A polygenic risk score for alcohol-associated cirrhosis among heavy drinkers with European ancestry.

BACKGROUND: Polygenic Risk Scores (PRS) based on results from genome-wide association studies offer the prospect of risk stratification for many common and complex diseases. We developed a PRS for alcohol-associated cirrhosis by comparing single-nucleotide polymorphisms among patients with alcohol-associated cirrhosis (ALC) versus drinkers who did not have evidence of liver fibrosis/cirrhosis. METHODS: Using a data-driven approach, a PRS for ALC was generated using a meta-genome-wide association study of ALC (N=4305) and an independent cohort of heavy drinkers with ALC and without significant liver disease (N=3037). It was validated in 2 additional independent cohorts from the UK Biobank with diagnosed ALC (N=467) and high-risk drinking controls (N=8981) and participants in the Indiana Biobank Liver cohort with alcohol-associated liver disease (N=121) and controls without liver disease (N=3239). RESULTS: A 20-single-nucleotide polymorphisms PRS for ALC (PRSALC) was generated that stratified risk for ALC comparing the top and bottom deciles of PRS in the 2 validation cohorts (ORs: 2.83 [95% CI: 1.82 -4.39] in UK Biobank; 4.40 [1.56 -12.44] in Indiana Biobank Liver cohort). Furthermore, PRSALC improved the prediction of ALC risk when added to the models of clinically known predictors of ALC risk. It also stratified the risk for metabolic dysfunction -associated steatotic liver disease -cirrhosis (3.94 [2.23 -6.95]) in the Indiana Biobank Liver cohort -based exploratory analysis. CONCLUSIONS: PRSALC incorporates 20 single-nucleotide polymorphisms, predicts increased risk for ALC, and improves risk stratification for ALC compared with the models that only include clinical risk factors. This new score has the potential for early detection of heavy drinking patients who are at high risk for ALC.

A genetic risk score and diabetes predict development of alcohol-related cirrhosis in drinkers.

BACKGROUND & AIMS: Only a minority of excess alcohol drinkers develop cirrhosis. We developed and evaluated risk stratification scores to identify those at highest risk. METHODS: Three cohorts (GenomALC-1: n = 1,690, GenomALC-2: n = 3,037, UK Biobank: relevant n = 6,898) with a history of heavy alcohol consumption (≥80 g/day (men), ≥50 g/day (women), for ≥10 years) were included. Cases were participants with alcohol-related cirrhosis. Controls had a history of similar alcohol consumption but no evidence of liver disease. Risk scores were computed from up to 8 genetic loci identified previously as associated with alcohol-related cirrhosis and 3 clinical risk factors. Score performance for the stratification of alcohol-related cirrhosis risk was assessed and compared across the alcohol-related liver disease spectrum, including hepatocellular carcinoma (HCC). RESULTS: A combination of 3 single nucleotide polymorphisms (SNPs) (PNPLA3:rs738409, SUGP1-TM6SF2:rs10401969, HSD17B13:rs6834314) and diabetes status best discriminated cirrhosis risk. The odds ratios (ORs) and (95% CIs) between the lowest (Q1) and highest (Q5) score quintiles of the 3-SNP score, based on independent allelic effect size estimates, were 5.99 (4.18-8.60) (GenomALC-1), 2.81 (2.03-3.89) (GenomALC-2), and 3.10 (2.32-4.14) (UK Biobank). Patients with diabetes and high risk scores had ORs of 14.7 (7.69-28.1) (GenomALC-1) and 17.1 (11.3-25.7) (UK Biobank) compared to those without diabetes and with low risk scores. Patients with cirrhosis and HCC had significantly higher mean risk scores than patients with cirrhosis alone (0.76 ± 0.06 vs. 0.61 ± 0.02, p = 0.007). Score performance was not significantly enhanced by information on additional genetic risk variants, body mass index or coffee consumption. CONCLUSIONS: A risk score based on 3 genetic risk variants and diabetes status enables the stratification of heavy drinkers based on their risk of cirrhosis, allowing for the provision of earlier preventative interventions. LAY SUMMARY: Excessive chronic drinking leads to cirrhosis in some people, but so far there is no way to identify those at high risk of developing this debilitating disease. We developed a genetic risk score that can identify patients at high risk. The risk of cirrhosis is increased >10-fold with just two risk factors - diabetes and a high genetic risk score. Risk assessment using this test could enable the early and personalised management of this disease in high-risk patients.

