Nonalcoholic fatty liver disease (NAFLD) is defined as the accumulation of fat in the liver in the absence of excessive alcohol consumption or other known liver pathologies. NAFLD has increased in prevalence and becomes the most common cause of chronic liver disease in children. NAFLD includes a wide spectrum of liver pathology ranging from steatosis to nonalcoholic steatohepatitis (NASH). Genetic and environmental factors are involved in the development and progression of the disease. NAFLD is associated with development of metabolic syndrome, type 2 diabetes, and cardiovascular disease. Studies on the long-term prognosis in the adult population demonstrate that simple steatosis follows a relatively benign clinical course, whereas steatohepatitis associated with increased fibrosis may progress to end-stage liver disease [1]. However, the natural history and prognosis of NAFLD in children remains scant. This article reviews new perspectives in epidemiology, genetics, gut microbiomes, diagnostic modalities, and natural history of NAFLD in children.

The pooled global prevalence of NAFLD was 7.6% in children from general population studies and 34.2% in obese children. In general population studies, there was no evidence that prevalence differed by geographical region. The prevalence of NAFLD in the pediatric populations from Asia, Europe, and North America was 5.9%, 5.7%, and 6.5% respectively. In clinical studies of obese populations, prevalence was higher in Asia than Europe and North America [2]. Using elevated serum ALT as a surrogate marker of NAFLD, data from Korean National Health and Nutrition Examination Surveys showed prevalence of NAFLD among adolescents was 5.3% and stable from 2001 to 2014. The risk factors for NAFLD were male gender, greater body mass index (BMI), and increased waist circumference [3]. Vitamin D deficiency was associated with pediatric NAFLD, independent of obesity and metabolic syndrome [4]. Studies using alanine aminotransferase (ALT) had a lower mean prevalence estimate than studies using ultrasound (US) or magnetic resonance imaging (MRI). In general population studies using US, prevalence estimates were 7.6% [2]. The prevalence of biopsy proven NAFLD in autopsy samples from children and adolescents was 9.6% in the United States [5].

NAFLD has a strong genetic component. Large multi-ethnic population-based studies showed ethnic difference in the prevalence of NAFLD, independent of adiposity, and insulin resistance [6]. Histologically confirmed NAFLD was present in 11.8% of Hispanic children, 10.2% of Asian children, 8.6% of European children, and 1.5% of African children [5]. There is the evidence that NAFLD tends to cluster in families. A familial aggregation study showed that in children without NAFLD, 17% of siblings and 37% of parents had NAFLD compared with 59% of siblings and 78% of parents of children with NAFLD. The heritability of fatty liver was 1.0 and of liver fat fraction was 0.39 after adjusting for age, gender, race, and BMI [7].

Patatin-like phospholipase domain–containing protein 3 (PNPLA3) encodes a transmembrane protein known as adiponutrin. PNPLA3 is expressed predominantly in adipose tissue and may be involved in the hydrolysis of triglycerides (TG). The rs738409 (G/C) single nucleotide polymorphism (SNP), which causes a I148M protein variant, had a strong association with increased hepatic fat content. This allele frequency was 0.48 in Hispanics, 0.23 in Europeans and 0.17 in Africans, which is consistent with different prevalence of NAFLD by ethnicity [8]. The minor allele frequency of rs738409 is 0.51 in Koreans [9]. Studies in adults have shown that I148M is associated with histologic severity of steatosis, NASH, and fibrosis [1011]. PNPLA3 SNP appears to be more important in the development of NAFLD in the non-obese population. In a study in Hong Kong, PNPLA3 variant was found in 78.4% of the non-obese NAFLD group and in 59.8% of the obese NAFLD group [12]. Genetic susceptibility rather than metabolic risk factors might play an important role in the development of NAFLD in the Asian population [9]. Several pediatric studies have demonstrated association of the I148M genotype with elevated ALT in Asian, Hispanic, and European children who are obese [1314151617]. A nonsynonymous SNP in the transmembrane 6 superfamily member 2 (TM6SF2) gene is associated with NAFLD but a clinically favorable lipid profile. This variant (rs58542926) is resulting in glutamic acid to lysine substitution at codon 167 (E167K). TM6SF2 is required for secretion of very low-density lipoprotein (VLDL) from the liver. The E167K variant results in impaired VLDL secretion and intrahepatic TG accumulation [18]. This variant is associated with pediatric NAFLD but may protect cardiovascular disease by lowering total cholesterol and TG in obese children [1920]. Glucokinase regulatory protein (GCKR) regulates de novo lipogenesis by controlling the influx of glucose in hepatocytes. The GCKR rs780094 SNP is associated with an increased risk of NAFLD, and this allele frequency is higher in Asian population [21]. Membrane bound O-acyl transferase domain-containing protein 7 (MBOAT7) is involved in phospholipid remodeling [22]. Variants in GCKR and MBOAT7 gene contribute to the risk of pediatric NAFLD [2324].

Studies evaluating multiple genetic polymorphisms have demonstrated that different ethnic groups have different risk genes [22232526]. Synergistic effects of the risk alleles of PNPLA3, TM6SF2, and GCKR on intrahepatic fat accumulation suggested that multiple risk genotypes have an additive effect on hepatic fat content [19]. Addition of genotypes to clinical risk factors including ethnicity, weight gain, and insulin resistance improved prediction of NALFD in obese children [2627]. Adiposity amplifies the effect of the risk alleles of PNPLA3, TM6SF2, and GCKR, indicating gene-adiposity interaction has a major role in the development and progression of NAFLD [28].

