NAS) was used to grade and stage all liver biopsy samples. The HEV-IgG prevalence was compared to a healthy cohort of 997 subjects. RESULTS: One hundred sixty-seven patients with NAFLD were included with the following characteristics: age, 50 ± 13 years; NAS ≤4, 89 (53.3%); NAS 5-8, 78 (46.7%); cirrhosis, 16 (9.6%). Two patients (1.2%) were HEV-IgM-positive, however HEV polymerase chain reaction remained negative and no signs of acute hepatitis were seen. Forty-four patients (26.3%) were HEV-IgG-positive and they were significantly older (55 ± 10 years vs. 48 ± 13 years, P < 0.001) and predominantly men (31 [70.5%] vs.13 [29.5%], P = 0.022). Distribution across NAS (P = 0.610) was not different. However, HEV-IgG-positive patients were significantly more often found with cirrhosis (8 [18.2%] vs. 8 [6.5%], P = 0.024) and liver stiffness values >10 kPa (14 [58.2%] vs. 29 [43.3%], P = 0.026). Multivariable analyses revealed age (odds ratio [OR], 1.054 [1.022-1.086]) and male sex (OR 2.77 [1.27-6.04]) associated with HEV-IgG positivity. Presence of diabetes (OR 3.86 [1.18-12.59]), higher aspartate aminotransferase levels (OR, 1.02 [1.006-1.033]), and HEV-IgG seropositivity (OR 3.52 [1.11-11.13]) were independently linked to cirrhosis. Finally, HEV-IgG positivity was not independently associated with NAFLD patients in a case-control study including healthy subjects. CONCLUSIONS: Prevalence of anti-HEV-IgG antibodies in patients with NAFLD is linked to age and male sex. Furthermore, previous HEV infection was an independent risk factor for cirrhosis. Whether this finding is causal or solely associative is unclear and should be elucidated in future studies.

Ultra-high-field magnetic resonance spectroscopy in non-alcoholic fatty liver disease: Novel mechanistic and diagnostic insights of energy metabolism in non-alcoholic steatohepatitis and advanced fibrosis.

BACKGROUND & AIMS: With the rising prevalence of non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) non-invasive tools obtaining pathomechanistic insights to improve risk stratification are urgently needed. We therefore explored high- and ultra-high-field magnetic resonance spectroscopy (MRS) to obtain novel mechanistic and diagnostic insights into alterations of hepatic lipid, cell membrane and energy metabolism across the spectrum of NAFLD. METHODS: MRS and liver biopsy were performed in 30 NAFLD patients with NAFL (n=8) or NASH (n=22). Hepatic lipid content and composition were measured using 3-Tesla proton (1 H)-MRS. 7-Tesla phosphorus (31 P)-MRS was applied to determine phosphomonoester (PME) including phosphoethanolamine (PE), phosphodiester (PDE) including glycerophosphocholine (GPC), phosphocreatine (PCr), nicotinamide adenine dinucleotide phosphate (NADPH), inorganic phosphate (Pi), γ-ATP and total phosphorus (TP). Saturation transfer technique was used to quantify hepatic ATP flux. RESULTS: Hepatic steatosis in 1 H-MRS highly correlated with histology (P<.001) showing higher values in NASH than NAFL (P<.001) without differences in saturated or unsaturated fatty acid indices. PE/TP ratio increased with advanced fibrosis (F3/4) (P=.002) whereas GPC/PME+PDE decreased (P=.05) compared to no/mild fibrosis (F0-2). γ-ATP/TP was lower in advanced fibrosis (P=.049), while PCr/TP increased (P=.01). NADPH/TP increased with higher grades of ballooning (P=.02). Pi-to-ATP exchange rate constant (P=.003) and ATP flux (P=.001) were lower in NASH than NAFL. CONCLUSIONS: Ultra-high-field MRS, especially saturation transfer technique uncovers changes in energy metabolism including dynamic ATP flux in inflammation and fibrosis in NASH. Non-invasive profiling by MRS appears feasible and may assist further mechanistic and therapeutic studies in NAFLD/NASH.

Low hepatic copper content and PNPLA3 polymorphism in non-alcoholic fatty liver disease in patients without metabolic syndrome.

