Nonalcoholic fatty liver disease (NAFLD) has emerged as the most common cause of pediatric liver disease in the developed world. Children have a form of NAFLD that is pathologically distinct from adults. Although NAFLD remains a pathologic diagnosis, biomarkers and imaging studies hold promise as noninvasive means of both establishing the diagnosis and following the disease course. Significant advancements have recently been made in genetics, pathophysiology, and the treatment of NAFLD. The purpose of this article is to provide a clinically relevant review of pediatric NAFLD with an emphasis on recent developments in the field.
Nonalcoholic fatty liver disease (NAFLD) is the most common cause of liver disease in children and its increase is coincident with the obesity epidemic. NAFLD is defined as the presence of macrovesicular steatosis in greater than 5% of hepatocytes in the absence of significant alcohol consumption, drug use, or other recognized disorders that may result in fatty liver. The disease includes a range of disease severity from simple steatosis, which is thought to have a relatively benign prognosis, to nonalcoholic steatohepatitis (NASH), which can progress to cirrhosis. Among children, it is estimated that 5% of normal or overweight and 38% of children who are obese have evidence of NAFLD. Given that obesity rates in the United States approach 30% in many areas, the prevalence of the disease is staggering. Obesity trends in children are not isolated to the United States and are found worldwide. Among those patients with NAFLD, a subset will develop NASH. Current evidence suggests that 1% to 3% of the Western population has NASH, making this disease a significant public health problem.
Clinically, NAFLD is primarily a silent disease that is often suspected incidentally on physical examination or on routine blood testing. On physical examination, most patients will be overweight or obese and will commonly have acanthosis nigricans on the back of the neck, intertrigenous areas, or joints. Hepatomegaly can usually be appreciated, although palpating the liver is often challenging in obese individuals. Alanine aminotransferase (ALT) and aspartate aminotransferases (AST) can be abnormal in NAFLD, usually less than 200 U/L. Patients can also complain of abdominal pain as a presenting symptom, which may relate to stretching of the liver capsule as the liver expands or may be the result of other known obesity-related gastrointestinal comorbidities, such as reflux, constipation, or biliary tract disease. Abdominal pain often prompts the clinician to order an abdominal ultrasound, which sometimes demonstrates echogenicity of the liver that is highly suggestive of fatty infiltration.
Although the diagnosis of NAFLD may be strongly suspected based on clinical parameters, liver function tests and ultrasound, staging and grading of the disease still requires liver biopsy. Liver biopsy not only can confirm NAFLD but can differentiate between simple steatosis and NASH, which is relevant given differences in natural history. Laboratory testing to exclude other forms of liver disease, such as viral hepatitis, alpha-1 antitrypsin deficiency, Wilsons disease, hemochromatosis, and autoimmune hepatitis should be performed before liver biopsy as part of a general hepatitis evaluation in patients with persistently abnormal liver function tests.
This review endeavors to provide a clinically relevant general overview of pediatric NAFLD by using the most up-to-date literature on the topic. Our understanding of this disease has improved significantly in the last several years, and there have been many interesting advancements in the areas of diagnosis, pathophysiology, genetics, and management.
Epidemiology and natural history
The true prevalence of pediatric NAFLD is difficult to determine because screening guidelines are not established and the diagnosis can only be made definitively by liver biopsy. ALT is a nonspecific marker of liver injury in NAFLD, which can be easily obtained; unfortunately normal ALT values have not been clearly established in children. Furthermore, it has been shown that up to 23% of children with NAFLD can have a normal ALT with liver fibrosis. Although abnormal ALT is seen in numerous other liver diseases, most abnormal ALT levels in large populations are attributable to NAFLD. Despite limitations in sensitivity and specificity, ALT can be a valuable screening tool and has been used in numerous studies looking at the prevalence of NAFLD. Data collected from the National Health and Nutrition Examination Survey on 5586 adolescents found elevated ALT in 8% of the study population. Elevated ALT correlated with male sex, Mexican American ethnicity, waist circumference, and fasting insulin levels. The metabolic syndrome, which includes overweight or obesity, insulin resistance, elevated blood pressure, and abnormal waist circumference, has been strongly correlated with the development of NAFLD and disease severity. In a European cohort of 16,390 overweight children and adolescents, 11% of the study population was found to have abnormal liver function tests that significantly correlated with high insulin levels, older age, increasing obesity, and male gender. NAFLD prevalence in Asia seems to be as high or higher, although pediatric data is limited. No official position statement by the major pediatric professional societies recommend routine screening for NAFLD, but plasma ALT may be an easily obtainable, although nonspecific, screening tool in patients considered high risk.
