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Recent cross-sectional studies have examined the association between nonalcoholic fatty liver disease (NAFLD) and bone mineral density (BMD) in children or adolescents, but these have produced conflicting results. We performed a systematic review and meta-analysis of these published studies to quantify the magnitude of the association, if any, between NAFLD and BMD. We searched publication databases from January 2000 to September 2018, using predefined keywords to identify relevant observational studies conducted in children or adolescents in whom NAFLD was diagnosed either by imaging or by histology and BMD Z score was measured by dual-energy X-ray absorptiometry. Data from selected studies were extracted, and a meta-analysis was performed using random-effects modeling. A total of eight observational cross-sectional or case-control studies enrolling 632 children and adolescents (mean age 12.8 years), 357 of whom had NAFLD, were included in the final analysis. Meta-analysis showed significant differences in whole-body or lumbar BMD Z scores between children/adolescents with and without NAFLD (n = 6 studies; pooled weighted mean difference [WMD], -0.48; 95% confidence interval [CI], -0.74 to -0.21; I 2 = 55.5%), as well as between those with biopsy-confirmed nonalcoholic steatohepatitis (NASH) and those with no-NASH (n = 4 studies; pooled WMD, -0.27; 95% CI, -0.40 to -0.13; I 2 = 0%). The aforementioned WMDs in BMD Z scores were independent of common clinical risk factors, such as age, sex, race/ethnicity, and body mass index. Sensitivity analyses did not modify these findings. Funnel plot and Egger test did not reveal significant publication bias. Conclusion: This meta-analysis shows that the presence and severity of NAFLD are significantly associated with reduced whole-body BMD Z scores in children and adolescents; however, the observational design of the studies included does not allow for proving causality., (© 2019 by the American Association for the Study of Liver Diseases.)

Emerging evidence suggests that patatin-like phospholipase domain-containing protein-3 (PNPLA3) rs738409 genotype (the major genetic variant associated with susceptibility to nonalcoholic fatty liver disease [NAFLD]) is associated with decreased kidney function in adults. Currently, it is uncertain whether this association also occurs in children/adolescents and whether any association is independent of liver disease severity. We enrolled a sample of 142 Caucasian children and adolescents with biopsy-proven NAFLD, presenting to the Liver Unit of the "Bambino Gesù" Children's Hospital. The glomerular filtration rate (e-GFR) was estimated using the Bedside Schwartz equation, whereas 24-hour proteinuria was measured using a radioimmunoassay method. Genotyping for the PNPLA3 rs738409 genotype was undertaken using the single-nucleotide polymorphism genotyping allelic discrimination method. Overall, 45 children had G/G, 56 had G/C, and 41 had C/C PNPLA3 rs738409 genotype, respectively. Children with G/G genotype had significantly lower e-GFR (107.5 ± 20 versus 112.8 ± 18 versus 125.3 ± 23 mL/min/1.73 m 2 , P = 0.002) and higher 24-hour proteinuria (58.5 ± 21 versus 53.9 ± 22 versus 42.9 ± 20 mg/day, P = 0.012) compared with those with either G/C or C/C genotypes. After adjustment for age, sex, systolic blood pressure, measures of adiposity, homeostasis model assessment-estimated insulin resistance and biopsy-confirmed nonalcoholic steatohepatitis and stage of liver fibrosis, the presence of rs738409 G/G genotype was independently associated with both lower e-GFR (β coefficient: -23.6; 95% confidence interval [CI]: -36.3 to -10.8; P < 0.001) and higher 24-hour proteinuria (β coefficient: 15.3; 95% CI: 1.12 to 30.5; P = 0.046). Conclusion: Regardless of established renal risk factors and the histological severity of NAFLD, the PNPLA3 G/G genotype was strongly associated with decreasing kidney function and increasing 24-hour proteinuria in children/adolescents with histologically confirmed NAFLD., (© 2019 by the American Association for the Study of Liver Diseases.)