Macrophage scavenger receptor 1 mediates lipid-induced inflammation in non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Obesity-associated inflammation is a key player in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). However, the role of macrophage scavenger receptor 1 (MSR1, CD204) remains incompletely understood. METHODS: A total of 170 NAFLD liver biopsies were processed for transcriptomic analysis and correlated with clinicopathological features. Msr1-/- and wild-type mice were subjected to a 16-week high-fat and high-cholesterol diet. Mice and ex vivo human liver slices were treated with a monoclonal antibody against MSR1. Genetic susceptibility was assessed using genome-wide association study data from 1,483 patients with NAFLD and 430,101 participants of the UK Biobank. RESULTS: MSR1 expression was associated with the occurrence of hepatic lipid-laden foamy macrophages and correlated with the degree of steatosis and steatohepatitis in patients with NAFLD. Mice lacking Msr1 were protected against diet-induced metabolic disorder, showing fewer hepatic foamy macrophages, less hepatic inflammation, improved dyslipidaemia and glucose tolerance, and altered hepatic lipid metabolism. Upon induction by saturated fatty acids, MSR1 induced a pro-inflammatory response via the JNK signalling pathway. In vitro blockade of the receptor prevented the accumulation of lipids in primary macrophages which inhibited the switch towards a pro-inflammatory phenotype and the release of cytokines such as TNF-ɑ. Targeting MSR1 using monoclonal antibody therapy in an obesity-associated NAFLD mouse model and human liver slices resulted in the prevention of foamy macrophage formation and inflammation. Moreover, we identified that rs41505344, a polymorphism in the upstream transcriptional region of MSR1, was associated with altered serum triglycerides and aspartate aminotransferase levels in a cohort of over 400,000 patients. CONCLUSIONS: Taken together, our data suggest that MSR1 plays a critical role in lipid-induced inflammation and could thus be a potential therapeutic target for the treatment of NAFLD. LAY SUMMARY: Non-alcoholic fatty liver disease (NAFLD) is a chronic disease primarily caused by excessive consumption of fat and sugar combined with a lack of exercise or a sedentary lifestyle. Herein, we show that the macrophage scavenger receptor MSR1, an innate immune receptor, mediates lipid uptake and accumulation in Kupffer cells, resulting in liver inflammation and thereby promoting the progression of NAFLD in humans and mice.

Obesity, Diabetes, Coffee, Tea, and Cannabis Use Alter Risk for Alcohol-Related Cirrhosis in 2 Large Cohorts of High-Risk Drinkers.

INTRODUCTION: Sustained high alcohol intake is necessary but not sufficient to produce alcohol-related cirrhosis. Identification of risk factors, apart from lifetime alcohol exposure, would assist in discovery of mechanisms and prediction of risk. METHODS: We conducted a multicenter case-control study (GenomALC) comparing 1,293 cases (with alcohol-related cirrhosis, 75.6% male) and 754 controls (with equivalent alcohol exposure but no evidence of liver disease, 73.6% male). Information confirming or excluding cirrhosis, and on alcohol intake and other potential risk factors, was obtained from clinical records and by interview. Case-control differences in risk factors discovered in the GenomALC participants were validated using similar data from 407 cases and 6,573 controls from UK Biobank. RESULTS: The GenomALC case and control groups reported similar lifetime alcohol intake (1,374 vs 1,412 kg). Cases had a higher prevalence of diabetes (20.5% (262/1,288) vs 6.5% (48/734), P = 2.27 × 10-18) and higher premorbid body mass index (26.37 ± 0.16 kg/m2) than controls (24.44 ± 0.18 kg/m2, P = 5.77 × 10-15). Controls were significantly more likely to have been wine drinkers, coffee drinkers, smokers, and cannabis users than cases. Cases reported a higher proportion of parents who died of liver disease than controls (odds ratio 2.25 95% confidence interval 1.55-3.26). Data from UK Biobank confirmed these findings for diabetes, body mass index, proportion of alcohol as wine, and coffee consumption. DISCUSSION: If these relationships are causal, measures such as weight loss, intensive treatment of diabetes or prediabetic states, and coffee consumption should reduce the risk of alcohol-related cirrhosis.

Genome-wide Association Study and Meta-analysis on Alcohol-Associated Liver Cirrhosis Identifies Genetic Risk Factors.

