Nonalcoholic fatty liver disease (NAFLD) has rapidly become the most common form of chronic liver disease in children and adolescents. Over the past 5 years, developments have revolutionized our understanding of the genetic factors, natural history, diagnostic modalities and therapeutic targets for this disease. New polymorphisms, such as those in PNPLA3, TM6SF2, MBOAT7 and GCKR, have been identified and used to predict the development and severity of NAFLD in both adults and children, and their interaction with environmental factors has been elucidated. Studies have demonstrated the true burden of paediatric NAFLD and its progression to end-stage liver disease in adulthood. In particular, nonalcoholic steatohepatitis can progress to advanced fibrosis and cirrhosis, emphasizing the importance of early diagnosis. Non-invasive imaging tests, such as transient elastography, will probably replace liver biopsy for the diagnosis of nonalcoholic steatohepatitis and the assessment of fibrosis severity in the near future. The therapeutic landscape is also expanding rapidly with the development of drugs that can modify liver steatosis, inflammation and fibrosis, indicating that pharmacotherapy for NAFLD will become available in the future. In this Review, we summarize current knowledge and new advances related to the pathogenesis and management of paediatric NAFLD.
Nonalcoholic fatty liver disease (NAFLD), the hepatic manifestation of the obesity and metabolic syndrome epidemics, is now the most common form of chronic liver disease in children and adolescents.
Knowledge on the pathogenic role of genetic and epigenetic factors in NAFLD has expanded tremendously over the past decade.
The most important risk factors for NAFLD in children are insulin resistance and central obesity.
New clinical practice guidelines for the management of NAFLD, published in 2017, provide some guidance to clinicians on how to screen, diagnose and treat NAFLD in children.
The landscape of therapeutic developments in paediatric NAFLD is expanding, bringing the identification of safe and effective treatments closer.
The global obesity epidemic of the 21st century is increasing the burden of several non-communicable diseases, including nonalcoholic fatty liver disease (NAFLD), which is now recognized as the most frequent cause of chronic liver disease in adults and children worldwide1. NAFLD is a multi-faceted disease that includes a broad spectrum of liver conditions, ranging from fat accumulation in >5% of hepatocytes (nonalcoholic fatty liver) to nonalcoholic steatohepatitis (NASH), which is characterized by tissue necro-inflammation, hepatocyte injury (hepatocellular ballooning) and eventual fibrosis at different stages associated with steatosis (nonalcoholic steatofibrosis) or NASH2. Current lines of evidence suggest that NASH can progress to cirrhosis and end-stage liver disease requiring liver transplantation3. Whether nonalcoholic fatty liver and NASH follow an aggressive course in children, with many children progressing to advanced fibrosis and cirrhosis either in childhood or in early adulthood, remains unexplored in cohort studies1. However, the rising prevalence of childhood obesity foreshadows a large burden of NAFLD that will exert a marked strain on both social care and health systems4.
A substantial amount of literature has established the clinical association between NAFLD and other obesity related comorbidities such as dyslipidaemia, insulin resistance, metabolic syndrome and obstructive sleep apnoea5,6,7,8,9. However, children affected by NAFLD are typically asymptomatic, with few or no signs of the metabolic syndrome, and paediatricians search for symptoms such as abdominal pain, fatigue, irritability, headaches and difficulty concentrating, which are not specific to NAFLD. In up to 50% of cases, hepatomegaly can be appreciated on manual palpation but this procedure is often difficult owing to central obesity. Acanthosis nigricans, a clinical marker of hyperinsulinaemia, is observed in 33–50% of children with biopsy-proven NAFLD. The absence of specific clinical symptoms makes it difficult to obtain an early diagnosis and, therefore, the diagnosis of paediatric NAFLD is often incidental, at a mean age of 11–13 years10,11. This issue also makes the prevalence of NAFLD in children difficult to estimate; however, depending on the type of diagnostic tool and the invasiveness of the test, it is currently estimated that NAFLD affects ~3–10% of children worldwide1.
Despite considerable progress in understanding the complexity of the disease, the pathophysiological mechanisms involved in the onset and progression of liver damage in paediatric NAFLD remain unclear. It is conceivable that genetic predisposition acts in conjunction with epigenetic factors in multiple ways to precipitate the development of liver disease12. On this background, an unhealthy lifestyle, mainly characterized by high intake of certain fats and/or carbohydrates coupled with sedentary behaviours, seems to be the main trigger of NAFLD in children12,13.
The focus of this Review is to provide an overview of current concepts and new scientific advancements in our understanding of the epidemiology, pathogenesis, diagnosis and treatment of paediatric NAFLD.
Prevalence of NAFLD in both children and adults can vary with diagnostic methods, which include screening by serum alanine aminotransferase (ALT) and ultrasonography or confirmation by liver biopsy. Epidemiological data on paediatric NAFLD are still scarce, but the overall prevalence of the disease is estimated to be of 3–10% in children in general paediatric populations14. At a global level there are regional differences in the prevalence of NAFLD, with the lowest prevalence being ~5% in Africa and the highest up to 25% in certain South American and Middle Eastern countries1,15 (Fig. 1). As stated before, part of the variability is explained by the method used to diagnose NAFLD and the fact that NAFLD is a diagnosis of exclusion of liver damage resulting from alcohol, viruses, inborn errors of metabolism, autoimmunity and/or drugs. However, NAFLD is a heritable disease and, according to the available data, genetic factors have a major role in determining prevalence variability among different ethnic groups16,17. Moreover, NAFLD prevalence differences between some groups within national populations, such as the American Hispanic and African American populations who exhibit the same risk factors (obesity and insulin resistance), might be associated with differences in body fat distribution16.
A pooled analysis of published studies established the global prevalence of NAFLD at 7.6% (95% CI 5.5–10.3%) in the general paediatric population and 34.2% (95% CI 27.8–41.2%) in children who were obese18.