NAFLD is a clinicopathologic diagnosis and, therefore, liver tissue is required to determine true prevalence data in children. The only practical means to obtain this data has been to obtain autopsy specimens from children who had an accidental death. In a landmark article using 742 autopsy specimens from children in San Diego County, 17.3% of the children were aged from 15 to 19 years, were found to have the disease. NAFLD was also found to be more common in boys and children of Asian (10.2%) and Hispanic (11.8%) background. African Americans had the lowest rates of NAFLD at 1.5%. Of note, most Hispanic subjects in this study were of Mexican background. Multiple studies have confirmed that male gender and Asian and Mexican ethnicity are risk factors for NAFLD, whereas African Americans seem to be protected. It is worth noting that although boys are more likely to have NAFLD, boys and girls with NAFLD have an equal chance of developing NASH.
The natural history of NAFLD in the pediatric population is not clearly understood because of a lack of prospective studies evaluating children over time. It seems that those patients with simple steatosis have a benign course, whereas those with NASH can progress to severe liver disease. In one study that retrospectively analyzed 66 pediatric patients with NAFLD over a period of 20 years, 4 patients developed type II diabetes and 4 progressed to cirrhosis. Adult data indicate that one-third of patients with early NASH will progress to cirrhosis in 5 to 10 years, and it is a risk factor for hepatocellular carcinoma (HCC). A recent study identified 4406 cases of HCC from a health care claims database and found that NASH was the leading etiologic risk factor (53%) followed by diabetes (36%).
In addition to liver disease, NAFLD has been associated with the development of risk factors for cardiovascular disease and impaired quality of life. In adults with NASH, death is more likely to result from cardiovascular disease than from liver disease. NAFLD in children is associated with multiple cardiovascular risk factors, including abnormal waist circumference, dyslipidemia, hypertension, and insulin resistance. Carotid intima media thickness (cIMT) is increased in children with NAFLD. In one study, cIMT and flow-mediated dilatation of the brachial artery (FMD) were measured in 150 children with NAFLD and were compared with 100 obese controls. Those children with NAFLD had significantly worse cIMT and FMD, demonstrating that NAFLD is a risk factor independent of obesity in the development of cardiovascular disease.
Pathology
NAFLD encompasses a range of disease severity spanning simple steatosis to NASH. Simple steatosis refers to the accumulation of liver fat without apparent inflammation. Simple steatosis has a benign prognosis compared with NASH. However, emerging adult data suggest that a significant, albeit diminished, proportion of patients with simple steatosis may progress to NASH. NASH describes a pattern in which there is both hepatic steatosis and inflammation. With prolonged liver inflammation, the liver responds with collagen deposition resulting in fibrosis and eventually cirrhosis. There exists both an adult pattern of NASH (type 1) and a unique pediatric pattern (type 2). Type 1 NASH is characterized by inflammatory changes and fat accumulation around the central hepatic venule ( Fig. 1 ). In type 2 NASH, inflammation and fibrosis are found around the portal tract ( Fig. 2 ). Type II NASH has been associated more strongly with male sex and Hispanic and Asian background. Many patients have been noted to have overlapping features.