INTRODUCTION: The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is multifactorial including metabolic, genetic (e.g. PNPLA3 [patatin-like phospholipase domain-containing 3 gene]), viral factors and drugs. Besides, there is evidence for a role of copper deficiency. Aim of the study was to evaluate the role of hepatic copper content, PNPLA3 in NAFLD patients with and without metabolic syndrome (MetS). METHODS: One-hundred seventy-four NAFLD patients, who underwent liver biopsy for diagnostic work-up, were studied. Diagnosis of MetS was based on the WHO Clinical Criteria. Steatosis was semiquantified as percentage of fat containing hepatocytes and was graded according to Brunt. Histological features of non-alcoholic steatohepatitis (NASH) were assessed using the Bedossa classification. Hepatic copper content (in µg/g dry weight) was measured by flame atomic absorption spectroscopy. SNP rs738409 in PNPLA3 was investigated by RT-PCR. RESULTS: Mean hepatic copper content was 22.3 (19.6-25.1) µg/g. The mean percentage of histologically lipid containing hepatocytes was 42.2% (38.3-46.0) and correlated inversely with hepatic copper content (ρ=-0.358, P<0.001). By subgroup analysis this inverse correlation remained significant only in patients without MetS (OR: 0.959 [CI95%: 0.926-0.944], P=0.020). Presence of minor allele (G) of PNPLA3 was also associated with moderate/severe steatosis (≥33%) both in patients with (OR: 2.405 [CI95%: 1.220-4.744], P=0.011) and without MetS (OR: 2.481 [CI95%: 1.172-5.250], P=0.018), but was only associated with NASH (OR: 2.002 [CI95%: 1.062-3.772], P=0.032) and liver fibrosis (OR: 2.646 [CI95%: 1.299-5.389], P=0.007) in patients without MetS. CONCLUSION: Hepatic copper content and PNPLA3 mutations are associated with disease activity in NAFLD patients without MetS. Presence of MetS appears to mask the effects of hepatic copper and PNPLA3.

Vitamin D3 Loading Is Superior to Conventional Supplementation After Weight Loss Surgery in Vitamin D-Deficient Morbidly Obese Patients: a Double-Blind Randomized Placebo-Controlled Trial.

BACKGROUND: Bariatric patients often suffer from vitamin D deficiency (VDD), and both, morbid obesity and VDD, are related to non-alcoholic fatty liver disease. However, limited data are available regarding best strategies for treating VDD, particularly, in bariatric patients undergoing omega-loop gastric bypass (OLGB). Therefore, we examined the efficacy and safety of a forced vitamin D dosing regimen and intervention effects in liver fibrotic patients. METHODS: In this double-blind, randomized, placebo-controlled trial, 50 vitamin D-deficient patients undergoing OLGB were randomly assigned to receive, in the first month postoperatively, oral vitamin D3 (≤3 doses of 100,000 IU; intervention group) or placebo as loading dose (control group) with subsequent maintenance dose (3420 IU/day) in both groups until 6-month visit. RESULTS: Compared with control group, higher increase of 25(OH)D (67.9 (21.1) vs. 55.7 nmol/L (21.1); p = 0.049) with lower prevalence of secondary hyperparathyroidism (10 vs. 24 %; p = 0.045) was observed in intervention group. No (serious) adverse events related to study medication were found. The loading dose regimen was more effective in increasing 25(OH)D in patients with significant liver fibrosis while this was not the case for conventional supplementation (placebo with maintenance dose) (71.5 (20.5) vs. 22.5 nmol/L (13.8); p = 0.022; n = 14). CONCLUSIONS: Our findings indicate that a high vitamin D3 loading dose, in the first month postoperatively, with subsequent maintenance dose is effective and safe in achieving higher vitamin D concentrations in OLGB patients. Unexpectedly, it is more effective in patients with significant liver fibrosis which is of potentially high clinical relevance and requires further investigation.

Farnesoid X Receptor Agonists and Other Bile Acid Signaling Strategies for Treatment of Liver Disease.

The intracellular nuclear receptor farnesoid X receptor (FXR) and the transmembrane G protein-coupled receptor 5 (TGR5) respond to bile acids (BAs) by activating transcriptional networks and/or signaling cascades. These cascades affect the expression of a great number of target genes relevant for BA, cholesterol, lipid and carbohydrate metabolism, as well as genes involved in inflammation, fibrosis and carcinogenesis. FXR activation in the liver tissue and beyond, such as the gut-liver axis, kidney and adipose tissue, plays a role in metabolic diseases. These BA receptors activators hold promise to become a new class of drugs to be used in the treatment of chronic liver disease, hepatocellular cancer and extrahepatic inflammatory and metabolic diseases. This review discusses the relevant BA receptors, the new drugs that target BA transport and signaling and their possible applications.