In clinical research, the most used predictive marker of paediatric NAFLD is elevation in ALT levels. Therefore, high ALT levels, in the absence of other causes of liver injury, often reflect the prevalence of NAFLD. Unfortunately, there is no consensus on the ideal ALT threshold to diagnose NAFLD, especially in terms of variability related to age, gender, ethnic groups, lifestyle and eating habits. As a result, guidelines produced by the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN)11 and by the European Association for the Study of the Liver, the European Association for the Study of Diabetes, and the European Association for the Study of Obesity19 suggest the use of both ALT and ultrasonography for the screening of NAFLD in children and adults.
Using elevated serum ALT as a surrogate marker of NAFLD, data from the National Health and Nutrition Examination Surveys programme in the USA showed a NAFLD prevalence of 6.0–11.5% among adolescents17. The main determinants of ALT elevation were ethnic differences, gender, waist circumference and hyperinsulinaemia. A similar prevalence has been described in cohorts from Europe, Korea, Japan and China, whereas in other regions, such as in India, prevalence seems to be higher20,21,22,23,24.
The prevalence of histologically confirmed NAFLD in autopsy samples from children and adolescents (2–19 years of age) was 9.6% in the Study of Child and Adolescent Liver Epidemiology in the USA. In children who were obese, the rate of fatty liver rose to 38%25.
Two studies assessed the prevalence of NAFLD among Australian adolescents26,27. Booth et al. reported that 10% of Australian adolescents at age 15 had elevated ALT levels26, whereas Ayonrinde et al.27 showed a NAFLD prevalence of 13% in individuals who underwent ultrasonography at age 17.
Insulin resistance, hypertension and abdominal obesity are typical features of children with NAFLD28. Weight gain during childhood and adolescence has been shown to increase the risk of not only NAFLD but also of end-stage liver disease and hepatocellular carcinoma later in life29,30,31,32,33,34,35. This aspect is clearly highlighted by the fact that, in the USA, NASH has now become the most common indication for liver transplantation in women and the second most common indication in men36. Children with NAFLD also have high rates of extrahepatic comorbidities such as cardiovascular disease, obstructive sleep apnoea, polycystic ovarian syndrome and osteopenia. Given the association of NAFLD with both hepatic and extrahepatic manifestations, epidemiological data are essential to understand its true effect on healthcare systems37,38,39.
Genetic and epigenetic factors
Several lines of evidence suggest that NAFLD has a strong heritable component38. Twin studies conducted at the population level led to the estimation that more than half of the variability of ALT levels and hepatic fat content are accounted for by heritable factors, and the heritability of liver fat and fibrosis are mostly shared40,41. Furthermore, large multi-ethnic cohort studies have highlighted substantial interethnic variability in NAFLD susceptibility, with risk being high in Hispanics, intermediate in Europeans and low in African Americans, independent of adiposity, insulin resistance and socioeconomic factors42. However, NAFLD risk is reduced in Caribbean Hispanic children as compared with American Hispanic children, probably due to admixture with African ancestry43. Family studies have also shown that the risk of progressive NAFLD is 12.5-fold higher in family members of individuals with NAFLD-related cirrhosis than in the general population, independently of several confounders44.
During the past decade, the advent of genome-wide association studies led to identification of the most important common specific genetic determinants of hepatic fat and NAFLD (Fig. 2). The major inherited risk factor for NAFLD in all ethnic groups is the rs738409 C>G single nucleotide polymorphism that results in the I148M protein variant of patatin-like phospholipase domain-containing 3 (PNPLA3)45. This variant is the main common genetic determinant of hepatic fat content and is more frequent in Hispanic individuals, accounting for a large fraction (more than half) of the increased NAFLD risk in this ethnic group45. The PNPLA3 I148M variant has been shown to increase susceptibility to the whole spectrum of liver damage related to NAFLD and to be a general modifier of liver disease progression46. Carriage of the I148M variant increases the risk of liver disease, particularly in children (<18 years), and interacts with dietary factors such as intake of fructose-enriched drinks47,48,49. The mechanism by which the I148M variant promotes liver disease is related to accumulation of the mutated protein on the surface of lipid droplets in hepatocytes, which alters lipid remodeling50,51,52. Furthermore, the I148M protein alters retinol release from hepatic stellate cells, promoting inflammation and fibrogenesis53,54,55.
Another major contributor to NAFLD risk is the rs58542926 C>T single nucleotide polymorphism that gives rise to the E167K variant in transmembrane 6 superfamily member 2 (TM6SF2). This variant protein favours hepatic fat accumulation by decreasing VLDL-mediated lipid secretion and increases susceptibility to liver damage in both adults and children56,57,58. Variants in the genes encoding glucokinase regulator (GCKR) and membrane bound O-acyl transferase 7 (MBOAT7) also contribute to the risk of NAFLD. For GCKR, the underlying mechanism is probably related to a lack of inhibition of glucokinase enzymatic activity by fructose-6-phoshate and unrestrained lipogenesis. Altered remodelling of phospholipids owing to reduced MBOAT7 protein expression probably underlies the effects of the rs641738 MBOAT7 variant59. In addition, a variant of the gene encoding protein phosphatase 1 regulatory subunit 3B (PPP1R3B) seems to have a protective effect against NAFLD by shifting substrate utilization in hepatocytes from de novo lipogenesis to glycogen synthesis60,61,62,63. However, variants in GCKR, MBOAT7 and PPP1R3B have a smaller effect size on the risk of NAFLD than PNPLA3 and TM6SF2 variants, and their effect on the risk of paediatric NAFLD is currently not well established64.
A general concept emerging from these genetic studies is that the risk of progressive NAFLD is strongly related to the effect of the five aforementioned variants on hepatic fat accumulation, suggesting accumulation of lipids in intracellular droplets is a major driver of liver damage64. Furthermore, these genetic risk factors have an additive effect on hepatic fat accumulation, enabling a genetic risk score for the disease to be developed62.
