Noninvasive scores are poorly predictive of histological fibrosis in paediatric fatty liver disease.

OBJECTIVES: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in children. Roughly a quarter of paediatric patients with NAFLD develop nonalcoholic steatohepatitis and fibrosis. Here, we evaluated the diagnostic accuracy of previously published noninvasive fibrosis scores to predict liver fibrosis in a large European cohort of paediatric patients with NAFLD. METHODS: The 457 patients with biopsy-proven NAFLD from 10 specialized centers were included. We assessed diagnostic accuracy for the prediction of any (F ≥ 1), moderate (F ≥ 2) or advanced (F ≥ 3) fibrosis for the AST/platelet ratio (APRI), Fibrosis 4 score (FIB-4), paediatric NAFLD fibrosis score (PNFS) and paediatric NAFLD fibrosis index (PNFI). RESULTS: Patients covered the full spectrum of fibrosis (F0: n = 103; F1: n = 230; F2: n = 78; F3: n = 44; F4: n = 2). None of the scores were able to accurately distinguish the presence of any fibrosis from no fibrosis. For the detection of moderate fibrosis, area under the receiver operating characteristic curve (AUROC) were: APRI: 0.697, FIB-4: 0.663, PNFI: 0.515, PNFS: 0.665, while for detection of advanced fibrosis AUROCs were: APRI: 0.759, FIB-4: 0.611, PNFI: 0.521, PNFS: 0.712. Fibrosis scores showed no diagnostic benefit over using ALT ≤ 50/ > 50 IU/L as a cut-off. CONCLUSIONS: Established fibrosis scores lack diagnostic accuracy to replace liver biopsy for staging of fibrosis, giving similar results as compared to using ALT alone. New diagnostic tools are needed for Noninvasive risk-stratification in paediatric NAFLD.

Insights into genetic variants associated with NASH-fibrosis from metabolite profiling.

Several genetic discoveries robustly implicate five single-nucleotide variants in the progression of non-alcoholic fatty liver disease to non-alcoholic steatohepatitis and fibrosis (NASH-fibrosis), including a recently identified variant in MTARC1. To better understand these variants as potential therapeutic targets, we aimed to characterize their impact on metabolism using comprehensive metabolomics data from two population-based studies. A total of 9135 participants from the Fenland study and 9902 participants from the EPIC-Norfolk cohort were included in the study. We identified individuals with risk alleles associated with NASH-fibrosis: rs738409C>G in PNPLA3, rs58542926C>T in TM6SF2, rs641738C>T near MBOAT7, rs72613567TA>T in HSD17B13 and rs2642438A>G in MTARC1. Circulating levels of 1449 metabolites were measured using targeted and untargeted metabolomics. Associations between NASH-fibrosis variants and metabolites were assessed using linear regression. The specificity of variant-metabolite associations were compared to metabolite associations with ultrasound-defined steatosis, gene variants linked to liver fat (in GCKR, PPP1R3B and LYPLAL1) and gene variants linked to cirrhosis (in HFE and SERPINA1). Each NASH-fibrosis variant demonstrated a specific metabolite profile with little overlap (8/97 metabolites) comprising diverse aspects of lipid metabolism. Risk alleles in PNPLA3 and HSD17B13 were both associated with higher 3-methylglutarylcarnitine and three variants were associated with lower lysophosphatidylcholine C14:0. The risk allele in MTARC1 was associated with higher levels of sphingomyelins. There was no overlap with metabolites that associated with HFE or SERPINA1 variants. Our results suggest a link between the NASH-protective variant in MTARC1 to the metabolism of sphingomyelins and identify distinct molecular patterns associated with each of the NASH-fibrosis variants under investigation.

A Systematic Review of Animal Models of NAFLD Finds High-Fat, High-Fructose Diets Most Closely Resemble Human NAFLD.