Prevalence of Liver Fibrosis and its Association with Non-invasive Fibrosis and Metabolic Markers in Morbidly Obese Patients with Vitamin D Deficiency.

BACKGROUND: Morbidly obese patients are at risk for non-alcoholic fatty liver disease (NAFLD) and vitamin D deficiency (VDD). Non-alcoholic steatohepatitis (NASH) is the progressive variant of NAFLD and can advance to fibrosis, cirrhosis, and liver cancer. We aimed to examine prevalence of liver fibrosis and its non-invasive predictors in bariatric patients with VDD (<75 nmol/l). METHODS: Baseline liver biopsy of a randomized controlled trial was performed in 46 patients with omega loop gastric bypass. Clinical, laboratory, and histological data were examined and tested with univariate and multivariable analysis. RESULTS: In total, 80 % were females, aged 42 (SD 13) years with BMI 44 (4) kg/m(2). Twenty-six percent had diabetes mellitus (DM) and 44 % metabolic syndrome (MeS). Seventy-two percent had NASH, 11 % simple steatosis, and 17 % normal liver. In total, 30 % demonstrated significant fibrosis (F ≥ 2) with 9 % of advanced (F3) and 4 % cirrhosis (F4). Increased stages of fibrosis were primarily associated with higher levels of HOMA2-insulin resistance (IR), procollagen type I propeptide (P1NP), lower osteocalcin, albumin-corrected calcium, parathyroid hormone, vitamin D, male sex, and higher age. Other independent risk factors for advanced fibrosis were MeS (OR = 9.3 [0.99-87.5], p = 0.052) and DM (OR = 12.8 [1.2-137.4], p = 0.035). The fibrosis FIB-4 index <10.62 and NAFLD fibrosis score <-26.93 had a negative predictive value of 100 and 96 %, respectively. CONCLUSIONS: Liver fibrosis is frequent in morbidly obese patients with concurrent DM and/or MeS. Increased serum levels of IR, P1NP, lower osteocalcin, and VDD are clinically relevant predictors of fibrosis. Consequently, we suggest that patients with preoperative presence of these markers are at increased risk for liver fibrosis and should be monitored closely.

Difficult case of Cronkhite-Canada syndrome with small intestinal bacterial overgrowth, Clostridium difficile infection and polymyalgia rheumatica.

A 64-year-old woman presented with heavy diarrhoea, nausea and weight loss accompanied by alopecia and dystrophic fingernails and toenails. The preceding diagnosis of an inflammatory bowel disease, a common pitfall, was excluded by endoscopic work up. Instead, Cronkhite-Canada syndrome (CCS), a rare polyposis condition, was identified as the reason for this almost pathognomonic combination of diagnostic findings including various polyps throughout the entire intestine and ectodermal abnormalities. This case exemplifies common risks and complications in terms of gastrointestinal malabsorption, infections and small intestinal bacterial overgrowth (SIBO), including its treatment as well as a hereto unreported association with polymyalgia rheumatica. In CCS, long-term immunosuppressive therapy and close endoscopic cancer screening of the patient is essential. The treatment of vitamin deficiency and recurring SIBO helps to reduce symptoms.

The link between obesity and vitamin D in bariatric patients with omega-loop gastric bypass surgery - a vitamin D supplementation trial to compare the efficacy of postoperative cholecalciferol loading (LOAD): study protocol for a randomized controlled trial.