However, other genetic variants, such as those in the genes encoding Mer tyrosine kinase (MERTK), interferon-λ4(IFNL4) and 17-β hydroxysteroid dehydrogenase 13 (HSD17B13), might modify the effect of fat accumulation on inflammation and fibrosis65,66,67. Although there is not yet robust evidence of genetic variants that specifically affect NAFLD development in children, data published in 2017 highlighted new candidate genes in Hispanic boys68.
Rare genetic mutations with a strong effect on the function of proteins involved in NAFLD pathogenesis seem to be involved in determining predisposition to advanced NAFLD and disease clustering in specific families. For example, mutations in apolipoprotein B also favour disease progression by causing lipid compartmentalization in hepatocytes69. Another mechanism leading to progressive NAFLD is telomere shortening and cell senescence70; mutations in the telomerase reverse transcriptase gene (TERT) have been associated with progressive NAFLD71,72. Finally, severe genetic disorders, such as lysosomal acid lipase deficiency (caused by mutation of the lysosomal acid lipase gene, which determines accumulation of cholesteryl esters and triglycerides in hepatocytes), can present as NAFLD, especially in children and young adults. These conditions should be promptly recognized as they require specific treatment and follow-up73.
Epigenetic changes are relatively stable alterations that can be transmitted through cell division, thereby explaining the effect of environmental factors on phenotype and part of the missing heritability of common diseases such as NAFLD74. Methylation of cytosine nucleotides at CpG-rich regulatory or promoter regions represents the first level of epigenetic regulation of gene expression. Several post-translational histone modifications also modulate the access of transcription and regulatory factors to DNA.
An important role of epigenetic factors in modulating the susceptibility to NAFLD is demonstrated by the effect of intrauterine exposure to a high-fat diet in experimental models, which leads to more severe hepatic fat accumulation and the development of NASH in the offspring75. In keeping, epidemiological studies have highlighted that both intrauterine growth restriction and accelerated fetal growth are associated with an increased risk of NAFLD in later life76,77,78,79. Hepatic DNA tends to be demethylated in patients with NAFLD78, with genes involved in DNA methylation, lipid metabolism (including PNPLA3), inflammation and fibrogenesis showing disease stage-dependent regulation, suggesting that epigenetic changes are involved in the progression of liver disease80,81. In a study by Suomela et al. of childhood factors associated with NAFLD in adulthood in 2,042 individuals, birth weight, adiposity and insulin resistance during adolescence and PNPLA3 and TM6SF2 variant status improved stratification of NAFLD risk over traditional risk factors78.
Another layer of epigenetic regulation is provided by non-coding RNAs. NAFLD is associated with deregulation of many hepatic microRNAs (miRNAs)82,83. The most robustly validated alteration is downregulation of miR-122 (refs82,83,84,85,86), which promotes lipogenesis and is associated with spontaneous development of NASH and hepatocellular carcinoma in experimental mice models83,84. A meta-analysis published in 2018 highlighted that miR-122, miR-34a and miR-192 are potential diagnostic markers for NAFLD and NASH with moderate diagnostic accuracy87. However, the correlation between miRNA expression in serum and liver tissue was inconsistent or inverse; additionally, considerable age, ethnic and methodological heterogeneity in the cohorts analysed, lack of standardization of sample processing and miRNA analysis, and the need for replication in larger independent cohorts remain substantial issues87. Furthermore, in these initial studies, the associations between epigenetic markers and NAFLD were not adjusted for lifestyle and environmental factors.
In sum, the available evidence indicates that, in the future, disease management could be improved by considering common and rare inherited risk variants, developmental risk factors associated with epigenetic alterations and metabolic risk factors during childhood (Fig. 2). These aspects could improve disease risk stratification (the first likely application), selection of the most appropriate treatment on the basis of genetic background, or even the development of pharmacological approaches able to counteract the effect of deleterious genetic mutations such as the PNPLA3 I148M protein variant88.
Establishing the diagnosis and disease severity as well as monitoring the disease over time remain major challenges for paediatricians caring for the growing number of children with NAFLD. The early diagnosis of NAFLD is an important issue at all ages but especially in at-risk children who are obese because of the tendency of the disease to progress in both adolescents or young adults. Moreover, although not yet explored, it is reasonable to assume that early detection of disease can strongly influence the choice of treatment, leading to better long-term outcomes11. Current guidelines by the American Association for the Study of Liver Diseases and by the North American Society of Pediatric Gastroenterology, Hepatology and Nutrition (NASPGHAN) for the diagnosis of paediatric NAFLD are reported in Table 1 (refs89,90).
Children who are obese, most of whom present traits of the metabolic syndrome, should be prioritized for assessment of NAFLD but, certainly, the first line of diagnostic screening should be the use of less-invasive and non-invasive tests. The primary aim of screening tests, such as the evaluation of genetic susceptibility and circulating biomarkers, should be to detect early disease in large numbers of individuals with high-risk factors. Conversely, the aim of a diagnostic test, such as imaging, should be to establish the presence or absence of disease. The cost-effectiveness of these tests could be an area of interest for future research. Unfortunately, owing to the lack of robust natural history data on disease progression and long-term outcomes in children, it is impossible to perform any cost-effectiveness analysis for widespread NAFLD screening in children who are obese.
Liver biopsy remains a key method for the diagnosis of NAFLD and for grading and staging disease severity. However, the invasiveness of the procedure, which can result in pain as the most common complication and, more rarely, dangerous events such internal bleeding, leak of bile from the liver or gallbladder, and pneumothorax, is the primary limitation for its use. Liver biopsy is also subject to sampling errors owing to the small area of the organ sampled91. Moreover, pathologist interpretation of liver histology can be variable, with inter-observer and intra-observer variability presenting a serious diagnostic issue92. However, despite these limitations, this procedure is currently the most consistent approach to evaluate liver damage and stage NAFLD. In children with suspected NAFLD, NASPGHAN recommends the exclusion of alternative aetiologies for elevated ALT and/or hepatic steatosis and investigation of the coexistence with other chronic liver diseases. However, NASPGHAN guidelines recommend performing a liver biopsy in patients who have increased risk of NASH and/or advanced fibrosis such as those with high ALT levels (>80 U/l), splenomegaly or an aspartate aminotransferase (AST) to ALT ratio of >1 (ref.89).