BACKGROUND AND AIMS: Animal models of human disease are a key component of translational hepatology research, yet there is no consensus on which model is optimal for NAFLD. APPROACH AND RESULTS: We generated a database of 3,920 rodent models of NAFLD. Study designs were highly heterogeneous, and therefore, few models had been cited more than once. Analysis of genetic models supported the current evidence for the role of adipose dysfunction and suggested a role for innate immunity in the progression of NAFLD. We identified that high-fat, high-fructose diets most closely recapitulate the human phenotype of NAFLD. There was substantial variability in the nomenclature of animal models: a consensus on terminology of specialist diets is needed. More broadly, this analysis demonstrates the variability in preclinical study design, which has wider implications for the reproducibility of in vivo experiments both in the field of hepatology and beyond. CONCLUSIONS: This systematic analysis provides a framework for phenotypic assessment of NAFLD models and highlights the need for increased standardization and replication.

A minority of somatically mutated genes in pre-existing fatty liver disease have prognostic importance in the development of NAFLD.

BACKGROUND: Understanding the genetics of liver disease has the potential to facilitate clinical risk stratification. We recently identified acquired somatic mutations in six genes and one lncRNA in pre-existing fatty liver disease. We hypothesised that germline variation in these genes might be associated with the risk of developing steatosis and contribute to the prediction of disease severity. METHODS: Genome-wide association study (GWAS) summary statistics were extracted from seven studies (>1.7 million participants) for variants near ACVR2A, ALB, CIDEB, FOXO1, GPAM, NEAT1 and TNRC6B for: aminotransferases, liver fat, HbA1c, diagnosis of NAFLD, ARLD and cirrhosis. Findings were replicated using GWAS data from multiple independent cohorts. A phenome-wide association study was performed to examine for related metabolic traits, using both common and rare variants, including gene-burden testing. RESULTS: There was no evidence of association between rare germline variants or SNPs near five genes (ACVR2A, ALB, CIDEB, FOXO1 and TNRC6B) and risk or severity of liver disease. Variants in GPAM (proxies for p.Ile43Val) were associated with liver fat (p = 3.6 × 10-13 ), ALT (p = 2.8 × 10-39 ) and serum lipid concentrations. Variants in NEAT1 demonstrated borderline significant associations with ALT (p = 1.9 × 10-11 ) and HbA1c, but not with liver fat, as well as influencing waist-to-hip ratio, adjusted for BMI. CONCLUSIONS: Despite the acquisition of somatic mutations at these loci during progressive fatty liver disease, we did not find associations between germline variation and markers of liver disease, except in GPAM. In the future, larger sample sizes may identify associations. Currently, germline polygenic risk scores will not capture data from genes affected by somatic mutations.

Comparison of the Lipidomic Signature of Fatty Liver in Children and Adults: A Cross-Sectional Study.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is an increasingly common condition in children characterised by insulin resistance and altered lipid metabolism. Affected patients are at increased risk of cardiovascular disease (CVD) and children with NAFLD are likely to be at risk of premature cardiac events. Evaluation of the plasma lipid profile of children with NAFLD offers the opportunity to investigate these perturbations and understand how closely they mimic the changes seen in adults with cardiometabolic disease. METHODS: We performed untargeted liquid chromatography-mass spectrometry (LC-MS) plasma lipidomics on 287 children: 19 lean controls, 146 from an obese cohort, and 122 NAFLD cases who had undergone liver biopsy. Associations between lipid species and liver histology were assessed using regression adjusted for age and sex. Results were then replicated using data from 9500 adults with metabolic phenotyping. RESULTS: More severe paediatric NAFLD was associated with lower levels of long chain, polyunsaturated phosphatidylcholines (pC) and triglycerides (TG). Similar trends in pC and TG chain length and saturation were seen in adults with hepatic steatosis; however, many of the specific lipids associated with NAFLD differed between children and adults. Five lipids replicated in adults (including PC(36:4)) have been directly linked to death and cardiometabolic disease, as well as indirectly via genetic variants. CONCLUSION: These findings suggest that, whilst similar pathways of lipid metabolism are perturbed in paediatric NAFLD as in cardiometabolic disease in adults, the specific lipid signature in children is different.

An AGTR1 Variant Worsens Nonalcoholic Fatty Liver Disease and the Metabolic Syndrome.