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Pathology
NAFLD encompasses a range of disease severity spanning simple steatosis to NASH. Simple steatosis refers to the accumulation of liver fat without apparent inflammation. Simple steatosis has a benign prognosis compared with NASH. However, emerging adult data suggest that a significant, albeit diminished, proportion of patients with simple steatosis may progress to NASH. NASH describes a pattern in which there is both hepatic steatosis and inflammation. With prolonged liver inflammation, the liver responds with collagen deposition resulting in fibrosis and eventually cirrhosis. There exists both an adult pattern of NASH (type 1) and a unique pediatric pattern (type 2). Type 1 NASH is characterized by inflammatory changes and fat accumulation around the central hepatic venule ( Fig. 1 ). In type 2 NASH, inflammation and fibrosis are found around the portal tract ( Fig. 2 ). Type II NASH has been associated more strongly with male sex and Hispanic and Asian background. Many patients have been noted to have overlapping features.
Imaging
There are 2 primary imaging modalities used in the assessment of NAFLD, including ultrasound and magnetic resonance imaging (MRI). Computerized tomography is not used because of radiation considerations. With ultrasound, a NAFLD liver seems echogenic or bright and is usually enlarged. Although readily available and of comparatively low cost, ultrasound has a few significant limitations. The first limitation is that liver echogenicity is only seen when approximately 30% or more of hepatocytes are steatotic, which results in diminished sensitivity. Other limitations of ultrasound include that it only visualizes part of the liver, provides no information on liver injury, and interpretation is operator dependent. Nevertheless, ultrasound is abundantly used both in clinical practice and some research studies because of its low cost and noninvasiveness. MRI shows the most promise as an imaging technique not only to assess liver fat content but to also assess liver fibrosis. MR spectroscopy provides the most accurate assessment of liver fat and a technique called MR elastography can be used to quantify fibrosis. The use of MR in NAFLD is still primarily a research tool. Another device that may prove useful in the staging of liver disease involves transient elastography (known as FibroScan, Echosens, Paris, France). This device assesses liver stiffness, which correlates with hepatic fibrosis, using vibration through a topically applied transducer similar to an ultrasound probe. Elastography has been found in adult studies of NAFLD to correlate well with histologic fibrosis scoring. In one study of 52 pediatric patients with biopsy proven NAFLD, this technique identified patients without fibrosis and those with significant fibrosis relatively well but was less useful for discriminating intermediate grades of fibrosis. The utility of transient elastography is often limited by the large amount of subcutaneous fat in affected patients.
Biomarkers
Considering the high prevalence of NAFLD and that the current diagnosis requires a liver biopsy, which can be associated with significant, although uncommon, complications, the need for noninvasive biomarkers to diagnose and follow liver injury is critical. In the previous section, the authors briefly discussed the use of imaging studies for this purpose, but serum biomarkers offer the potential of diagnosis and monitoring disease activity at a low cost and with a simple blood test. The investigation of biomarkers has revolved around 3 different approaches. The first approach is to use known markers of liver disease in isolation or in combination as an algorithm that can be correlated with NAFLD disease severity. The second is to investigate new markers based on what is known about NAFLD pathophysiology. The third method is to perform nonhypothesis-driven genome-wide association studies (GWAS) or proteomic studies to uncover genes or proteins that can be used clinically as markers of disease or disease activity. Most studies have been conducted in adult cohorts. For a full review of this topic, the authors recommend referral to recently published articles.
The enhanced liver fibrosis test (ELF) comprises a panel of serum markers of liver fibrosis, including hyaluronic acid, amino terminal propeptide of collagen type III, and tissue inhibitor of metalloproteinase, combined in an algorithm to predict liver fibrosis. In a study limited by the paucity of patients with moderate or severe fibrosis, the ELF test was used on 112 pediatric patients with biopsy-proven NAFLD. Another pediatric study attempted to improve on the ELF by combining it with the NAFLD fibrosis index (age, waist circumference, and triglycerides) and the results showed promise in predicting the absence or presence of fibrosis. Maffeis and colleagues used a model combining the height-to-waist ratio, Homeostasis Model of Assessment-Insulin Resistance, adiponectin, and ALT in 56 obese 10-year-old children and found this algorithm to be predictive of NAFLD, although the study population was small. In adults, other biomarker profiles have also been used, including the FIB-4 (AST, ALT, platelet count, patient age), which has shown encouraging results.