The limitations of liver biopsy highlight an urgent need to find non-invasive markers for the assessment of NAFLD and NASH, yet no currently available non-invasive or minimally invasive tests provide the data consistency obtained using liver biopsy.
The four key components of liver histology involved in the diagnosis and staging of NAFLD are steatosis, hepatocyte ballooning, inflammation and fibrosis. Steatosis is defined as abnormal fat accumulation within hepatocytes, and the minimum criterion for NAFLD diagnosis is the presence of macrovesicular steatosis (a single large fat vacuole displacing the nucleus at periphery) or microvesicular steatosis (small lipid vesicles in the cytoplasm) in >5% of hepatocytes93. Ballooning is a manifestation of hepatocellular damage or apoptosis and is defined as twofold enlargement of the normal diameter of the cell. The cytoplasm also becomes clarified and can contain eosinophilic inclusion bodies composed of cytoskeletal peptide aggregates known as Mallory–Denk bodies94. Inflammatory infiltrates comprising mixed mononuclear and polymorphonuclear leukocytes are another characteristic histological feature of NASH. Finally, fibrosis is a histological marker of chronic liver damage and forms a characteristic chicken-wire pattern1.
The histological diagnosis of NASH in children can also be challenging as the pattern of features found in liver biopsy samples often differ from those commonly seen in adults1,95. The typical adult pattern (termed NASH type 1) is characterized by the presence of steatosis (mainly macrovesicular) with ballooning degeneration and/or perisinusoidal fibrosis (zone 3 lobular involvement) and relative sparing of the portal tracts. Paediatric NASH (NASH type 2) is described as the presence of steatosis with portal inflammation and/or fibrosis in the absence of ballooning degeneration and perisinusoidal fibrosis1,95. However, a large proportion of paediatric patients have overlapping features of both type 1 and type 2 NASH96,97,98 (Fig. 3). Although the mechanisms underlying the differences between the adult and paediatric NASH patterns have yet to be fully elucidated, data indicate that genetics play a pivotal role in the variation of NASH patterns among children of different races and ethnicities. Moreover, it has been reported that the Hedgehog pathway could be involved in some differences observed between the two types of NASH in children. In particular, it was demonstrated that Hedgehog signalling is activated in the portal and peri-portal compartment of pre-pubescent male livers, thereby causing a fibroductular reaction and local fibrogenesis, resulting in type 2 NAFLD99.
Several scoring systems have been developed to standardize the histological grading of NAFLD, including the Brunt score for NASH100 and the NAFLD activity score (NAS)101, developed by the National Institute of Diabetes and Digestive and Kidney Diseases and the NASH Clinical Research Network, respectively. The NAS score captures the spectrum of histological patterns of NAFLD; therefore, in the paediatric non-mixed and mixed setting (children and adolescents), this score remains the most appropriate. Owing to the previously discussed histological differences between adult and paediatric NAFLD, the Pediatric NAFLD Histological Score was developed102. However, this score is less relevant in clinical practice than in clinical research because, unlike the NAS, it evaluates the degree of portal inflammation, typical of paediatric NASH, and its main purpose is to determine histological improvement in NASH severity in the context of clinical trials.
Identifying and validating potential novel non-invasive biomarkers is a critical area of research in paediatric NAFLD. Diagnostics development in the area of NAFLD research can be divided into two major groups: modalities to detect and quantify the presence of fibrosis and modalities directed at establishing the diagnosis of NASH. Markers for the diagnosis of NAFLD and NASH are used more often than markers of fibrosis because of easier clinical use, although liver biopsy remains the gold standard for the diagnosis of NASH and to define the degree of fibrosis in the ESPGHAN guidelines11.
Biomarkers for detection of fibrosis
The Pediatric NAFLD Fibrosis Index (PNFI), which is obtained from three simple measures (age, waist circumference and circulating triglyceride levels), was developed to predict liver fibrosis in children with NAFLD103. This index is easy to calculate with no additional cost to the patient and a PNFI ≥9 has a strong positive predictive value (98.5%) to rule-in fibrosis; however, its negative predictive value to exclude fibrosis (75%) is suboptimal. These limitations can be overcome when used in a sequential algorithm with the enhanced liver fibrosis (ELF) test, which calculates a score on the basis of serum levels of three extracellular matrix components: tissue inhibitor of metalloproteinases 1, amino-terminal propeptide of type III procollagen and hyaluronic acid104. When starting from the PNFI score, which is based on data easily obtainable after the first clinical evaluation, a value ≥9 indicates the presence of fibrosis, whereas a value <3 excludes it. If the PNFI is within 3.47–8.99, then the ELF score can be used to differentiate between patients with or without fibrosis. An ELF >8.49 indicates the presence of fibrosis in 97% of patients. Overall, the combined use of PNFI and ELF tests as a first-line approach predicted the presence or absence of fibrosis in 86.4% of children with NAFLD104. To date, based on our experience, this algorithm enables the progression of liver damage towards elevated degrees of fibrosis (F3 and F4/cirrhosis) to be monitored as well as the evaluation of clinical improvements, both at the laboratory level (PNFI) and in terms of hepatic fibrosis progression (ELF). However, future studies are needed to externally validate these findings before the combination of PNFI and ELF can be recommended by guidelines for children with NAFLD.