BACKGROUND: Beyond its classical role in calcium homoeostasis and bone metabolism, vitamin D deficiency has been found to be associated with several diseases, including diabetes, non-alcoholic fatty liver disease, and even obesity itself. Importantly, there are limited data on therapeutic strategies for vitamin D deficiency in bariatric patients, and the procedure-specific guidelines may not be sufficient. To improve long-term outcomes, nutritional screening and appropriate supplementation to prevent nutrient deficiencies are urgently needed. Therefore, the aim of this study is to examine effects and safety of a forced dosing regimen of vitamin D versus conventional dose supplementation on vitamin D levels and other parameters in bariatric patients. METHODS/DESIGN: The study includes loading plus repeat dosing compared with repeated administration of vitamin D without a loading dose, according to guidelines, in a prospective, double-blind, randomized controlled trial. Up to a triple oral loading dose is given on day 1, then 2 and 4 weeks after surgery (100,000 IU dose each time), followed by an oral maintenance dose (3420 IU/day). The control group (n = 25) will receive placebo, followed by administration of a standard dose (3420 IU/day). We hypothesize that a significant increase in vitamin D levels will occur in patients in the treatment group (n = 25) by 24 weeks after surgery. Further measurements are aimed at evaluating changes in inflammation, bone turnover, insulin resistance, blood pressure, liver, mental health, and gut microbiota of patients undergoing omega-loop gastric bypass surgery. Furthermore, possible associations between concentrations of vitamin D, the involved enzymes, or vitamin D receptor in adipose and/or liver tissues will be determined. DISCUSSION: To our knowledge, this trial is the first of its kind with this type of vitamin D supplementation in bariatric patients. Its major strength is the design and implementation of evaluation of influencing factors such as liver function, bone health, inflammation, insulin resistance, blood pressure, symptoms of depression, or microbiota. This alternative vitamin D dosing regimen has the potential to be a safe, fast, evidence-based treatment of vitamin D deficiency in bariatric patients. Owing to the increasing number of bariatric patients, it is also of interest to elucidate the link between obesity and vitamin D. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02092376 . Registered on 17 March 2014.

Challenges and Management of Liver Cirrhosis: Practical Issues in the Therapy of Patients with Cirrhosis due to NAFLD and NASH.

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome and comprises a liver disease spectrum ranging from steatosis to nonalcoholic steatohepatitis (NASH) with risk of progression to liver cirrhosis and hepatocellular carcinoma (HCC). Associated metabolic conditions and comorbidities such as obesity, diabetes and cardiovascular diseases are common and require concerted management. Adiponutrin (PNPLA3) variants may help to identify NAFLD patients at higher risk for liver disease progression towards advanced fibrosis and HCC. The therapeutic options in NAFLD/NASH include lifestyle modification, pharmacological treatment, bariatric surgery for patients with morbid obesity and treatment of complications of liver cirrhosis and HCC, including liver transplantation. Insulin sensitizers and antioxidative treatment strategies with vitamin E are among the best-established pharmacological approaches, but both drugs have long-term safety issues and there is limited evidence in cirrhotic patients. Treatment of concomitant/underlying metabolic conditions with statins or metformin may also have beneficial effects on portal hypertension, complications of liver cirrhosis and HCC prevention. The bile acid receptor FXR may be a promising novel therapeutic target for the treatment of NAFLD/NASH, fibrosis and portal hypertension, but the prognostic implications of associated changes in low- and high-density lipoprotein cholesterol require further studies. Morbidly obese NASH patients can benefit from bariatric surgery which may reduce liver fibrosis but carries a risk of decompensation in patients with advanced liver cirrhosis. When carefully selected, patients with NASH cirrhosis undergoing liver transplantation have a good outcome. This review summarizes recent progress in the management of patients with liver cirrhosis due to NASH.

Hepatic steatosis in Wilson disease--Role of copper and PNPLA3 mutations.

BACKGROUND & AIMS: The earliest characteristic alterations of the liver pathology in Wilson disease (WD) include steatosis, which is sometimes indistinguishable from non-alcoholic fatty liver disease (NAFLD). Steatosis in WD may reflect copper-induced mitochondrial dysfunction. A genetic polymorphism in rs738409, in the patatin-like phospholipase domain-containing 3 gene (PNPLA3), is strongly associated with appearance of in NAFLD. This study evaluated the role of PNPLA3 and hepatic copper content for development of steatosis in patients with WD. METHODS: Liver biopsies obtained at diagnosis and the PNPLA3 genotype were analyzed in 98 Caucasian patients with WD (male: 52 [53.1%]; mean age: 27.6 years [CI 95%: 24.8-30.4, range: 5.8-61.5]). Steatosis was graded as percentage of lipid containing hepatocytes by an expert hepatopathologist unaware of the results of genetic testing. RESULTS: Moderate/severe steatosis (>33% of hepatocytes) was observed in 28 patients (pediatric: n=13/26 [50.0%], adult: n=15/72 [20.8%]; p=0.01). Forty-six patients (46.9%; pediatric: n=7, adult: n=39; p=0.022) had cirrhosis. Multivariate logistic regression identified PNPLA3 G allele (OR: 2.469, CI 95%: 1.203-5.068; p=0.014) and pediatric age (OR: 4.348; 1.577-11.905; p=0.004) as independent variables associated with moderate/severe steatosis. In contrast, hepatic copper content did not impact on moderate/severe steatosis (OR: 1.000, CI 95%: 1.000-1.001; p=0.297). CONCLUSIONS: Steatosis is common in WD and the PNPLA3 G allele contributes to its pathogenesis. The role of hepatic copper concentration and ATP7B mutations in steatosis development deserve further investigations.

