Deafness associated with pigmentation phenotypes in North American yaks (Bos grunniens).

OBJECTIVE: To assess the presence of suspected pigment-associated deafness in North American yaks (Bos grunniens). ANIMALS: 12 North American yaks, including 11 with the homozygous piebald Royal pigmentation phenotype and 1 with the heterozygous piebald Trim phenotype. PROCEDURES: Hearing was assessed using the brainstem auditory evoked response (BAER) on yaks restrained in the head gate of a grooming chute. RESULTS: Five of the Royal yaks and the Trim yak had hearing in both ears. Six Royal yaks were affected; 3 were deaf in 1 ear and 3 were deaf in both ears. CLINICAL RELEVANCE: For the first time, probable sensorineural deafness has been confirmed to be present in Royal yaks. The disorder is assumed to be congenital and associated with white pigmentation, based on the pattern of occurrence in other species.

Interstitial Cystitis Can Be Improved With Intravesical Instillation of Platelet-Rich Plasma.

Intravesical instillation of platelet-rich plasma has the potential to improve symptoms and reduce pain in patients who have interstitial cystitis and painful bladder syndrome by utilizing the body's own growth factors found in platelets. Interstitial cystitis is a disease of the bladder that causes pain, urinary frequency, urgency, and nocturia. It is difficult to treat and has unknown etiology. Patients who have interstitial cystitis have high rates of anxiety as a comorbid condition. People with anxiety are known to have dysregulation of serotonin. These concepts are interrelated.

Implementation of a care bundle improves the management of patients with non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is common and is associated with liver-related and cardiovascular-related morbidity. Our aims were: (1) to review the current management of patients with NAFLD attending hospital clinics in North East England (NEE) and assess the variability in care; (2) develop a NAFLD 'care bundle' to standardise care; (3) to assess the impact of implementation of the NAFLD care bundle. METHODS: A retrospective review was conducted to determine baseline management of patients with NAFLD attending seven hospitals in NEE. A care bundle for the management of NAFLD was developed including important recommendations from international guidelines. Impact of implementation of the bundle was evaluated prospectively in a single centre. RESULTS: Baseline management was assessed in 147 patients attending gastroenterology, hepatology and a specialist NAFLD clinic. Overall, there was significant variability in the lifestyle advice given and management of metabolic risk factors, with patients attending an NAFLD clinic significantly more likely to achieve >10% body weight loss and have metabolic risk factors addressed. Following introduction of the NAFLD bundle 50 patients were evaluated. Use of the bundle was associated with significantly better documentation and implementation of most aspects of patient management including management of metabolic risk factors, documented lifestyle advice and provision of NAFLD-specific patient advice booklets. CONCLUSION: The introduction of an outpatient 'care bundle' led to significant improvements in the assessment and management of patients with NAFLD in the NEE and could help improve and standardise care if used more widely.

Drug-induced hepatocellular injury due to herbal supplement ashwagandha.

A 39-year-old female presented with a one-week history of jaundice and nausea after taking an over-the-counter herbal supplement containing ashwagandha root extract. Initial investigations revealed a hepatocellular pattern of liver enzyme abnormality with jaundice. Investigations, including viral serology, liver specific autoantibodies and an ultrasound scan of the abdomen, were unremarkable. Liver biopsy showed an acute cholestatic hepatitis with confluent necrosis but no features of chronicity. These histopathological findings differ to that of a previously reported case. Review of recent literature revealed that some clinical features and the time course of liver injury were similar to previous reports of ashwagandha drug-induced liver injury (DILI). The patient received treatment with ursodeoxycholic acid. We compare this case to previous reported cases of ashwagandha DILI and discuss the biochemical and histopathological features of ashwagandha DILI, therapeutic strategies and the importance of recognising herbal supplements as a possible cause of DILI.

RNA sequencing reveals changes in the microRNAome of transdifferentiating hepatic stellate cells that are conserved between human and rat.