Nonalcoholic fatty liver disease and hypertension are closely related but there has been little genetic evidence to link them. In this issue, Musso et al. provide evidence that a common variant in AGTR1 (A1166C) is associated with both incident hypertension and nonalcoholic fatty liver disease, as well as nonalcoholic steatohepatitis, fibrosis, dyslipidemia, and insulin resistance. AGTR1 is strongly expressed in adipose, liver, and arteries. The mechanism of this gain-of-function variant is unclear but may include adipose or endothelial dysfunction and immune activation. Despite previous unsuccessful clinical trials of angiotensin receptor blockers in nonalcoholic steatohepatitis, individuals with the rs5186A>C variant may have greater benefit from this therapy.

The global prevalence of Wilson disease from next-generation sequencing data.

PURPOSE: Wilson disease (WD) is an autosomal recessive disorder of copper metabolism, caused by pathogenic variants in ATP7B. We aimed to (1) perform a meta-analysis of previous WD prevalence estimates, (2) estimate the prevalence of WD from population sequencing data, and (3) generate an ATP7B gene variant database. METHODS: MEDLINE and EMBASE were systematically searched. Previous prevalence estimates were subjected to meta-analysis. All previously reported pathogenic ATP7B variants were compiled and annotated with gnomAD allele frequencies. Pooled global and ethnicity-specific genetic prevalences for WD were generated using the Hardy-Weinberg equation. RESULTS: Meta-analysis of genetic studies of WD prevalence gave an estimate 12.7 per 100,000 (95% confidence interval [CI]: 6.3-23.0). We developed a referenced, searchable ATP7B database comprising 11,520 variants including 782 previously reported disease variants, which can be found at http://www.wilsondisease.tk/ ; 216/782 of these were present in gnomAD, remained after filtering by allele frequency, and met American College of Medical Genetics and Genomics criteria. Based on these, the genetic prevalence of WD was 13.9 per 100,000 (95% CI: 12.9-14.9), or 1 per 7194. Combining this with 60 predicted pathogenic variants gave a birth prevalence of 15.4 per 100,000 (95% CI: 14.4-16.5). CONCLUSION: The genetic prevalence of Wilson disease may be greater than previous estimates.

Nonalcoholic Fatty Liver Disease in Children.

Nonalcoholic steatohepatitis, a progressive form of nonalcoholic fatty liver disease (NAFLD), is one of the most common hepatic diseases in children who present with particular risk factors including obesity, sedentary lifestyle, and/or a predisposing genetic background. The worldwide prevalence of NAFLD in children is a worrying phenomenon because this disease is closely associated with the development of both cirrhosis and cardiometabolic syndrome in adulthood. To date, the etiopathogenesis of primary NAFLD in children is unknown. Understanding the pathogenetic mechanisms provides the basis to characterize early predictors of the disease and noninvasive diagnostic tools and to design novel specific treatments and possible management strategies. Despite a few clinical trials on the use of antioxidants combined with lifestyle intervention for NAFLD, no treatment exists for children with NAFLD. In this review, the authors provide an overview of current concepts in epidemiology, histological features, etiopathogenesis, diagnosis, and treatment of NAFLD in pediatric population.

Fifteen-minute consultation: liver disease in children.

Liver disease in children can present in many ways from the frequently encountered prolonged neonatal jaundice to the comparatively rare acute liver failure. In this article, we will discuss 'red flags' of liver disease, the initial investigations required and when to refer to a specialist liver centre. Across all presentations, the degree of elevation of alanine aminotransferase or aspartate aminotransferase provides only little diagnostic information. Measurement of clotting is vital, and coagulopathy should be followed by a trial of intravenous vitamin K before being repeated.

Portal inflammation is independently associated with fibrosis and metabolic syndrome in pediatric nonalcoholic fatty liver disease.