Biomarkers for diagnosis of NASH
Hepatocyte apoptosis is a prominent feature in patients with NASH, making it an appealing focus for biomarker development and therapeutic intervention105. A large body of evidence has demonstrated the utility of measuring plasma levels of caspase-generated cytokeratin 18 (CK-18) fragments, a specific by-product of hepatic apoptosis, in diagnosing NASH. Fitzpatrick et al. demonstrated that children with biopsy-proven NAFLD also showed considerably elevated levels of CK-18 fragments compared with healthy control individuals106. In addition, plasma levels of CK-18 fragments correlated with disease severity, as children with established NASH had higher levels than those with hepatic steatosis or borderline disease. The dynamic changes in the serum levels of CK-18 fragments over time and their association with histological improvement have been studied in 231 adults with NASH and 152 children with NAFLD who participated in two separate prospective randomized clinical trials107. This study found that decreases in serum levels of CK-18 fragments are strongly associated with improved liver histology in both adults and children with NAFLD, but that these measurements do not perform better than serum ALT levels in identifying histological changes in NAFLD. These results suggest that measuring CK-18 fragments might be useful in the work-up of children suspected of having NASH. However, in general, we believe that the studies evaluating CK-18 were done with CK-18 measurement assays that are not robust for clinical use, which might account for some of the differences among studies. Notably, a 2017 meta-analysis found that levels of caspase-cleaved CK-18 (M30) have moderate accuracy for the diagnosis of NASH: 75% of patients with suspected NASH will be identified by CK-18 and will avoid a liver biopsy, whereas 23% of patients are initially diagnosed as non-NASH on the basis of CK-18 levels and would therefore require a liver biopsy for diagnosis. Full-length CK-18 (M65) is more useful as a biomarker to identify NASH than as a screening tool, owing to its high specificity and sensitivity (area under the receiver operating characteristics (AUROC) = 0.80; 95% CI 0.76–0.83)108. Thus, although the CK-18 circulating fragment might specifically reflect the degree of hepatocellular apoptosis characteristic of NASH, it has no major clinical implications as it does not enable NASH severity to be determined. Large validation studies are needed to continue assessing the clinical applicability of CK-18 biomarkers.
Numerous other biomarkers of inflammation, oxidative stress, apoptosis and fibrosis are currently under investigation. For example, Perito et al. determined that plasma cytokine levels were associated with liver histology among 235 paediatric patients from the NASH Clinical Research Network109. Specifically, both total and activated plasminogen activator inhibitor serum levels were elevated in paediatric patients with definite NASH and lobular inflammation compared with those with non-NASH and borderline NASH, and increased levels of IL-8 and soluble IL-2 receptor-α were associated with stage 3–4 fibrosis and portal inflammation. However, adipocytokines such as plasminogen activator inhibitor, IL-8 or soluble IL-2 receptor-α are inconsistent biomarkers of NAFLD severity because they are not liver specific and can be related to low-grade systemic inflammation or adipose tissue inflammation110.
Several radiological techniques can be used to quantify hepatic steatosis (CT, MRI or magnetic resonance spectroscopy) and fibrosis (transient elastography). These instrumental exams have been used to research valid instrumental alternatives to liver biopsy, which remains the gold standard of NASH diagnosis to date.
Ultrasonography is the most commonly used imaging modality for detection of steatosis predominantly because it is inexpensive, widely available and user friendly. Several studies in adults have demonstrated that this technique is highly sensitive and specific for the detection of NAFLD111. Moreover, liver ultrasonography can provide a good estimate of the degree or extent of hepatic steatosis present on the basis of a series of ultrasonographic characteristics, including hepatorenal echo contrast, liver echogenicity and visualization of intrahepatic vessels, liver parenchyma and the diaphragm. Using these characteristics, liver ultrasonography was shown to be a useful tool for quantifying steatosis in paediatric patients with suspected NAFLD, with the score strongly correlating with grade of steatosis on liver biopsy; the sensitivity and specificity for diagnosing moderate-to-severe steatosis were 79.7% and 86.2%, respectively112.
The sensitivity of ultrasonography to diagnose NAFLD is low when the liver contains <30% fat or when a patient has a BMI >40 kg/m2. Furthermore, ultrasonography cannot rule out the presence of NASH or fibrosis. On the basis of these factors and the vulnerability of the technique to operator variability, the results obtained from hepatic ultrasonography should be interpreted cautiously113. Ultrasonography is recommended by ESPGHAN for screening children who are obese and with signs of insulin resistance and/or hypertransaminasaemia.
Techniques based on CT and MRI, especially magnetic resonance spectroscopy and magnetic resonance proton density fat fraction (MRI-PDFF), are more sensitive techniques for the quantification of steatosis than ultrasonography.
Schwimmer et al. evaluated the accuracy of MRI and MRI-PDFF compared with liver biopsy in children with NAFLD. The mean MRI-PDFF of the liver was 2.6% for grade 0 steatosis (as defined by histology), 9.2% for grade 1, 15.1% for grade 2 and 26.8% for grade 3. The overall accuracy of MRI-PDFF for predicting histologically determined steatosis was 56% (95% CI 54–60%), with an AUROC of 0.82 in distinguishing a steatosis grade 0 from steatosis grade 1 (ref.114). A later study published in 2018 concluded that MRI-PDFF has high diagnostic accuracy for both the classification of histological steatosis grade and the prediction changes in histological steatosis grade in children with NAFLD. MRI-PDFF was able to distinguish between histological grade 1 steatosis and grade 2–3 steatosis with an AUROC of 0.87 (95% CI = 0.80–0.94). This study also found that an MRI-PDFF cut-off of 17.5% provides a sensitivity of 74% for discriminating grade 1 histological steatosis from grades 2–3 (ref.115). MRI-PDFF values can have utility in distinguishing histological steatosis grades, yet MRI is currently not sufficient to replace liver biopsy in children as it is unable to evaluate fibrosis and the presence of fibrosis can lead to slightly underestimated steatosis. This method could replace liver biopsy in children if validated in other cohort studies.