MicroRNAs are small (~ 22nt long) noncoding RNAs (ncRNAs) that regulate gene expression at the post-transcriptional level. Over 2000 microRNAs have been described in humans and many are implicated in human pathologies including tissue fibrosis. Hepatic stellate cells (HSC) are the major cellular contributors to excess extracellular matrix deposition in the diseased liver and as such are important in the progression of liver fibrosis. We employed next generation sequencing to map alterations in the expression of microRNAs occurring across a detailed time course of culture-induced transdifferentiation of primary human HSC, this a key event in fibrogenesis. Furthermore, we compared profiling of human HSC microRNAs with that of rat HSC so as to identify those molecules that are conserved with respect to modulation of expression. Our analysis reveals that a total of 229 human microRNAs display altered expression as a consequence of HSC transdifferentiation and of these 104 were modulated early during the initiation phase. Typically modulated microRNAs were targeting kinases, transcription factors, chromatin factors, cell cycle regulators and growth factors. 162 microRNAs changed in expression during transdifferentiation of rat HSC, however only 17 underwent changes that were conserved in human HSC. Our study therefore identifies widespread changes in the expression of HSC microRNAs in fibrogenesis, but suggests a need for caution when translating data obtained from rodent HSC to events occurring in human cells.

The European NAFLD Registry: A real-world longitudinal cohort study of nonalcoholic fatty liver disease.

Non-Alcoholic Fatty Liver Disease (NAFLD), a progressive liver disease that is closely associated with obesity, type 2 diabetes, hypertension and dyslipidaemia, represents an increasing global public health challenge. There is significant variability in the disease course: the majority exhibit only fat accumulation in the liver but a significant minority develop a necroinflammatory form of the disease (non-alcoholic steatohepatitis, NASH) that may progress to cirrhosis and hepatocellular carcinoma. At present our understanding of pathogenesis, disease natural history and long-term outcomes remain incomplete. There is a need for large, well characterised patient cohorts that may be used to address these knowledge gaps and to support the development of better biomarkers and novel therapies. The European NAFLD Registry is an international, prospectively recruited observational cohort study that aims to establish a large, highly-phenotyped patient cohort and linked bioresource. Here we describe the infrastructure, data management and monitoring plans, and the standard operating procedures implemented to ensure the timely and systematic collection of high-quality data and samples. Already recruiting subjects at secondary/tertiary care centres across Europe, the Registry is supporting the European Union IMI2-funded LITMUS 'Liver Investigation: Testing Marker Utility in Steatohepatitis' consortium, which is a major international effort to robustly validate biomarkers that diagnose, risk stratify and/or monitor NAFLD progression and liver fibrosis stage. The European NAFLD Registry has the demonstrable capacity to support research and biomarker development at scale and pace.

Continuous chromosome-scale haplotypes assembled from a single interspecies F1 hybrid of yak and cattle.

BACKGROUND: The development of trio binning as an approach for assembling diploid genomes has enabled the creation of fully haplotype-resolved reference genomes. Unlike other methods of assembly for diploid genomes, this approach is enhanced, rather than hindered, by the heterozygosity of the individual sequenced. To maximize heterozygosity and simultaneously assemble reference genomes for 2 species, we applied trio binning to an interspecies F1 hybrid of yak (Bos grunniens) and cattle (Bos taurus), 2 species that diverged nearly 5 million years ago. The genomes of both of these species are composed of acrocentric autosomes. RESULTS: We produced the most continuous haplotype-resolved assemblies for a diploid animal yet reported. Both the maternal (yak) and paternal (cattle) assemblies have the largest 2 chromosomes in single haplotigs, and more than one-third of the autosomes similarly lack gaps. The maximum length haplotig produced was 153 Mb without any scaffolding or gap-filling steps and represents the longest haplotig reported for any species. The assemblies are also more complete and accurate than those reported for most other vertebrates, with 97% of mammalian universal single-copy orthologs present. CONCLUSIONS: The high heterozygosity inherent to interspecies crosses maximizes the effectiveness of the trio binning method. The interspecies trio binning approach we describe is likely to provide the highest-quality assemblies for any pair of species that can interbreed to produce hybrid offspring that develop to sufficient cell numbers for DNA extraction.

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.

Barriers and Facilitators to Mediterranean Diet Adoption by Patients With Nonalcoholic Fatty Liver Disease in Northern Europe.