BACKGROUND AND AIMS: Only a minority of heavy drinkers progress to alcohol-associated cirrhosis (ALC). The aim of this study was to identify common genetic variants that underlie risk for ALC. APPROACH AND RESULTS: We analyzed data from 1,128 subjects of European ancestry with ALC and 614 heavy-drinking subjects without known liver disease from Australia, the United States, the United Kingdom, and three countries in Europe. A genome-wide association study (GWAS) was performed, adjusting for principal components and clinical covariates (alcohol use, age, sex, body mass index, and diabetes). We validated our GWAS findings using UK Biobank. We then performed a meta-analysis combining data from our study, the UK Biobank, and a previously published GWAS. Our GWAS found genome-wide significant risk association of rs738409 in patatin-like phospholipase domain containing 3 (PNPLA3) (odds ratio [OR] = 2.19 [G allele], P = 4.93 × 10-17 ) and rs4607179 near HSD17B13 (OR = 0.57 [C allele], P = 1.09 × 10-10 ) with ALC. Conditional analysis accounting for the PNPLA3 and HSD17B13 loci identified a protective association at rs374702773 in Fas-associated factor family member 2 (FAF2) (OR = 0.61 [del(T) allele], P = 2.56 × 10-8 ) for ALC. This association was replicated in the UK Biobank using conditional analysis (OR = 0.79, P = 0.001). Meta-analysis (without conditioning) confirmed genome-wide significance for the identified FAF2 locus as well as PNPLA3 and HSD17B13. Two other previously known loci (SERPINA1 and SUGP1/TM6SF2) were also genome-wide significant in the meta-analysis. GeneOntology pathway analysis identified lipid droplets as the target for several identified genes. In conclusion, our GWAS identified a locus at FAF2 associated with reduced risk of ALC among heavy drinkers. Like the PNPLA3 and HSD17B13 gene products, the FAF2 product has been localized to fat droplets in hepatocytes. CONCLUSIONS: Our genetic findings implicate lipid droplets in the biological pathway(s) underlying ALC.

Transcriptomic profiling across the nonalcoholic fatty liver disease spectrum reveals gene signatures for steatohepatitis and fibrosis.

The mechanisms that drive nonalcoholic fatty liver disease (NAFLD) remain incompletely understood. This large multicenter study characterized the transcriptional changes that occur in liver tissue across the NAFLD spectrum as disease progresses to cirrhosis to identify potential circulating markers. We performed high-throughput RNA sequencing on a discovery cohort comprising histologically characterized NAFLD samples from 206 patients. Unsupervised clustering stratified NAFLD on the basis of disease activity and fibrosis stage with differences in age, aspartate aminotransferase (AST), type 2 diabetes mellitus, and carriage of PNPLA3 rs738409, a genetic variant associated with NAFLD. Relative to early disease, we consistently identified 25 differentially expressed genes as fibrosing steatohepatitis progressed through stages F2 to F4. This 25-gene signature was independently validated by logistic modeling in a separate replication cohort (n = 175), and an integrative analysis with publicly available single-cell RNA sequencing data elucidated the likely relative contribution of specific intrahepatic cell populations. Translating these findings to the protein level, SomaScan analysis in more than 300 NAFLD serum samples confirmed that circulating concentrations of proteins AKR1B10 and GDF15 were strongly associated with disease activity and fibrosis stage. Supporting the biological plausibility of these data, in vitro functional studies determined that endoplasmic reticulum stress up-regulated expression of AKR1B10, GDF15, and PDGFA, whereas GDF15 supplementation tempered the inflammatory response in macrophages upon lipid loading and lipopolysaccharide stimulation. This study provides insights into the pathophysiology of progressive fibrosing steatohepatitis, and proof of principle that transcriptomic changes represent potentially tractable and clinically relevant markers of disease progression.

Genome-wide association study of non-alcoholic fatty liver and steatohepatitis in a histologically characterised cohort☆.

BACKGROUND & AIMS: Genetic factors associated with non-alcoholic fatty liver disease (NAFLD) remain incompletely understood. To date, most genome-wide association studies (GWASs) have adopted radiologically assessed hepatic triglyceride content as the reference phenotype and so cannot address steatohepatitis or fibrosis. We describe a GWAS encompassing the full spectrum of histologically characterised NAFLD. METHODS: The GWAS involved 1,483 European NAFLD cases and 17,781 genetically matched controls. A replication cohort of 559 NAFLD cases and 945 controls was genotyped to confirm signals showing genome-wide or close to genome-wide significance. RESULTS: Case-control analysis identified signals showing p values ≤5 × 10-8 at 4 locations (chromosome [chr] 2 GCKR/C2ORF16; chr4 HSD17B13; chr19 TM6SF2; chr22 PNPLA3) together with 2 other signals with p <1 × 10-7 (chr1 near LEPR and chr8 near IDO2/TC1). Case-only analysis of quantitative traits showed that the PNPLA3 signal (rs738409) had genome-wide significance for steatosis, fibrosis and NAFLD activity score and a new signal (PYGO1 rs62021874) had close to genome-wide significance for steatosis (p = 8.2 × 10-8). Subgroup case-control analysis for NASH confirmed the PNPLA3 signal. The chr1 LEPR single nucleotide polymorphism also showed genome-wide significance for this phenotype. Considering the subgroup with advanced fibrosis (≥F3), the signals on chr2, chr19 and chr22 maintained their genome-wide significance. Except for GCKR/C2ORF16, the genome-wide significance signals were replicated. CONCLUSIONS: This study confirms PNPLA3 as a risk factor for the full histological spectrum of NAFLD at genome-wide significance levels, with important contributions from TM6SF2 and HSD17B13. PYGO1 is a novel steatosis modifier, suggesting that Wnt signalling pathways may be relevant in NAFLD pathogenesis. LAY SUMMARY: Non-alcoholic fatty liver disease is a common disease where excessive fat accumulates in the liver and may result in cirrhosis. To understand who is at risk of developing this disease and suffering liver damage, we undertook a genetic study to compare the genetic profiles of people suffering from fatty liver disease with genetic profiles seen in the general population. We found that particular sequences in 4 different areas of the human genome were seen at different frequencies in the fatty liver disease cases. These sequences may help predict an individual's risk of developing advanced disease. Some genes where these sequences are located may also be good targets for future drug treatments.