Imaging to assess fibrosis
Transient elastography is a non-invasive ultrasound-based method that uses shear wave velocity to measure liver stiffness116. Transient elastography provides a high level of accuracy for detecting clinically significant liver fibrosis, advanced fibrosis and cirrhosis in adult NAFLD, and a meta-analysis has indicated that transient elastography is excellent for ruling out the presence of fibrosis, differentiating advanced (≥F3) and F4 (cirrhosis) fibrosis from the earlier stages of fibrosis, and that it has moderate accuracy (78%) for diagnosis of ≥F2 fibrosis in adults with NAFLD117.
Transient elastography has also been validated as a method to predict the presence of moderate-to-severe fibrosis in paediatric NAFLD118. In particular, the study demonstrated that liver stiffness measurements of 7– 9 kPa were able to predict fibrosis stages 1 or 2, whereas values of at least 9 kPa were associated with the presence of advanced fibrosis. The addition of a controlled attenuation parameter to transient elastography enables the simultaneous measurement of liver fat and liver fibrosis in patients with NAFLD. In a study conducted in Japan, the controlled attenuation parameter score in children with liver diseases was higher (202 ± 62 dB/m) than in healthy children without obesity (179 ± 41 dB/m), and liver stiffness was also higher in children who are obese (5.5 ± 2.3 kPa) than in the control group of healthy children who were not obese (3.9 ± 0.9 kPa)119.
Further study of transient elastography in paediatric patients with NAFLD is needed to validate this imaging modality, even if it seems the most appropriate for this population given its non-invasive nature and ease of use.
Magnetic resonance elastography has also been used to estimate liver fibrosis in children with NAFLD120. However, the results of this study did not show the degree of diagnostic performance reported in adult-based studies and further technical advances remain a major need. Magnetic resonance elastography was not effective in children probably as a result of a small sample size and greater degrees of steatosis in paediatric NAFLD, which can influence liver stiffness readings.
Finally, acoustic radiation force impulse imaging is an ultrasonographic technique for the assessment of fibrosis. This approach uses short bursts of high-intensity acoustic pulses that produce shear waves through the liver tissue, the velocity of which correlates with liver stiffness. An acoustic radiation force impulse cut-off value of <2 m/s has 100% sensitivity in distinguishing between mild and severe (>F2) fibrosis in children121.
Management of paediatric NAFLD
Lifestyle modifications to induce weight reduction through diet and physical activity remain the mainstay of paediatric NAFLD management122 (Fig. 4). The effect of weight loss on the severity of NAFLD has been widely studied in adults123,124. The amount of weight loss needed to induce improvement in paediatric NAFLD is unknown, although reduction in central abdominal fat compared with subcutaneous fat might halt the progression of NAFLD and a host of NAFLD-related co-morbidities and is an important outcome that should be monitored in primary care settings by following waist circumference percentiles125. Paediatric studies evaluating lifestyle dietary changes and weight loss have suggested that interventions resulting in persistent weight loss are associated with improvement of ALT and AST levels, liver echogenicity and liver histology126,127. A multidisciplinary approach with a team that includes a dietician, a psychologist and an exercise physiologist might result in higher success rates of lifestyle interventions128.
For compliance purposes, we find it beneficial to encourage the participation of other family members in dietary and lifestyle changes. Several approaches to weight loss maintenance have been attempted, including cognitive behavioural therapy, outpatient after care with the physician, ongoing face-to-face family therapy and problem-solving approaches. This area requires further research to establish to most effective methods for maintaining weight loss129.
Dietary advice should be based on the pathological mechanisms of NAFLD progression, favouring nutrients with anti-inflammatory potential and beneficial effects on metabolic syndrome parameters. Consumption of sugary drinks and carbohydrates should be limited, especially drinks and foods that contain high-fructose corn syrup, as recommended in the NASPGHAN guidelines89. Trans-fats should be restricted in favour of monounsaturated fatty acids, and a balanced ratio of omega-6 to omega-3 polyunsaturated fatty acids should be sought130. Finally, bariatric surgery is now widely accepted as a treatment option for adolescents (aged 12 years and older) with a BMI ≥35 kg/m2 with a major comorbidity, such as type 2 diabetes or NASH with advanced fibrosis, or BMI ≥40 kg/m2 with milder comorbidities131. A prospective, multicentre observational study in 242 adolescents with severe obesity (median BMI 50.5 kg/m2) observed a favourable short-term (30 days postoperative) complication profile, supporting the early postoperative safety of weight loss surgery in selected adolescents132. More recently, a prospective study from our group in Italy evaluated the efficacy of laparoscopic sleeve gastrectomy in 20 severely obese (BMI ≥35 kg/m2) adolescents with biopsy-proven NAFLD. All patients had stage 2 fibrosis at baseline and 30% (n = 6) had NASH. At 12 months after surgery, disappearance of NASH occurred in 100% (6/6) and reversal of stage 2 fibrosis occurred in 90% of patients (18/20)133.
Lifestyle interventions are largely unsuccessful at producing and maintaining clinically meaningful weight loss128, leading to a focus on pharmacological interventions in adults and children with NAFLD4 (Fig. 4).
Vitamin E and metformin
The Treatment of NAFLD in Children trial was a multicentre randomized placebo-controlled trial that evaluated the efficacy of the antioxidant vitamin E (800 units daily) and the insulin sensitizer metformin (500 mg twice daily) in paediatric NAFLD (age 8–17 years)134. The primary outcome of substantial and persistent reduction in ALT levels (defined as 50% or less of the baseline level or 40 U/l or less at visits every 12 weeks from 48 to 96 weeks of treatment) was not achieved by either metformin or vitamin E. However, patients on vitamin E had greater resolution of NASH than the other groups (58% with vitamin E versus 28% with placebo; P = 0.006) with statistically significant improvements in their histological activity scores (−0.7 with placebo versus −1.8 with vitamin E; P = 0.02). Hepatocyte ballooning was improved in 38% of children taking metformin, which was higher than in those receiving placebo, but there was no difference in steatosis, inflammation or fibrosis between these groups133. Major criticisms of this study include the low dose of metformin used, the lack of systematic assessment of metformin adherence and the lack of improvement in insulin sensitivity relative to comparable studies. Our approach as a tertiary care centre is to consider using vitamin E (800 units daily) only in patients with biopsy-proven NASH after discussing potential adverse effects, including increased risk of overall mortality, haemorrhagic stroke and prostate cancer, as suggested by data from adult studies135,136. The 2018 American Association for the Study of Liver Diseases practice guidance supports the use of vitamin E but not metformin in children with biopsy-proven NASH90.