The interferon (IFN) lambda 3/4 (IFNL3/4) locus, influencing innate immunity regulation, has been associated with the severity of hepatitis and fibrosis progression during chronic hepatitis C infection, while contrasting results were reported in nonalcoholic fatty liver disease. In this study, we examined whether rs12979860 and the linked causal rs368234815 variant encoding for the alternative IFNL4 protein variant are associated with liver fibrosis and damage in a large multicenter cohort of patients at risk of nonalcoholic steatohepatitis. To clarify the mechanism, we also evaluated the impact on IFN-stimulated gene hepatic expression in a subset of patients. We considered 946 consecutive Italian individuals at risk of nonalcoholic steatohepatitis with liver histology evaluated according to Kleiner. The rs368234815 TT>δG, rs12979860 C>T, and patatin-like phospholipase-3 rs738409 C>G polymorphisms were genotyped; and IFN-stimulated gene hepatic expression (n = 16) was tested by TaqMan assays. We found that the rs368234815 TT allele was independently associated with severe F3-F4 fibrosis (odds ratio, 1.53; 95% confidence interval, 1.15-2.31; P = 0.005) and with severe (grade 2-3) lobular necroinflammation (odds ratio, 1.47; 95% confidence interval, 1.14-1.88; P = 0.002). The impact of rs368234815 on liver damage was generally more marked in nonobese individuals, where association with severe fibrosis, necroinflammation, and nonalcoholic steatohepatitis was observed (P < 0.05). IFN-stimulated genes were hypo-expressed in the liver of patients carrying the IFNL4 rs368234815 TT/TT genotype (P < 0.05). Similar results were observed when considering the rs12979860 polymorphism, which was in high linkage disequilibrium with rs368234815 (R 2 = 0.87)., Conclusion: The IFNL4 genotype is associated with severity of fibrosis in nonalcoholic fatty liver disease patients of European ancestry, likely by modulating the activation of innate immunity and necroinflammation. (Hepatology 2017;66:1885-1893)., (© 2017 by the American Association for the Study of Liver Diseases.)

There is evidence that nonalcoholic fatty liver disease (NAFLD) is affected by gut microbiota. Therefore, we investigated its modifications in pediatric NAFLD patients using targeted metagenomics and metabolomics. Stools were collected from 61 consecutive patients diagnosed with nonalcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), or obesity and 54 healthy controls (CTRLs), matched in a case-control fashion. Operational taxonomic units were pyrosequenced targeting 16S ribosomal RNA and volatile organic compounds determined by solid-phase microextraction gas chromatography-mass spectrometry. The α-diversity was highest in CTRLs, followed by obese, NASH, and NAFL patients; and β-diversity distinguished between patients and CTRLs but not NAFL and NASH. Compared to CTRLs, in NAFLD patients Actinobacteria were significantly increased and Bacteroidetes reduced. There were no significant differences among the NAFL, NASH, and obese groups. Overall NAFLD patients had increased levels of Bradyrhizobium, Anaerococcus, Peptoniphilus, Propionibacterium acnes, Dorea, and Ruminococcus and reduced proportions of Oscillospira and Rikenellaceae compared to CTRLs. After reducing metagenomics and metabolomics data dimensionality, multivariate analyses indicated a decrease of Oscillospira in NAFL and NASH groups and increases of Ruminococcus, Blautia, and Dorea in NASH patients compared to CTRLs. Of the 292 volatile organic compounds, 26 were up-regulated and 2 down-regulated in NAFLD patients. Multivariate analyses found that combination of Oscillospira, Rickenellaceae, Parabacteroides, Bacteroides fragilis, Sutterella, Lachnospiraceae, 4-methyl-2-pentanone, 1-butanol, and 2-butanone could discriminate NAFLD patients from CTRLs. Univariate analyses found significantly lower levels of Oscillospira and higher levels of 1-pentanol and 2-butanone in NAFL patients compared to CTRLs. In NASH, lower levels of Oscillospira were associated with higher abundance of Dorea and Ruminococcus and higher levels of 2-butanone and 4-methyl-2-pentanone compared to CTRLs., Conclusion: An Oscillospira decrease coupled to a 2-butanone up-regulation and increases in Ruminococcus and Dorea were identified as gut microbiota signatures of NAFL onset and NAFL-NASH progression, respectively. (Hepatology 2017;65:451-464)., (© 2016 by the American Association for the Study of Liver Diseases.)