BACKGROUND & AIMS: Dietary interventions for weight loss are effective therapies for nonalcoholic fatty liver disease (NAFLD). The Mediterranean diet might benefit these patients, but it is not followed consistently in Northern European countries. We examined factors that determine Mediterranean diet adoption and maintenance in a northern European population. METHODS: We used a mixed-methods approach to investigate the effects of a 12-week Mediterranean diet intervention and perceived barriers and facilitators. Nineteen adults with NAFLD were recruited from a tertiary hepatology center in England. Participants were taught behavioral strategies through the provision of shopping lists, meal planners, and recipes; no advice was given on calorie allowances or physical activities. We used the 14-point Mediterranean diet assessment tool to assess dietary intake, based on a small number of foods in servings per day or servings per week, at baseline and after 12 weeks; participants were assigned scores of low (<5 points), moderate (6-9 points), or high (10-14 points). Semistructured interviews were audiorecorded, transcribed, and analyzed using the framework method. RESULTS: Twelve weeks after the dietary advice, Mediterranean diet adoption significantly increased from moderate to high (mean increase, 2.2 points; from 7.6 ± 2.5 at baseline to 9.8 ± 2.8 at 12 wk) (P = .006). This increase was associated with a mean reduction in body weight of 2.4 kg (from 99.2 ± 17.0 kg at baseline to 96.8 ± 17.5 kg at 12 wk) (P = .001) and increased serum concentrations of high-density lipoprotein cholesterol in 72% of participants (from 1.10 ± 0.8 at baseline to 1.20 ± 1.30 vs 1.00 ± 0.5 at 12 wk) (P = .009). Increased nutrition knowledge and skills, family support, Mediterranean diet promotion in media and clinical settings, and nutritional care facilitated diet changes. Barriers to Mediterranean diet uptake included an obesogenic environment, life stressors, and demand for convenience. Poor understanding of the causes and significance of NAFLD adversely affected readiness to change dietary habits. CONCLUSIONS: In an analysis of patients with NAFLD in the northern United Kingdom, we found a 12-week Mediterranean diet intervention was acceptable and associated with significant reductions in body weight and increased serum levels of high-density lipoprotein. We identified barriers and facilitators that could support appropriate treatment adaptations and guide personalized intervention approaches.

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.

Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention.

NAFLD is one of the most important causes of liver disease worldwide and will probably emerge as the leading cause of end-stage liver disease in the coming decades, with the disease affecting both adults and children. The epidemiology and demographic characteristics of NAFLD vary worldwide, usually parallel to the prevalence of obesity, but a substantial proportion of patients are lean. The large number of patients with NAFLD with potential for progressive liver disease creates challenges for screening, as the diagnosis of NASH necessitates invasive liver biopsy. Furthermore, individuals with NAFLD have a high frequency of metabolic comorbidities and could place a growing strain on health-care systems from their need for management. While awaiting the development effective therapies, this disease warrants the attention of primary care physicians, specialists and health policy makers.

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 Proof-of-Concept for Epigenetic Therapy of Tissue Fibrosis: Inhibition of Liver Fibrosis Progression by 3-Deazaneplanocin A.

The progression of fibrosis in chronic liver disease is dependent upon hepatic stellate cells (HSCs) transdifferentiating to a myofibroblast-like phenotype. This pivotal process is controlled by enzymes that regulate histone methylation and chromatin structure, which may be targets for developing anti-fibrotics. There is limited pre-clinical experimental support for the potential to therapeutically manipulate epigenetic regulators in fibrosis. In order to learn if epigenetic treatment can halt the progression of pre-established liver fibrosis, we treated mice with the histone methyltransferase inhibitor 3-deazaneplanocin A (DZNep) in a naked form or by selectively targeting HSC-derived myofibroblasts via an antibody-liposome-DZNep targeting vehicle. We discovered that DZNep treatment inhibited multiple histone methylation modifications, indicative of a broader specificity than previously reported. This broad epigenetic repression was associated with the suppression of fibrosis progression as assessed both histologically and biochemically. The anti-fibrotic effect of DZNep was reproduced when the drug was selectively targeted to HSC-derived myofibroblasts. Therefore, the in vivo modulation of HSC histone methylation is sufficient to halt progression of fibrosis in the context of continuous liver damage. This discovery and our novel HSC-targeting vehicle, which avoids the unwanted effects of epigenetic drugs on parenchymal liver cells, represents an important proof-of-concept for epigenetic treatment of liver fibrosis.

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.

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.

Epigenetics in liver disease: from biology to therapeutics.