The degree of hepatic steatosis associates with impaired cardiac and autonomic function.

BACKGROUND & AIMS: Cardiovascular disease is the principle cause of death in patients with elevated liver fat unrelated to alcohol consumption, more so than liver-related morbidity and mortality. The aim of this study was to evaluate the relationship between liver fat and cardiac and autonomic function, as well as to assess how impairment in cardiac and autonomic function is influenced by metabolic risk factors. METHODS: Cardiovascular and autonomic function were assessed in 96 sedentary individuals: i) non-alcoholic fatty liver disease (NAFLD) (n = 46, hepatic steatosis >5% by magnetic resonance spectroscopy), ii) Hepatic steatosis and alcohol (dual aetiology fatty liver disease [DAFLD]) (n = 16, hepatic steatosis >5%, consuming >20 g/day of alcohol) and iii) CONTROL (n = 34, no cardiac, liver or metabolic disorders, <20 g/day of alcohol). RESULTS: Patients with NAFLD and DAFLD had significantly impaired cardiac and autonomic function when compared with controls. Diastolic variability and systolic variability (LF/HF-sBP [n/1]; 2.3 (1.7) and 2.3 (1.5) vs. 3.4 (1.5), p <0.01) were impaired in patients with NAFLD and DAFLD when compared to controls, with DAFLD individuals showing a decrease in diastolic variability relative to NAFLD patients. Hepatic steatosis and fasting glucose were negatively correlated with stroke volume index. Fibrosis stage was significantly negatively associated with mean blood pressure (r = -0.47, p = 0.02), diastolic variability (r = -0.58, p ≤0.01) and systolic variability (r = -0.42, p = 0.04). Hepatic steatosis was independently associated with cardiac function (p ≤0.01); TNF-α (p ≤0.05) and CK-18 (p ≤0.05) were independently associated with autonomic function. CONCLUSION: Cardiac and autonomic impairments appear to be dependent on level of liver fat, metabolic dysfunction, inflammation and fibrosis staging, and to a lesser extent alcohol intake. Interventions should be sought to moderate the excess cardiovascular risk in patients with NAFLD or DAFLD. LAY SUMMARY: Increased levels of fat in the liver impair the ability of the cardiovascular system to work properly. The amount of fat in the liver, metabolic control, inflammation and alcohol are all linked to the degree that the cardiovascular system is affected.

Modeling NAFLD disease burden in China, France, Germany, Italy, Japan, Spain, United Kingdom, and United States for the period 2016-2030.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are increasingly a cause of cirrhosis and hepatocellular carcinoma globally. This burden is expected to increase as epidemics of obesity, diabetes and metabolic syndrome continue to grow. The goal of this analysis was to use a Markov model to forecast NAFLD disease burden using currently available data. METHODS: A model was used to estimate NAFLD and NASH disease progression in eight countries based on data for adult prevalence of obesity and type 2 diabetes mellitus (DM). Published estimates and expert consensus were used to build and validate the model projections. RESULTS: If obesity and DM level off in the future, we project a modest growth in total NAFLD cases (0-30%), between 2016-2030, with the highest growth in China as a result of urbanization and the lowest growth in Japan as a result of a shrinking population. However, at the same time, NASH prevalence will increase 15-56%, while liver mortality and advanced liver disease will more than double as a result of an aging/increasing population. CONCLUSIONS: NAFLD and NASH represent a large and growing public health problem and efforts to understand this epidemic and to mitigate the disease burden are needed. If obesity and DM continue to increase at current and historical rates, both NAFLD and NASH prevalence are expected to increase. Since both are reversible, public health campaigns to increase awareness and diagnosis, and to promote diet and exercise can help manage the growth in future disease burden. LAY SUMMARY: Non-alcoholic fatty liver disease and non-alcoholic steatohepatitis can lead to advanced liver disease. Both conditions are becoming increasingly prevalent as the epidemics of obesity and diabetes continue to increase. A mathematical model was built to understand how the disease burden associated with non-alcoholic fatty liver disease and non-alcoholic steatohepatitis will change over time. Results suggest increasing cases of advanced liver disease and liver-related mortality in the coming years.