Unlabelled: The patatin-like phosholipase domain-containing 3 (PNPLA3) rs738409 polymorphism (I148M) is a major determinant of hepatic fat and predisposes to the full spectrum of liver damage in nonalcoholic fatty liver disease (NAFLD). The aim of this study was to evaluate whether additional PNPLA3 coding variants contribute to NAFLD susceptibility, first in individuals with contrasting phenotypes (with early-onset NAFLD vs. very low aminotransferases) and then in a large validation cohort. Rare PNPLA3 variants were not detected by sequencing coding regions and intron-exon boundaries either in 142 patients with early-onset NAFLD nor in 100 healthy individuals with alanine aminotransferase <22/20 IU/mL. Besides rs738409 I148M, the rs2294918 G>A polymorphism (E434K sequence variant) was over-represented in NAFLD (adjusted P = 0.01). In 1,447 subjects with and without NAFLD, the 148M-434E (P < 0.0001), but not the 148M-434K, haplotype (P > 0.9), was associated with histological NAFLD and steatohepatitis. Both the I148M (P = 0.0002) and E434K variants (P = 0.044) were associated with serum ALT levels, by interacting with each other, in that the 434K hampered the association with liver damage of the 148M allele (P = 0.006). The E434K variant did not affect PNPLA3 enzymatic activity, but carriers of the rs2294918 A allele (434K) displayed lower hepatic PNPLA3 messenger RNA and protein levels (P < 0.05)., Conclusions: Rare loss-of-function PNPLA3 variants were not detected in early-onset NAFLD. However, PNPLA3 rs2294918 E434K decreased PNPLA3 expression, lessening the effect of the I148M variant on the predisposition to steatosis and liver damage. This suggests that the PNPLA3 I148M variant has a codominant negative effect on triglycerides mobilization from lipid droplets, mediated by inhibition of other lipases., (© 2015 by the American Association for the Study of Liver Diseases.)

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., (© 2015 by the American Association for the Study of Liver Diseases.)

Unlabelled: Excess hepatic storage of triglycerides is considered a benign condition, but nonalcoholic steatohepatitis (NASH) may progress to fibrosis and promote atherosclerosis. Carriers of the TM6SF2 E167K variant have fatty liver as a result of reduced secretion of very-low-density lipoproteins (VLDLs). As a result, they have lower circulating lipids and reduced risk of myocardial infarction. In this study, we aimed to assess whether TM6SF2 E167K affects liver damage and cardiovascular outcomes in subjects at risk of NASH. Liver damage was evaluated in 1,201 patients who underwent liver biopsy for suspected NASH; 427 were evaluated for carotid atherosclerosis. Cardiovascular outcomes were assessed in 1,819 controls from the Swedish Obese Subjects (SOS) cohort. Presence of the inherited TM6SF2 E167K variant was determined by TaqMan assays. In the liver biopsy cohort, 188 subjects (13%) were carriers of the E167K variant. They had lower serum lipid levels than noncarriers (P < 0.05), had more-severe steatosis, necroinflammation, ballooning, and fibrosis (P < 0.05), and were more likely to have NASH (odds ratio [OR]: 1.84; 95% confidence interval [CI]: 1.23-2.79) and advanced fibrosis (OR, 2.08; 95% CI: 1.20-3.55), after adjustment for age, sex, body mass index, fasting hyperglycemia, and the I148M PNPLA3 risk variant. However, E167K carriers had lower risk of developing carotid plaques (OR, 0.49; 95% CI: 0.25-0.94). In the SOS cohort, E167K carriers had higher alanine aminotransferase ALT and lower lipid levels (P < 0.05), as well as a lower incidence of cardiovascular events (hazard ratio: 0.61; 95% CI: 0.39-0.95)., Conclusions: Carriers of the TM6SF2 E167K variant are more susceptible to progressive NASH, but are protected against cardiovascular disease. Our findings suggest that reduced ability to export VLDLs is deleterious for the liver., (© 2014 by the American Association for the Study of Liver Diseases.)