Omega-3 fatty acids
The omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid might improve hepatic fatty acid metabolism by inhibiting lipogenesis and stimulating fatty acid oxidation. Our team reported the results of a 6-month randomized controlled trial that tested the efficacy of DHA (250 mg/day and 500 mg/day versus placebo) on liver fat content as detected by ultrasonography in a group of 60 children who were overweight or obese137. Liver steatosis improved in both groups receiving DHA compared with placebo. The odds of more severe versus less severe liver steatosis after treatment were lower in children treated with 250 mg DHA per day (OR 0.01, 0.002–0.110; P < 0.001) and 500 mg DHA per day (OR 0.04, 0.002–0.460; P = 0.01) compared with placebo but there was no difference between the DHA groups (P = 0.4). However, ALT levels did not improve. In a long-term follow-up study that assessed the same cohort of patients at 12, 18 and 24 months, ALT levels decreased in both DHA groups from month 12 onwards versus placebo138. In a study published in 2015, Pacifico et al. randomly assigned 51 children to DHA or placebo and showed that DHA reduced the hepatic fat fraction as estimated by MRI (by 53.4% in the DHA group compared with 22.6% in the placebo group; P = 0.040) and decreased fasting insulin and triglyceride levels, although ALT levels were not improved139. However, a more recent randomized trial of DHA and eicosapentaenoic acid (450–1300 mg per day) in 64 children with NAFLD who were obese or overweight (defined by ALT ≥30 U/l and ultrasonographic evidence of steatosis) showed no effect on ALT, steatosis or insulin resistance compared with placebo140. Interestingly, response to DHA might vary according to PNPLA3 polymorphisms, with patients with the I148M variant being less responsive to DHA, making the case for personalized medicine in guiding the optimal treatment option141. Although omega-3 fatty acids (specifically DHA) might improve NAFLD in children, given the small sample size and heterogeneity of trial design, the quality of the evidence remains insufficient to fully recommend their use.
Gut dysbiosis might have a role in the development and progression of NAFLD; therefore, the manipulation of gut microbiota with probiotics might prove an effective treatment strategy. In a randomized controlled trial involving 20 children with elevated ALT levels and ultrasonographic evidence of steatosis, 8-week treatment with Lactobacillus rhamnosus strain GG (12 billion colony forming units (CFU) per day) led to improved ALT levels compared with placebo, independent of changes in BMI, although hepatic fat levels were not quantified142. A randomized clinical trial conducted by our team demonstrated improved steatosis (defined by the odds ratio of having more severe versus less severe steatosis on ultrasonography) in children with biopsy-proven NAFLD after 4 months of treatment with the probiotic VSL#3 (Lactobacillus plantarum, Lactobacillus delbrueckii subsp. Bulgaricus, Lactobacillus casei, Lactobacillus acidophilus, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis and Streptococcus salivarius subsp. thermophilus) compared with placebo143. In this study, the probability that children supplemented with VSL#3 had none, light, moderate or severe steatosis at the end of the study was 21%, 70%, 9% and 0%, respectively, compared with rates of 0%, 7%, 76% and 17% for the placebo group (P <0.001). More recently, another randomized placebo-controlled trial showed that probiotic treatment (containing Lactobacillus acidophilus ATCC B3208, 3 × 109 CFU; Bifidobacterium lactis DSMZ 32269, 6 × 109 CFU; Bifidobacterium bifidum ATCC SD6576, 2 × 109 CFU; L. rhamnosus DSMZ 21690, 2 × 109 CFU) for 12 weeks improved serum liver enzyme levels (mean levels of ALT decreased from 32.8 to 23.1 U/l (P = 0.02) and mean AST levels decreased from 32.2 to 24.3 U/l (P = 0.02)) and circulating lipid profiles (levels of total cholesterol, LDL cholesterol and triglycerides) in children who are obese with fatty liver detected by ultrasonography144. Combined with minimal adverse effects, these promising results make probiotics an exciting treatment option. However, it is essential that efficacy is established based on liver histology, the durability of the response during long-term follow-up and cost-effectiveness before probiotics are routinely prescribed to children with NAFLD.
Drugs in late-phase development for adult NAFLD
The past few years have witnessed a marked increase in the number of clinical trials of novel drugs to treat NAFLD in adults. Here, we summarize the drugs in phase III development for adult NAFLD that might get FDA approval in the next 2–3 years, with the likelihood that some of these medications will then be tested in children.
Elafibranor (also known as GFT505, Genfit) is a dual peroxisome proliferator-activated receptor-α and -δ agonist that acts as an insulin sensitizer, leading to potential improvements in hepatic steatosis, inflammation and fibrosis. In a phase IIb trial in adult patients with NASH without cirrhosis, a dose of 120 mg for 52 weeks led to higher rates of NASH resolution than the placebo145. A phase 3 trial is underway to assess the effect of 120 mg daily on NASH resolution after 72 weeks of treatment, with long-term follow-up to evaluate the development of liver-related complications (NCT02704403)146.