Systematic review assessing the effectiveness of dietary intervention on gut microbiota in adults with type 2 diabetes.

AIMS/HYPOTHESIS: Despite improved understanding of the pathophysiology of type 2 diabetes mellitus, explanations for individual variability in disease progression and response to treatment are incomplete. The gut microbiota has been linked to the pathophysiology of type 2 diabetes mellitus and may account for this variability. We conducted a systematic review to assess the effectiveness of dietary and physical activity/exercise interventions in modulating the gut microbiota and improving glucose control in adults with type 2 diabetes mellitus. METHODS: A systematic search was conducted to identify studies reporting on the effect of dietary and physical activity/exercise interventions on the gut microbiota and glucose control in individuals with a confirmed diagnosis of type 2 diabetes mellitus. Study characteristics, methodological quality and details relating to interventions were captured using a data-extraction form. Meta-analyses were conducted where sufficient data were available, and other results were reported narratively. RESULTS: Eight studies met the eligibility criteria of the systematic review. No studies were found that reported on the effects of physical activity/exercise on the gut microbiota and glucose control. However, studies reporting on dietary interventions showed that such interventions were associated with modifications to the composition and diversity of the gut microbiota. There was a statistically significant improvement in HbA1c (standardised mean difference [SMD] -2.31 mmol/mol [95% CI -2.76, -1.85] [0.21%; 95% CI -0.26, -0.16]; I2 = 0%, p < 0.01), but not in fasting blood glucose (SMD -0.25 mmol/l [95% CI -0.85, 0.35], I2 = 87%, p > 0.05), fasting insulin (SMD -1.82 pmol/l [95% CI -7.23, 3.60], I2 = 54%, p > 0.05) or HOMA-IR (SMD -0.15 [95% CI -0.63, 0.32], I2 = 69%, p > 0.05) when comparing dietary interventions with comparator groups. There were no significant changes in the relative abundance of bacteria in the genera Bifidobacterium (SMD 1.29% [95% CI -4.45, 7.03], I2 = 33%, p > 0.05), Roseburia (SMD -0.85% [95% CI -2.91, 1.21], I2 = 79%, p > 0.05) or Lactobacillus (SMD 0.04% [95% CI -0.01, 0.09], I2 = 0%, p > 0.05) when comparing dietary interventions with comparator groups. There were, however, other significant changes in the gut microbiota, including changes at various taxonomic levels, including phylum, family, genus and species, Firmicutes:Bacteroidetes ratios and changes in diversity matrices (α and ß). Dietary intervention had minimal or no effect on inflammation, short-chain fatty acids or anthropometrics. CONCLUSIONS/INTERPRETATION: Dietary intervention was found to modulate the gut microbiota and improve glucose control in individuals with type 2 diabetes. Although the results of the included studies are encouraging, this review highlights the need for further well-conducted interventional studies to inform the clinical use of dietary interventions targeting the gut microbiota.

Evaluation of laboratory tests for cirrhosis and for alcohol use, in the context of alcoholic cirrhosis.

Laboratory tests can play an important role in assessment of alcoholic patients, including for evaluation of liver damage and as markers of alcohol intake. Evidence on test performance should lead to better selection of appropriate tests and improved interpretation of results. We compared laboratory test results from 1578 patients between cases (with alcoholic cirrhosis; 753 men, 243 women) and controls (with equivalent lifetime alcohol intake but no liver disease; 439 men, 143 women). Comparisons were also made between 631 cases who had reportedly been abstinent from alcohol for over 60 days and 364 who had not. ROC curve analysis was used to estimate and compare tests' ability to distinguish patients with and without cirrhosis, and abstinent and drinking cases. The best tests for presence of cirrhosis were INR and bilirubin, with areas under the ROC curve (AUCs) of 0.91 ± 0.01 and 0.88 ± 0.01, respectively. Confining analysis to patients with no current or previous ascites gave AUCs of 0.88 ± 0.01 for INR and 0.85 ± 0.01 for bilirubin. GGT and AST showed discrimination between abstinence and recent drinking in patients with cirrhosis, including those without ascites, when appropriate (and for GGT, sex-specific) limits were used. For AST, a cut-off limit of 85 units/L gave 90% specificity and 37% sensitivity. For GGT, cut-off limits of 288 units/L in men and 138 units/L in women gave 90% specificity for both and 40% sensitivity in men, 63% sensitivity in women. INR and bilirubin show the best separation between patients with alcoholic cirrhosis (with or without ascites) and control patients with similar lifetime alcohol exposure. Although AST and GGT are substantially increased by liver disease, they can give useful information on recent alcohol intake in patients with alcoholic cirrhosis when appropriate cut-off limits are used.