The composition of gut microbiome at phylum level demonstrated that a significant increase in Bacteroides and decrease in Firmicutes was found in the obese and NASH children, compared to the healthy children. Proteobacteria was significantly increased in NASH group, compared to obese group. Patients with NASH had not only more alcohol producing Escherichia, but also higher elevated blood ethanol levels. This study suggests that microbiomes rich in ethanol-producing Escherichia may be involved in the disease progression from obesity to NASH [29]. In a metagenomics and metabolomics study of children with simple steatosis, NASH, obesity, and healthy controls, 26 of 292 volatile organic compounds were increased in patients with NAFLD. The combination of a low abundance of Oscillospira with high levels of 2-butanone was intestinal microbiota signatures of liver steatosis, and high abundance of Ruminococcus and Dorea were associated with progression of simple steatosis to NASH [30]. Gut microbiota is associated with advanced fibrosis in adults with NAFLD and gut microbiome model can be used as a non-invasive test to accurately diagnose advanced fibrosis [31]. In a study of children with obesity or NAFLD, gut microbiomes of children with NAFLD had lower α-diversity than obese children without NAFLD. Increases in Prevotella copri were associated with more severe fibrosis. Bacterial genes for lipopolysaccharide biosynthesis were enriched in children with NASH, and genes for flagellar assembly were enriched in moderate to severe fibrosis [32]. Alterations of the intestinal microbiome might contribute to the pathogenesis of NAFLD and be used as markers of disease or severity.

There is growing evidence that fructose contributes to the development and severity of NAFLD. In an epidemiologic study, energy-adjusted higher fructose intake was associated with NAFLD in adolescents with obesity [33]. Fructose promotes increased hepatic de novo lipogenesis, production of uric acid, visceral adiposity, and decreased insulin sensitivity [3435]. Hepatic de novo lipogenesis is a major contributor of intrahepatic fat accumulation [36]. Visceral obesity and insulin resistance are the most important risk factors for pediatric NAFLD [37]. Children with NAFLD had higher fasting insulin and glucose compared with children with obesity alone [38]. Visceral adipose tissue is the main source of free fatty acids for hepatic TG synthesis [39]. Fructose may alter intestinal microbiome or its products such as endotoxin [40]. Adolescents with NAFLD had higher endotoxin levels than obese controls and the endotoxin level correlated with insulin resistance and inflammatory cytokines [41].

In a retrospective longitudinal adult studies of biopsy confirmed NAFLD, fibrosis stage at baseline, but no other histologic features of steatohepatitis, were associated independently with long-term overall mortality, liver transplantation, and liver-related events [61]. Adolescents with NAFLD may have more advanced fibrosis compared with adults [62]. There are few longitudinal studies on natural history of pediatric NAFLD. In a retrospective study with a follow-up liver biopsy after a mean interval of 28 months, 7 of 18 patients had progression of fibrosis, and 3 patients had regression or disappearance of fibrosis after losing weight. Advanced fibrosis might be seen in patients in the absence of liver enzyme abnormalities [63]. The natural history of pediatric NAFLD in the setting of lifestyle counseling was represented by the placebo arm of a randomized control trial designed to compare vitamin E, metformin, and placebo with liver biopsies at baseline and at 2-year follow-up. In the placebo cohort, 28% had resolution of NASH, 40% improved fibrosis, 40% improved steatosis, and 43% improved lobular inflammation [64]. Lifestyle modification with diet and increased physical activity for 2 years improved grade of steatosis, lobular inflammation, hepatocyte ballooning, and NAFLD activity score [65]. Baseline hepatic fat fraction and weight loss predicted resolution of NAFLD at 2 years follow-up [26].

Children with NAFLD have a very high risk of type 2 diabetes and persistence of obesity at follow-up in young adulthood [66]. A retrospective longitudinal study over time periods up to 20 years showed that progression of fibrosis on follow-up liver biopsy was noted in 4 of 5 patients, and 2 patients with decompensated cirrhosis underwent liver transplantation. Children with NAFLD had a 13.8-fold higher risk of dying or requiring liver transplantation than the general population of the same age and sex [67]. Severe fibrosis and cirrhosis can occur within a few years of diagnosis in the most severe cases. There are insufficient data to predict which children are at higher risk of rapid disease progression [68]. Because death, cardiovascular disease, and liver transplantation from pediatric NAFLD typically do not occur in childhood, studies determining health outcomes from pediatric NAFLD will require longitudinal follow-up into adulthood.

NAFLD is the leading cause of chronic liver disease in children. Multiple genetic factors, intestinal microbiomes, and fructose intake have been identified as risk factors for pediatric NAFLD. A better understanding of NAFLD pathophysiology may help to identify children at risk of the progression of NAFLD and efficient personized therapeutic approaches. Noninvasive imaging modalities, such as US and MRI are used to estimate the steatosis and fibrosis, but they require further validation in children. Additional larger pediatric longitudinal studies are needed to investigate the progression and natural history of pediatric NAFLD.