Unlabelled: Hepatic progenitor cells (HPCs) play a major role in liver repair and regeneration. We evaluated HPC involvement in pediatric nonalcoholic fatty liver disease (pNAFLD). Thirty biopsies of consecutive children and adolescents with untreated NAFLD (19 with nonalcoholic steatohepatitis [NASH] and 11 without NASH) were studied using immunohistochemistry and immunofluorescence. HPCs and HPC-expressing adipokines (e.g., adiponectin, resistin, and glucagon-like peptide 1 [GLP-1]) were counted and correlated with steatosis, inflammation, hepatocyte ballooning, fibrosis, and NAFLD activity score (NAS). The HPC compartment was expanded in pNAFLD, especially in children with NASH, and was independently associated with degree of fibrosis (r = 0.303; P = 0.033). NASH livers were also characterized by increased hepatocyte apoptosis, cell-cycle arrest, and an expanded pool of intermediate hepatocytes. Adiponectin expression in HPCs of pNAFLD patients was down-regulated with respect to the healthy liver, and this expression was inversely correlated with NAS score (r = -0.792; P < 0.001) and steatosis (r = -0.769; P < 0.001). Resistin expression in HPCs increased in pNAFLD and was related to degree of fibrosis (r = 0.432; P < 0.05). GLP-1 was overexpressed in HPCs of pNAFLD patients, and GLP-1 expression was related to degree of steatosis (r = 0.577; P < 0.05) and NAS (r = 0.594; P < 0.01)., Conclusions: HPC activation is involved in the response of the liver to oxidative stress in pNAFLD and is correlated with fibrosis and the progression toward NASH. HPCs express adiponectin, resistin, and GLP-1, which become available to resident liver cells and are strongly associated with the severity of NAFLD. These results may have important pathophysiological implications in the modulation of hepatic insulin resistance and the progression of liver injury., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Unlabelled: Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease in children. Genetic variability, which is a main player in NAFLD, is especially characterized by polymorphisms in genes involved in the development and progression of the disease to nonalcoholic steatohepatitis (NASH). Recently, the rs738409 C>G adiponutrin/patatin-like phospholipase domain-containing 3 (PNPLA3) polymorphism, which encodes the I148M protein variant in the catalytic domain, has been associated with severe steatosis, NASH, and liver fibrosis in adults. In this study, we investigated the association between the rs738409 PNPLA3 gene polymorphism and NAFLD in 149 consecutive children and adolescents (age = 6-13 years) with biopsy-proven NAFLD. We analyzed the rs738409 polymorphism by a 5'-nuclease TaqMan assay and assessed its association with NASH: 41% of the subjects with NAFLD showed heterozygosity and 15% showed homozygosity for the at-risk G allele. The rs738409 genotype did not influence the body mass, adiposity, lipid levels, or insulin resistance and was not associated with alanine aminotransferase levels. Interestingly, the rs738409 G allele was strongly associated with the severity of steatosis (P < 0.0001), the presence of NASH (P < 0.0001), hepatocellular ballooning (P < 0.0001), lobular inflammation (P < 0.0001), and the presence of fibrosis (P = 0.01) independently of confounders. Individuals carrying two minor G alleles almost always had severe steatosis and NASH, heterozygotes were at intermediate risk, and patients negative for G alleles had milder and often uncomplicated steatosis., Conclusion: The PNPLA3 rs738409 polymorphism is associated with steatosis severity, hepatocellular ballooning, lobular inflammation, and perivenular fibrosis in pediatric NAFLD.

Unlabelled: Inherited factors play a major role in the predisposition to nonalcoholic fatty liver disease (NAFLD), and the rs738409 C-->G polymorphism of PNPLA3/adiponutrin, encoding for the isoleucine-to-methionine substitution at residue 148 (I148M) protein variant, has recently been recognized as a major determinant of liver fat content. However, the effect of the rs738409 polymorphism on the severity of liver fibrosis in patients with NAFLD is still unknown. In this study, we considered 253 Italian patients, 179 healthy controls, and 71 family trios with an affected child with NAFLD. Analyses were replicated in 321 patients from the United Kingdom. The rs738409 polymorphism was determined by TaqMan assays. Liver histology was scored according to Kleiner et al. Hepatic expression of genes regulating liver damage was assessed by real-time polymerase chain reaction in 52 patients. The rs738409 GG genotype was more prevalent in patients than in controls (14% versus 3%, adjusted odds ratio [OR] = 3.29, 95% confidence interval [CI] = 1.8-6.9), and in the family study, the G allele was overtransmitted to affected children (P = 0.001). In Italian and United Kingdom patients, adiponutrin genotype influenced alanine aminotransferase levels and the severity of steatosis. Adiponutrin genotype was associated with the expression of genes involved in the steatosis-related liver damage, including the proapoptotic molecule Fas ligand. In the whole series combined, adiponutrin genotype was associated with steatosis grade >1 (OR = 1.35, 95% CI = 1.04-1.76), nonalcoholic steatohepatitis (OR = 1.5, 95% CI = 1.12-2.04), and fibrosis stage >1 (OR = 1.5, 95% CI = 1.09-2.12), independent of age, body mass index, and diabetes. Adiponutrin genotype demonstrated a dose effect with heterozygote risk intermediate between CC and GG homozygotes., Conclusion: In patients with NAFLD, adiponutrin rs738409 C-->G genotype, encoding for I148M, is associated with the severity of steatosis and fibrosis and the presence of nonalcoholic steatohepatitis.