Obeticholic acid (OCA, Intercept Pharmaceuticals) is a synthetic chenodeoxycholic acid analogue with great potency for the farnesoid X receptor. This nuclear receptor is expressed in the liver and modulates bile acid, lipid and glucose metabolism147. In the FLINT trial, OCA (25 mg daily) for 72 weeks led to histological NASH improvement in 45% of patients, compared with 21% in the placebo arm (P = 0.0002). Those receiving OCA also had higher rates of fibrosis regression than those receiving placebo148. The safety and long-term effects of OCA are being evaluated in a phase III trial (NCT02548351). Promising top-line results were recently released demonstrating significant improvement in fibrosis by one stage in patients that received OCA 25 mg daily for 18 months compared with those in the placebo arm (23.1% versus 11.9%; P = 0.0002)148.
Selonsertib (also known as GS-4997, Gilead Sciences) is an apoptosis signal-regulating kinase 1 inhibitor that modulates hepatocyte apoptosis and liver fibrosis149. This drug was evaluated in a phase II trial that included 72 patients with biopsy-proven NASH (69% were female, the median age was 56 years, 71% had diabetes and the median BMI was 33 kg/m2). Selonsertib treatment led to improvement in fibrosis on biopsy, liver stiffness measured by magnetic resonance elastography and fat content in patients with moderate-to-severe liver fibrosis (F2 or F3)135. Two phase III trials assessed the efficacy of selonsertib in adult patients with NASH-associated advanced fibrosis or cirrhosis (NCT03053050 and NCT03053063)150,151. Top-line results from the trial in patients with cirrhosis were announced in 2019 and showed no statistically significant improvement in terms of fibrosis regression between those treated with selonsertib or placebo152. An interim analysis of data from the STELLAR-3 phase 3 trial of selonsertib in adult patients with NASH, released in April 2019, found that the drug did not outperform placebo for improving fibrosis without any worsening of NASH, and the trial was terminated153.
Cenicriviroc is a dual antagonist of CCR2 and CCR5. Activation of these receptors causes fibrogenesis by monocyte and macrophage recruitment to inflamed liver tissue and the activation of hepatic stellate cells154. These receptors are upregulated in the liver in patients with NASH who are obese. The CENTAUR phase IIb trial evaluated cenicriviroc in non-cirrhotic patients with NASH with fibrosis155. At the interim analysis conducted after 1 year, improvement in fibrosis by at least one stage was achieved in more patients on cenicriviroc than on placebo (20% versus 10%; P = 0.02), leading to the advancement of this compound to a phase III trial that is currently recruiting patients (NCT03028740)156.
Medications entering clinical trials for paediatric NAFLD
Historically speaking, drug development for liver diseases that affect both adults and children has typically followed a trickle-down pattern, with drugs being tested first in adults and then in the paediatric population. For instance, the development of antivirals for hepatitis B and hepatitis C followed this path. However, we and others in the field believe that addressing the unmet need for paediatric NAFLD treatment should not wait for the clinical development of effective drugs for adults. Thus, several pharmaceutical companies have already started planning for trials of new agents in children with NAFLD. Several trial design issues are important to consider for the successful development of future treatments in the paediatric population, including optimal primary endpoints beyond improvement in surrogate biomarkers such as progression to cirrhosis and end-stage liver disease, the effect of confounding variables such as puberty and paediatric-specific safety-related adverse events such as effects on growth and development.
Aramchol is a synthetic bile acid (cholic acid) and a saturated fatty acid (arachidic acid) conjugate that inhibits stearoyl-CoA desaturase 1, the rate-limiting enzyme in monounsaturated fatty acid synthesis, leading to downregulation of liver steatosis. A pilot phase IIa trial using aramchol (100 or 300 mg/day) in 60 adult patients with NAFLD for 3 months showed a dose-dependent decrease in hepatic fat157. Currently, a phase IIb trial is evaluating the efficacy of higher doses of aramchol (400 and 600 mg/day) in adults with non-cirrhotic NASH (NCT02279524)158, and there are plans to evaluate this drug in the paediatric population through the ARTISAN Study (ARamchol Trial to Improve Steatosis in Adolescent NAFLD), but the study is not yet listed on the clinicaltrials.gov website.
The angiotensin II receptor blocker losartan (100 mg orally daily for 24 weeks) is also being evaluated as a treatment for NAFLD in children of age 8–17 years and weighing 70–149 kg with the primary end-point being improvement in serum ALT levels (NCT03467217)159. The rationale behind the investigation of losartan is that the drug can reduce the production of plasminogen activator inhibitor 1 and block the renin angiotensin system, as both pathways are involved in liver inflammation and fibrosis. Losartan is currently approved by the FDA to lower blood pressure in children older than 6 years of age.
NAFLD has emerged as the leading cause of chronic liver disease in children worldwide. Given its complexity, NAFLD management in children requires a multilevel and integrated approach, from screening for genetic predisposition in individuals with risk factors, to personalized initiatives for diagnosis and treatment. The identification of common and rare inherited genetic variants on different epigenetic and risk factor backgrounds will help improve the prediction of the risk of severe progressive NAFLD, enabling preventive lifestyle approaches and efficient multi-targeted therapeutic approaches to be tailored to the patient.
Although liver biopsy is still considered the gold standard technique for the diagnosis and staging of NAFLD, this invasive procedure is not suitable for screening and risk stratification of paediatric patients. Unfortunately, the currently available non-invasive tests have two central limitations: the lack of sensitivity and specificity to distinguish hepatic steatosis from more severe NASH; and inadequate accuracy for staging owing to difficulties in determining the presence and extent of liver fibrosis. Consequently, there is a great necessity to identify reliable non-invasive biomarkers that enable the stratification of patients into those with mild and severe disease.
In the near future, ongoing and new clinical trials, as well as longitudinal studies, will permit accurate assessment of long-term outcomes of children with NAFLD, enabling the allocation of appropriate patients to receive treatments that will halt, reverse and potentially cure NASH and fibrosis.
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The authors declare no competing interests.
We wish to dedicate this article to the memory of Valerio Nobili, who died prematurely and unexpectedly after its acceptance, and to whom we are all profoundly indebted, not only for conceiving this manuscript, but most importantly for his significant personal contribution to the recent advancements in the field that are here described and for his friendship.
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