UNLABELLED: Pediatric nonalcoholic fatty liver disease (NAFLD) histology demonstrates variable amounts of portal inflammation, which may be associated with more severe liver disease and fibrosis. We assessed the relationship between portal inflammation, hepatic fibrosis, and the metabolic syndrome in pediatric NAFLD. Children with biopsy-proven NAFLD were eligible for inclusion. Histology was assessed using Kleiner fibrosis stage and the Nonalcoholic Steatohepatitis Clinical Research Network system for portal inflammation. Patients were divided by histology into type 1, type 2, and overlap NAFLD. Multivariable ordinal logistic regression was used to determine factors associated with fibrosis and portal inflammation. The 430 Caucasian children were divided into 52 with type 1, 95 with type 2, and 283 with overlap NAFLD. Those with type 2 had a more severe metabolic phenotype, with higher body mass index z score (2.0 versus 1.6, P < 0.0001), waist circumference centile (96th versus 90th, P < 0.0001), and triglycerides (84 versus 77 mg/dL, P = 0.01) and lower high-density lipoprotein (46 versus 60 mg/dL, P = 0.004) than those with type 1. Similarly, those with overlap NAFLD had a more severe phenotype. Stage 2-3 fibrosis was present in 69/283 (24%) with overlap NAFLD. Portal inflammation was associated with stage 2-3 fibrosis on multivariable analysis (95% confidence interval 1.4-5.2, odds ratio = 3.7). Waist circumference centile was associated with portal inflammation (95% confidence interval 1.2-3.4, odds ratio = 2.0). CONCLUSION: Portal inflammation is associated with more advanced pediatric NAFLD and features of the metabolic syndrome.

Update on lipid species and paediatric nonalcoholic fatty liver disease.

PURPOSE OF REVIEW: To describe the recent advances in our understanding of fatty acids and lipids in paediatric nonalcoholic fatty liver disease (NAFLD) and their future implications. RECENT FINDINGS: Data have been accumulated to suggest that ceramides are the main drivers of hepatic insulin resistance in NAFLD, and inhibition of ceramide synthesis improves histology in mice.Saturated fatty acids formed by de novo lipogenesis generate increased lipotoxicity compared with dietary-derived saturated fatty acids.Hepatic lipogenesis and associated insulin resistance have been found to be influenced by several novel proteins, including E2F1, cyclic AMP response element binding protein transcriptional coactivator 2, Raptor, and eukaryotic initiation factor 6. There are encouraging data from animal models that modulation of these could be therapeutic targets.Human and animal metabolomics and lipidomics data have been used to generate a lipid signature for NAFLD and nonalcoholic steatohepatitis. Serum lipidomics appears to correlate with hepatic lipidomics.Therapeutic trials of polyunsaturated fatty acids in children have had mixed results, with some reductions in noninvasive biomarkers. SUMMARY: Multiple new pathways for drug targets have been identified, and use of lipidomics is likely to become a noninvasive method for assessing disease. However, much of the data for paediatric NAFLD are extrapolated from adult or animal studies.

Management and outcomes of neonates with down syndrome admitted to neonatal units.

BACKGROUND: Neonates with Down syndrome have an increased risk of being admitted to a neonatal unit compared with unaffected neonates. We aimed to estimate the proportion of neonates with Down syndrome admitted to a neonatal unit and compare their management and outcomes with other neonatal admissions. METHODS: Case-control study of neonates born from 2009 to 2011 admitted to 122 NHS Neonatal Units in England using data from the National Down Syndrome Cytogenetic Register and the National Neonatal Research Database. For each neonate with Down syndrome, three neonates admitted to the same unit in the same month and born at the same gestation were identified. RESULTS: Forty-six percent of neonates with Down syndrome were admitted to a neonatal unit. Boys were more likely to be admitted than girls (odds ratio = 1.7; 95% confidence interval, 1.4-2.0). Neonates with Down syndrome required more intensive or high dependency care compared with unaffected neonates (37% vs. 27%. p < 0.01) and stayed in neonatal units for longer (11 days vs. 5 days, p < 0.01). A total of 31% of neonates with Down syndrome required respiratory support compared with 22% (p < 0.001) of unaffected neonates, and 11% were discharged requiring oxygen supplementation compared with 3% (p < 0.001) of unaffected neonates. A total of 3% of neonates with Down syndrome died in a neonatal unit compared with 1% (p = 0.01) of unaffected neonates. CONCLUSION: Neonates with Down syndrome are more likely than unaffected neonates to be admitted to a neonatal unit, have a prolonged stay, and be discharged home on supplemental oxygen. Birth Defects Research (Part A) 106:468-474, 2016. © 2016 Wiley Periodicals, Inc.

Common mechanisms in pediatric acute liver failure.