A potential marker of NAFLD severity is serum cytokeratin-18 (CK18), which is a protein filament cleaved by caspase-3 during apoptosis and released into the circulation. Feldstein and colleagues published data comparing CK18 levels in 139 patients with biopsy-proven NAFLD versus 150 healthy controls. Serum levels of CK18 were found to both predict the presence of NASH and its severity with the area under the receiver operator curve at 0.83 (0.74, 0.91) for the diagnosis of NASH. CK18 may only be useful as a measure of disease activity once the diagnosis of NAFLD has already been confirmed because apoptosis is not unique to NAFLD.
The use of biomarkers in the staging and diagnoses of NAFLD can only be advocated for research purposes, especially in pediatrics, but advancements in the field show great promise and the authors anticipate that biomarkers will be used in the future for staging disease.
Pathophysiology
Over the last several years, there have been many advances toward understanding the pathophysiology of NAFLD. From a simplistic point of view, the cause of NAFLD can be attributed to overnutrition, which can be defined as excessive caloric intake in the absence of appropriate caloric expenditure. The biologic mechanisms by which overnutrition leads to NAFLD are multifactorial and interrelated. Although the epidemic of obesity can largely be ascribed to the decreased activity levels and poor food choices (or options) typical of many modern lifestyles, dozens of genes have been implicated in the development of obesity primarily through appetite regulation. The effect of single genes is small but combinations may have additive effects whose end result may promote obesity development. However, given the rapid increase in obesity rates in the developing world, the role of genes in obesity development is likely small compared with the behavioral and environmental components.
Overnutrition is fundamental to the development of NAFLD, but excessive ingestion or deficiency of particular dietary components may play a significant role. One of the most widely discussed dietary factors is fructose, a constituent of both sucrose and high fructose corn syrup. Fructose, unlike glucose, is processed almost exclusively by the liver and is preferentially shunted into the lipogenesis pathway via glyceraldehyde-3-phosphate. Fructose consumption has been associated with increased central obesity, hepatic lipogenesis, dyslipidemia, insulin resistance, and increased uric acid levels. This finding is particularly relevant because fructose consumption in the form of sugar-sweetened beverages over the last several decades has increased considerably. As a percentage of total caloric intake in children aged 2 to 19 years, dietary surveys indicate that between 1977 and 2001 soft drink consumption increased from 3.0% to 6.9%, whereas fruit drink consumption increased from 1.8% to 3.4%. In adult studies, consumption of soft drinks has been shown to be a risk factor for the development of NAFLD and increased liver fibrosis. Although the deleterious effect of fructose consumption on NAFLD in pediatrics has still yet to be proven, one study has demonstrated a correlation between uric acid levels (a surrogate marker for fructose consumption) and NAFLD severity. Mouse models provide further data that fructose intake may be harmful in NAFLD. A recent murine study revealed that a high-fat diet accompanied with high fructose and carbohydrate intake worsened hepatic fibrosis when compared with mice of the same weight on a high-fat diet alone.
Other dietary components that may play a role in the development of NAFLD include saturated fatty acids and trans fatty acids. Both have been found to have a role in the development of the metabolic syndrome. Rodent models have demonstrated reasonable causality between these two types of fats and NAFLD.