Effects of Exercise on Liver Fat and Metabolism in Alcohol Drinkers.

BACKGROUND & AIMS: Exercise is an important component of obesity-associated disorders and has been shown to reduce markers of nonalcoholic fatty liver disease (NAFLD). However, little is known about how these effects are influenced by alcohol intake. The authors performed a randomized controlled trial to investigate the effects of exercise on hepatic triglyceride content (HTGC) and metabolism in overweight or obese patients who consume alcohol. METHODS: The authors performed a prospective study of 27 patients (mean 54 ± 11 years of age, body mass index [BMI] 31 ± 4 kg/m2) with >5% HTGC in the United Kingdom, consuming alcohol (mean 221 ± 75 g/week). Anthropometry, body composition, HTGC, and abdominal fat were measured using plethysmography and magnetic resonance imaging. Subjects were assigned to groups that exercised (3 times/week on nonconsecutive days) for 12 weeks (n = 14) or continued standard care (control group, n = 13), maintaining baseline weight and alcohol consumption. The exercise program consisted of aerobic exercise (static cycling) and a circuit of resistance exercise (free weights and machines). Patients were examined at baseline and at 12 weeks; data collected on HTGC, body composition, metabolic control, circulating inflammatory, and fibrosis markers were assessed at baseline and at 12 weeks. Between-group differences were evaluated using an unpaired t test and within-group differences using a paired t test. The primary outcomes for this study were changes in HTGC between baseline and 12 weeks. RESULTS: After 12 weeks, there was no significant difference between the exercise and control groups in HTGC (reduction of 0.1% ± 2.1% in exercisers vs increase of 0.5 ± 2.1% in control group; P > .05). At week 12, the exercise group had significant reductions in subcutaneous fat (loss of 23 ± 28 cm2 in the exercisers vs increase of 12 ± 19 cm2 in the control group; P < .01), and whole body fat (loss of 2.1 ± 1.1 kg in the exercisers vs increase of 0.2 ± 2.1 kg; P < .01). The exercise group also had a significantly greater increase in lean body mass (increase of 1.9 ± 1.4 kg for the exercisers vs increase of 0.7 ± 1.5 kg for the control group; P < .01) and a significantly greater reduction in level of cytokeratin 18 (reduction of 49 ± 82 U/L in exercisers vs increase of 17 ± 38 U/L in control group; P < .05). There were no differences between groups in changes in metabolic factors or markers of inflammation. CONCLUSIONS: In a randomized controlled trial of obese individuals who consume alcohol, exercise significantly improved body composition and reduced hepatocyte apoptosis (cytokeratin 18), but did not reduce HTGC. This finding could indicate that alcohol consumption reduces the effects of exercise on NAFLD observed in previous studies. Clinical care teams should look to use exercise as part of the management strategy for people consuming alcohol, but optimal benefit may be as an adjunct to alcohol reduction and weight management strategies. (ISRCTN.com, Number: ISRCTN90597099).

A randomised controlled trial of losartan as an anti-fibrotic agent in non-alcoholic steatohepatitis.

INTRODUCTION: Non-alcoholic fatty liver disease (NAFLD) is a common liver disease worldwide. Experimental and small clinical trials have demonstrated that angiotensin II blockers (ARB) may be anti-fibrotic in the liver. The aim of this randomised controlled trial was to assess whether treatment with Losartan for 96 weeks slowed, halted or reversed the progression of fibrosis in patients with non-alcoholic steatohepatitis (NASH). METHODS: Double-blind randomised-controlled trial of Losartan 50 mg once a day versus placebo for 96 weeks in patients with histological evidence of NASH. The primary outcome for the study was change in histological fibrosis stage from pre-treatment to end-of-treatment. RESULTS: The study planned to recruit 214 patients. However, recruitment was slower than expected, and after 45 patients were randomised (median age 55; 56% male; 60% diabetic; median fibrosis stage 2), enrolment was suspended. Thirty-two patients (15 losartan and 17 placebo) completed follow up period: one patient (6.7%) treated with losartan and 4 patients (23.5%) in the placebo group were "responders" (lower fibrosis stage at follow up compared with baseline). The major reason for slow recruitment was that 39% of potentially eligible patients were already taking an ARB or angiotensin converting enzyme inhibitor (ACEI), and 15% were taking other prohibited medications. CONCLUSIONS: Due to the widespread use of ACEI and ARB in patients with NASH this trial failed to recruit sufficient patients to determine whether losartan has anti-fibrotic effects in the liver. TRIAL REGISTRATION: ISRCTN 57849521.