Knowledge of the fundamental epigenetic mechanisms governing gene expression and cellular phenotype are sufficiently advanced that novel insights into the epigenetic control of chronic liver disease are now emerging. Hepatologists are in the process of shedding light on the roles played by DNA methylation, histone/chromatin modifications and non-coding RNAs in specific liver pathologies. Alongside these discoveries are advances in the technologies for the detection and quantification of epigenetic biomarkers, either directly from patient tissue or from body fluids. The premise for this review is to survey the recent advances in the field of liver epigenetics and to explore their potential for translation by industry and clinical hepatologists for the design of novel therapeutics and diagnostic/prognostic biomarkers. In particular, we present findings in the context of hepatocellular carcinoma, fibrosis and non-alcoholic fatty liver disease, where there is urgent unmet need for new clinical interventions and biomarkers.

DNA methylation profiling identifies novel markers of progression in hepatitis B-related chronic liver disease.

BACKGROUND: Chronic hepatitis B infection is characterized by hepatic immune and inflammatory response with considerable variation in the rates of progression to cirrhosis. Genetic variants and environmental cues influence predisposition to the development of chronic liver disease; however, it remains unknown if aberrant DNA methylation is associated with fibrosis progression in chronic hepatitis B. RESULTS: To identify epigenetic marks associated with inflammatory and fibrotic processes of the hepatitis B-induced chronic liver disease, we carried out hepatic genome-wide methylation profiling using Illumina Infinium BeadArrays comparing mild and severe fibrotic disease in a discovery cohort of 29 patients. We obtained 310 differentially methylated regions and selected four loci comprising three genes from the top differentially methylated regions: hypermethylation of HOXA2 and HDAC4 along with hypomethylation of PPP1R18 were significantly linked to severe fibrosis. We replicated the prominent methylation marks in an independent cohort of 102 patients by bisulfite modification and pyrosequencing. The timing and causal relationship of epigenetic modifications with disease severity was further investigated using a cohort of patients with serial biopsies. CONCLUSIONS: Our findings suggest a linkage of widespread epigenetic dysregulation with disease progression in chronic hepatitis B infection. CpG methylation at novel genes sheds light on new molecular pathways, which can be potentially exploited as a biomarker or targeted to attenuate inflammation and fibrosis.

Nonalcoholic Fatty Liver Disease: Pathogenesis and Disease Spectrum.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of liver dysfunction in the Western world and is increasing owing to its close association with obesity and insulin resistance. NAFLD represents a spectrum of liver disease that, in a minority of patients, can lead to progressive nonalcoholic steatohepatitis (NASH), fibrosis, and ultimately hepatocellular carcinoma and liver failure. NAFLD is a complex trait resulting from the interaction between environmental exposure and a susceptible polygenic background and comprising multiple independent modifiers of risk, such as the microbiome. The molecular mechanisms that combine to define the transition to NASH and progressive disease are complex, and consequently, no pharmacological therapy currently exists to treat NASH. A better understanding of the pathogenesis of NAFLD is critical if new treatments are to be discovered.

Differential DNA methylation of genes involved in fibrosis progression in non-alcoholic fatty liver disease and alcoholic liver disease.

BACKGROUND: Chronic liver injury can lead to the development of liver fibrosis and cirrhosis but only in a minority of patients. Currently, it is not clear which factors determine progression to fibrosis. We investigated whether DNA\methylation profile as determined by pyrosequencing can distinguish patients with mild from those with advanced/severe fibrosis in non-alcoholic liver disease (NAFLD) and alcoholic liver disease (ALD). To this end, paraffin-embedded liver biopsies were collected from patients with biopsy-proven NAFLD or ALD, as well as paraffin-embedded normal liver resections, genomic DNA isolated, bisulfite converted and pyrosequencing assays used to quantify DNA methylation at specific CpGs within PPARα, PPARα, TGFß1, Collagen 1A1 and PDGFα genes. Furthermore, we assessed the impact of age, gender and anatomical location within the liver on patterns of DNA methylation in the same panel of genes. RESULTS: DNA methylation at specific CpGs within genes known to affect fibrogenesis distinguishes between patients with mild from those with severe fibrosis in both NAFLD and ALD, although same CpGs are not equally represented in both etiologies. In normal liver, age, gender or anatomical location had no significant impact on DNA methylation patterns in the liver. CONCLUSIONS: DNA methylation status at specific CpGs may be useful as part of a wider set of patient data for predicting progression to liver fibrosis.