Application of localized ³¹P MRS saturation transfer at 7 T for measurement of ATP metabolism in the liver: reproducibility and initial clinical application in patients with non-alcoholic fatty liver disease.

OBJECTIVES: Saturation transfer (ST) phosphorus MR spectroscopy ((31)P MRS) enables in vivo insight into energy metabolism and thus could identify liver conditions currently diagnosed only by biopsy. This study assesses the reproducibility of the localized (31)P MRS ST in liver at 7 T and tests its potential for noninvasive differentiation of non-alcoholic fatty liver (NAFL) and steatohepatitis (NASH). METHODS: After the ethics committee approval, reproducibility of the localized (31)P MRS ST at 7 T and the biological variation of acquired hepato-metabolic parameters were assessed in healthy volunteers. Subsequently, 16 suspected NAFL/NASH patients underwent MRS measurements and diagnostic liver biopsy. The Pi-to-ATP exchange parameters were compared between the groups by a Mann-Whitney U test and related to the liver fat content estimated by a single-voxel proton ((1)H) MRS, measured at 3 T. RESULTS: The mean exchange rate constant (k) in healthy volunteers was 0.31 ± 0.03 s(-1) with a coefficient of variation of 9.0 %. Significantly lower exchange rates (p < 0.01) were found in NASH patients (k = 0.17 ± 0.04 s(-1)) when compared to healthy volunteers, and NAFL patients (k = 0.30 ± 0.05 s(-1)). Significant correlation was found between the k value and the liver fat content (r = 0.824, p < 0.01). CONCLUSIONS: Our data suggest that the (31)P MRS ST technique provides a tool for gaining insight into hepatic ATP metabolism and could contribute to the differentiation of NAFL and NASH. KEY POINTS: • 1D localized (31) P MRS saturation transfer in the liver is reproducible at 7 T • NASH patients have decreased hepatic Pi-to-ATP exchange rate • In this study, hepatic metabolic activity correlates with liver fat content.

Noninvasive differentiation of simple steatosis and steatohepatitis by using gadoxetic acid-enhanced MR imaging in patients with nonalcoholic fatty liver disease: a proof-of-concept study.

PURPOSE: To determine whether gadoxetic acid-enhanced magnetic resonance (MR) imaging can be used to distinguish between simple steatosis and nonalcoholic steatohepatitis (NASH) in patients with nonalcoholic fatty liver disease (NAFLD), defined according to the steatosis activity and fibrosis (SAF) scoring system, which is based on the semiquantitative scoring of steatosis activity and liver fibrosis. MATERIALS AND METHODS: The local institutional review committee approved this study and waived written informed consent. This was a retrospective study of gadoxetic acid-enhanced 3-T MR imaging performed in 81 patients with NAFLD (45 men [56%]; mean age, 56 years; range, 25-78 years). The MR images were analyzed by using the relative enhancement (the ratio of signal intensities of the liver parenchyma before and 20 minutes after intravenous administration of gadoxetic acid). Univariate and multiple regression analyses were applied to identify variables associated with relative enhancement measurements. The ability of relative enhancement to allow differentiation between simple steatosis and NASH was assessed by using area under the receiver operating characteristic (ROC) curve analysis. RESULTS: Relative enhancement negatively correlated with the degree of lobular inflammation (r = -0.59, P < .0001), ballooning (r = -0.44, P < .0001), and fibrosis (r = -0.59, P ≤ .0001), but not with steatosis (r = -0.16, P = .15). Patients with NASH had a significantly lower relative liver enhancement (0.82 ± 0.22) than those with simple steatosis (1.39 ± 0.52) (P < .001). Relative enhancement measurements performed well in the differentiation between simple steatosis and NASH, with an area under the ROC curve of 0.85 (95% confidence interval: 0.75, 0.91) (cutoff = 1.24, sensitivity = 97%, specificity = 63%). CONCLUSION: Gadoxetic acid relative enhancement was significantly lower in patients with NASH than in patients with simple steatosis, but further prospective studies are warranted.