Acute liver failure (ALF) is a rare but potentially fatal disease in children. The etiology is multifactorial, including infection, autoimmune, and genetic disorders, as well as indeterminate hepatitis, which has a higher requirement for liver transplantation. Activation of the innate and adaptive immune systems leads to hepatocyte-specific injury which is mitigated by T regulatory cell activation. Recovery of the native liver depends on activation of apoptotic and regenerative pathways, including the integrated stress response (ISR; e.g., PERK), p53, and HNF4α. Loss-of-function mutations in these pathways cause recurrent ALF in response to non-hepatotropic viruses. Deeper understanding of these mechanisms will lead to improved diagnosis, management, and outcomes for pediatric ALF.

A mouse model of human mitofusin-2-related lipodystrophy exhibits adipose-specific mitochondrial stress and reduced leptin secretion.

Mitochondrial dysfunction has been reported in obesity and insulin resistance, but primary genetic mitochondrial dysfunction is generally not associated with these, arguing against a straightforward causal relationship. A rare exception, recently identified in humans, is a syndrome of lower body adipose loss, leptin-deficient severe upper body adipose overgrowth, and insulin resistance caused by the p.Arg707Trp mutation in MFN2, encoding mitofusin 2. How the resulting selective form of mitochondrial dysfunction leads to tissue- and adipose depot-specific growth abnormalities and systemic biochemical perturbation is unknown. To address this, Mfn2R707W/R707W knock-in mice were generated and phenotyped on chow and high fat diets. Electron microscopy revealed adipose-specific mitochondrial morphological abnormalities. Oxidative phosphorylation measured in isolated mitochondria was unperturbed, but the cellular integrated stress response was activated in adipose tissue. Fat mass and distribution, body weight, and systemic glucose and lipid metabolism were unchanged, however serum leptin and adiponectin concentrations, and their secretion from adipose explants were reduced. Pharmacological induction of the integrated stress response in wild-type adipocytes also reduced secretion of leptin and adiponectin, suggesting an explanation for the in vivo findings. These data suggest that the p.Arg707Trp MFN2 mutation selectively perturbs mitochondrial morphology and activates the integrated stress response in adipose tissue. In mice, this does not disrupt most adipocyte functions or systemic metabolism, whereas in humans it is associated with pathological adipose remodelling and metabolic disease. In both species, disproportionate effects on leptin secretion may relate to cell autonomous induction of the integrated stress response.

Variants in mitochondrial amidoxime reducing component 1 and hydroxysteroid 17-beta dehydrogenase 13 reduce severity of nonalcoholic fatty liver disease in children and suppress fibrotic pathways through distinct mechanisms.

Genome-wide association studies in adults have identified variants in hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) and mitochondrial amidoxime reducing component 1 (MTARC1) as protective against nonalcoholic fatty liver disease (NAFLD). We aimed to test their association with pediatric NAFLD liver histology and investigate their function using metabolomics. A total of 1450 children (729 with NAFLD, 399 with liver histology) were genotyped for rs72613567T>TA in HSD17B13, rs2642438G>A in MTARC1, and rs738409C>G in patatin-like phospholipase domain-containing protein 3 (PNPLA3). Genotype-histology associations were tested using ordinal regression. Untargeted hepatic proteomics and plasma lipidomics were performed in a subset of children. We found rs72613567T>TA in HSD17B13 to be associated with lower odds of NAFLD diagnosis (odds ratio, 0.7; 95% confidence interval, 0.6-0.9) and a lower grade of portal inflammation (p < 0.001). rs2642438G>A in MTARC1 was associated with a lower grade of hepatic steatosis (p = 0.02). Proteomics found reduced expression of HSD17B13 in carriers of the protective -TA allele. MTARC1 levels were unaffected by genotype. Both variants were associated with down-regulation of fibrogenic pathways. HSD17B13 perturbs plasma phosphatidylcholines and triglycerides. In silico modeling suggested p.Ala165Thr disrupts the stability and metal binding of MTARC1. Conclusion: Both HSD17B13 and MTARC1 variants are associated with less severe pediatric NAFLD. These results provide further evidence for shared genetic mechanisms between pediatric and adult NAFLD.