Unlabelled: Transient elastography (TE) has received increasing attention as a means to evaluate disease progression in chronic liver disease patients. In this study, we assessed the value of TE for the prediction of fibrosis stage in a cohort of pediatric patients with nonalcoholic steatohepatitis. Furthermore, TE interobserver agreement was evaluated. TE was performed in 52 consecutive biopsy-proven nonalcoholic steatohepatitis patients (32 males, 20 females, age 13.6 +/- 2.44 years). The area under the receiver operating characteristic curves for the prediction of "any" (>or=1), significant (>or=2), or advanced fibrosis (>or=3) were 0.977, 0.992, and 1, respectively. Calculation of multilevel likelihood ratios showed that TE values <5, <7, and <9 kPa, suggest the presence of "any" fibrosis, significant fibrosis, and advanced fibrosis, respectively. TE values between 5 and 7 kPa predict a fibrosis stage of 1, but with some degree of uncertainty. TE values between 7 and 9 kPa predict fibrosis stages 1 or 2, but cannot discriminate between these two stages. TE values of at least 9 kPa are associated with the presence of advanced fibrosis. The intraclass correlation coefficient for absolute agreement was 0.961., Conclusion: TE is an accurate and reproducible methodology to identify pediatric subjects without fibrosis or significant fibrosis, or with advanced fibrosis. In patients in which likelihood ratios are not optimal to provide a reliable indication of the disease stage, liver biopsy should be considered when clinically indicated.

Unlabelled: No proven treatment exists for nonalcoholic fatty liver disease (NAFLD) in children and adolescents. We sought to determine the efficacy of lifestyle intervention with or without antioxidant therapy in pediatric NAFLD. A total of 53 patients (age 5.7-18.8 years, 37 boys) were included. Lifestyle intervention consisting of a diet tailored to the patient's calorie needs, and increased physical activity was prescribed in all. Patients were concomitantly randomized to alpha-tocopherol 600 IU/day plus ascorbic acid 500 mg/day (n = 25) or placebo (n = 28), and treated for 24 months. The study was an extension of a previous study aimed at evaluating the effect of 12-month lifestyle intervention and antioxidant therapy on serum levels of aminotransferases. The primary end point of the present study was change in liver histology on repeated biopsy at 24 months. Secondary end points were changes in body weight, liver enzymes, and insulin sensitivity indices on 2-hour oral glucose tolerance test. The amount of weight lost at 24 months was similar in the placebo and antioxidant groups (-4.75 [range, -16-4.0] versus -5.5 [range, -12.2-0.4] kg, respectively, P = 0.9). A significant improvement occurred in the grade of steatosis, lobular inflammation, and hepatocyte ballooning, and in the NAFLD activity score in both groups. Levels of aminotransferases, triglycerides, cholesterol, fasting glucose, and insulin, and insulin sensitivity indices improved significantly as well. The improvement in all these parameters was not significantly different between the two groups., Conclusion: Lifestyle intervention with diet and increased physical activity induces weight loss and is associated with a significant improvement in liver histology and laboratory abnormalities in pediatric NAFLD. Alpha-tocopherol plus ascorbic acid does not seem to increase the efficacy of lifestyle intervention alone.

Nonalcoholic fatty liver disease (NAFLD), a common cause of chronic liver disease in adults, is incompletely characterized in children. We conducted a prospective study to better characterize the clinical presentation of NAFLD in children and to determine the effect of lifestyle advice in the management of pediatric NAFLD. From June 2001 to April 2003, 84 children (age 3-18.8 yr) who had elevated aminotransferases and the diagnosis of NAFLD confirmed via liver biopsy underwent a 2-hour oral glucose tolerance test and a 12-month program of lifestyle advice consisting of diet and physical exercise. Thirty-four (40.5%) patients were obese (body mass index [BMI] >97th percentile), and 43 (51.2%) were overweight (BMI 85th-97th percentile). Ten (12%) had abnormal glucose tolerance; 10 (12%) had elevated triglycerides, cholesterol, or both; and all had normal blood pressure. Most children (67/84, 80%) were insulin-resistant, including the 7 children with normal BMI (<85th percentile). Increased liver fibrosis was present in 49 (58.1%) patients and was independently associated with obesity (OR 2.7, 95% CI 1.2-6.2) and age (1-year increase; OR 1.2, 95% CI 1.04-1.5). A 12-month program with diet and physical exercise resulted in a significant decrease in BMI, and levels of fasting glucose, insulin, lipids, and liver enzymes, as well as liver echogenicity on ultrasonography. In conclusion, children with NAFLD are almost always insulin-resistant regardless of BMI. Obesity and older age are independently associated with increased liver fibrosis. A simple lifestyle advice program significantly improves insulin resistance, and the liver disease in pediatric NAFLD.