Association of Liver Injury From Specific Drugs, or Groups of Drugs, With Polymorphisms in HLA and Other Genes in a Genome-Wide Association Study.

BACKGROUND & AIMS: We performed a genome-wide association study (GWAS) to identify genetic risk factors for drug-induced liver injury (DILI) from licensed drugs without previously reported genetic risk factors. METHODS: We performed a GWAS of 862 persons with DILI and 10,588 population-matched controls. The first set of cases was recruited before May 2009 in Europe (n = 137) and the United States (n = 274). The second set of cases were identified from May 2009 through May 2013 from international collaborative studies performed in Europe, the United States, and South America. For the GWAS, we included only cases with patients of European ancestry associated with a particular drug (but not flucloxacillin or amoxicillin-clavulanate). We used DNA samples from all subjects to analyze HLA genes and single nucleotide polymorphisms. After the discovery analysis was concluded, we validated our findings using data from 283 European patients with diagnosis of DILI associated with various drugs. RESULTS: We associated DILI with rs114577328 (a proxy for A*33:01 a HLA class I allele; odds ratio [OR], 2.7; 95% confidence interval [CI], 1.9-3.8; P = 2.4 × 10-8) and with rs72631567 on chromosome 2 (OR, 2.0; 95% CI, 1.6-2.5; P = 9.7 × 10-9). The association with A*33:01 was mediated by large effects for terbinafine-, fenofibrate-, and ticlopidine-related DILI. The variant on chromosome 2 was associated with DILI from a variety of drugs. Further phenotypic analysis indicated that the association between DILI and A*33:01 was significant genome wide for cholestatic and mixed DILI, but not for hepatocellular DILI; the polymorphism on chromosome 2 was associated with cholestatic and mixed DILI as well as hepatocellular DILI. We identified an association between rs28521457 (within the lipopolysaccharide-responsive vesicle trafficking, beach and anchor containing gene) and only hepatocellular DILI (OR, 2.1; 95% CI, 1.6-2.7; P = 4.8 × 10-9). We did not associate any specific drug classes with genetic polymorphisms, except for statin-associated DILI, which was associated with rs116561224 on chromosome 18 (OR, 5.4; 95% CI, 3.0-9.5; P = 7.1 × 10-9). We validated the association between A*33:01 terbinafine- and sertraline-induced DILI. We could not validate the association between DILI and rs72631567, rs28521457, or rs116561224. CONCLUSIONS: In a GWAS of persons of European descent with DILI, we associated HLA-A*33:01 with DILI due to terbinafine and possibly fenofibrate and ticlopidine. We identified polymorphisms that appear to be associated with DILI from statins, as well as 2 non-drug-specific risk factors.

Plasma DNA methylation: a potential biomarker for stratification of liver fibrosis in non-alcoholic fatty liver disease.

OBJECTIVE: Liver biopsy is currently the most reliable way of evaluating liver fibrosis in patients with non-alcoholic fatty liver disease (NAFLD). Its inherent risks limit its widespread use. Differential liver DNA methylation of peroxisome proliferator-activated receptor gamma (PPARγ) gene promoter has recently been shown to stratify patients in terms of fibrosis severity but requires access to liver tissue. The aim of this study was to assess whether DNA methylation of circulating DNA could be detected in human plasma and potentially used to stratify liver fibrosis severity in patients with NAFLD. DESIGN: Patients with biopsy-proven NAFLD and age-matched controls were recruited from the liver and gastroenterology clinics at the Newcastle upon Tyne Hospitals NHS Foundation Trust. Plasma cell-free circulating DNA methylation of PPARγ was quantitatively assessed by pyrosequencing. Liver DNA methylation was quantitatively assessed by pyrosequencing NAFLD explant tissue, subjected to laser capture microdissection (LCM). Patients with alcoholic liver disease (ALD) were also subjected to plasma DNA and LCM pyrosequencing. RESULTS: 26 patients with biopsy-proven NAFLD were included. Quantitative plasma DNA methylation of PPARγ stratified patients into mild (Kleiner 1-2) and severe (Kleiner 3-4) fibrosis (CpG1: 63% vs 86%, p<0.05; CpG2: 51% vs 65% p>0.05). Hypermethylation at the PPARγ promoter of plasma DNA correlated with changes in hepatocellular rather than myofibroblast DNA methylation. Similar results were demonstrated in patients with ALD cirrhosis. CONCLUSIONS: Differential DNA methylation at the PPARγ promoter can be detected within the pool of cell-free DNA of human plasma. With further validation, plasma DNA methylation of PPARγ could potentially be used to non-invasively stratify liver fibrosis severity in patients with NAFLD. Plasma DNA methylation signatures reflect the molecular pathology associated with fibrotic liver disease.

Age as a Confounding Factor for the Accurate Non-Invasive Diagnosis of Advanced NAFLD Fibrosis.

OBJECTIVES: Non-invasive fibrosis scores are widely used to identify/exclude advanced fibrosis in patients with non-alcoholic fatty liver disease (NAFLD). However, these scores were principally developed and validated in patients aged between 35 and 65 years of age. The objective of this study was to assess the effect of age on the performance of non-invasive fibrosis tests in NAFLD. METHODS: Patients were recruited from European specialist hepatology clinics. The cohort was divided into five age-based groups: ≤35 (n=74), 36-45 (n=96), 46-55 (n=197), 56-64 (n=191), and ≥65 years (n=76), and the performance of the aspartate aminotransferase (AST)/alanine transaminase (ALT) ratio, fibrosis 4 (FIB-4), and NAFLD fibrosis score (NFS) for advanced fibrosis (stage F3-F4) for each group was assessed using liver biopsy as the standard. RESULTS: Six hundred and thirty-four patients were included. The diagnostic accuracy of the AST/ALT ratio was lower than NFS and FIB-4 in all the age groups. The AST/ALT ratio, NFS, and FIB-4 score performed poorly for a diagnosis of advanced fibrosis in those aged ≤35 years (area under the receiver operating characteristic curves (AUROCs 0.52, 0.52, and 0.60, respectively). For all groups >35 years, AUROCs for advanced fibrosis were similar for the NFS and FIB-4 score (range 0.77-0.84). However, the specificity for advanced fibrosis using the FIB-4 and NFS declined with age, becoming unacceptably low in those aged ≥65 years (35% for FIB-4 and 20% for NFS). New cutoffs were derived (and validated) for those aged ≥65 years, which improved specificity to 70% without adversely affecting sensitivity (FIB-4 2.0, sensitivity 77%; NFS 0.12, sensitivity 80%). CONCLUSIONS: The NFS and FIB-4 scores have similar accuracy for advanced fibrosis in patients aged >35 years. However, the specificity for advanced fibrosis is unacceptably low in patients aged ≥65 years, resulting in a high false positive rate. New thresholds for use in patients aged ≥65 years are proposed to address this issue.

Exercise Reduces Liver Lipids and Visceral Adiposity in Patients With Nonalcoholic Steatohepatitis in a Randomized Controlled Trial.

BACKGROUND & AIMS: Pharmacologic treatments for nonalcoholic steatohepatitis (NASH) are limited. Lifestyle interventions are believed to be effective in reducing features of NASH, although the effect of regular exercise, independent of dietary change, is unclear. We performed a randomized controlled trial to study the effect of exercise on hepatic triglyceride content (HTGC) and biomarkers of fibrosis in patients with NASH. METHODS: Twenty-four patients (mean age, 52 ± 14 y; body mass index, 33 ± 6 kg/m2) with sedentary lifestyles (<60 min/wk of moderate-vigorous activity) and biopsy-proven NASH were assigned randomly to groups that exercised (n = 12) or continued standard care (controls, n = 12) for 12 weeks while maintaining their weight. The exercise (cycling and resistance training) was supervised at an accredited sports center and supervised by a certified exercise specialist and recorded 3 times per week on nonconsecutive days. We measured HTGC, body composition, circulating markers of inflammation, fibrosis, and glucose tolerance at baseline and at 12 weeks. RESULTS: Compared with baseline, exercise significantly reduced HTGC (reduction of 16% ± 24% vs an increase of 9% ± 15% for controls; P < .05), visceral fat (reduction of 22 ± 33 cm2 vs an increase of 14 ± 48 cm2 for controls; P < .05), plasma triglycerides (reduction of 0.5 ± 1.0 mmol/L vs an increase of 0.3 ± 0.4 mmol/L for controls; P < .05), and γ-glutamyltransferase (reduction of 10 ± 28 U/L-1 vs a reduction of 17 ± 38 U/L-1 for controls; P < .05). There were no effects of exercise on liver enzyme levels, metabolic parameters, circulatory markers of inflammation (levels of interleukin 6, tumor necrosis factor-α, or C-reactive protein) and fibrosis. CONCLUSIONS: In a randomized controlled trial, 12 weeks of exercise significantly reduced HTGC, visceral fat, and plasma triglyceride levels in patients with NASH, but did not affect circulating markers of inflammation or fibrosis. Exercise without weight loss therefore affects some but not all factors associated with NASH. Clinical care teams should consider exercise as part of a management strategy of NASH, but weight management strategies should be included. Larger and longer-term studies are required to determine the effects of exercise in patients with NASH. ISRCTN registry.com: ISRCTN16070927.