Overnutrition eventually leads to the development of obesity and the accumulation of fat stores. Previously it was thought that adipose tissue was merely an inert storage site for lipid but now it is clear that it is metabolically active. Central obesity resulting from the accumulation of visceral adipose tissue (VAT) is strongly linked to the metabolic syndrome and seems to be key in NAFLD development. Interestingly, VAT is the primary source of liver fat in adults with NAFLD, contributing 59% of the triglyceride (main fat component in NAFLD) found in the liver. VAT is also fundamental to the proinflammatory state seen in the metabolic syndrome through the production of inflammatory cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-6) and free fatty acids (FFA), all of which promote insulin resistance, hepatic fat accumulation, and steatohepatitis ( Fig. 3 ). The cause of the release of proinflammatory mediators from adipose tissue may relate to adipose tissue hypoxia. There is evidence to suggest that as the adipose bed expands, adipocytes suffer from a microhypoxic environment resulting in cellular injury and death, which can then lead to an upregulation of the inflammatory cascade. This finding is particularly interesting because there are data to suggest that hypoxia induced by obstructive sleep apnea in patients who are obese may worsen NAFLD.
In addition to the production of proinflammatory cytokines, VAT produces the adipocytokines leptin and adiponectin. Leptin is a peptide hormone that acts centrally as an appetite suppressant. High levels of leptin are often seen in obese individuals, who commonly develop leptin resistance. In this resistant state, some of the protective effects of leptin are lost, such as the promotion of fatty oxidation and the prevention of hepatic lipogenesis. There is also evidence that leptin directly promotes hepatic fibrogenesis and may play a role in the development of hepatocellular carcinoma. Adiponectin has been shown to improve insulin sensitivity as well as have an antiinflammatory effect. Increased VAT results in decreased adiponectin, low levels of which have been associated in the development of NAFLD in both epidemiologic studies and mouse models. The effects of adiponectin on glucose homeostasis seem to be at least partially mediated through AMP-activated protein kinase and its upstream regulator liver kinase B1. The antiinflammatory properties of adiponectin relate to the inhibition of TNF-α, upregulation of the antiinflammatory cytokine IL-10, and suppression of the lipopolysaccharide–induced inflammatory cascade. Both increased leptin and decreased adiponectin have been associated with increased steatosis in NAFLD.
One of the primary physiologic results of the proinflammatory cytokines and adipocytokines, which are produced in the obese state, is insulin resistance, which has been closely linked with the development of NAFLD, specifically NASH. Insulin is an anabolic hormone that promotes glucose uptake in liver, skeletal muscle, and adipose tissue as well as increasing hepatic and peripheral glycogenesis and lipogenesis. In obesity-related insulin resistance, glucose uptake is blunted, lipogenesis is increased, and there is increased breakdown and uptake of FFAs resulting in high serum FFAs. Much of these FFAs are taken up by the liver where they are invariably processed into triglyceride (TG). The increased fat deposition in the liver caused by insulin is mediated by sterol regulatory binding element (SREBP-1c), which upregulates many lipogenic genes. As insulin resistance develops, elevated serum glucose levels activate the carbohydrate responsive element binding protein, which also promotes lipogenesis. Chronic overnutrition resulting in obesity, therefore, creates an inflammatory cycle that promotes insulin resistance and hepatic lipid deposition.
As triglyceride accumulates within the hepatocyte, large fat vacuoles develop within the cytoplasm. The pathogenicity of this fat accumulation has been debated. Wanless and Shiota postulated that extracellular fat accumulation after hepatocyte necrosis might impair hepatic blood flow through the hepatic veins but this remains unproven. Choi and Diehl suggested that the formation of lipid droplets may actually be protective by sequestering away toxic FFAs in the form of triglyceride. When this sequestering process exceeds capacity, certain FFAs begin to exert their toxic effect. This concept is supported by mouse work done by the same group, which demonstrated that when the final step in triglyceride synthesis (DGAT2) was inhibited with antisense oligonucleotides, hepatic fat accumulation decreased but liver damage worsened as measured by necroinflammation and fibrosis. Conversely, upregulation of DGAT2 resulting in increased hepatic TG creation was associated with no significant increase in liver inflammatory markers. FFAs and their lipotoxic intermediates have been implicated in hepatocellular injury by promoting inflammation, endoplasmic reticulum stress, mitochondrial dysfunction, and oxidant stress. As a result of these processes, hepatocytes begin to die and release inflammatory cytokines and reactive oxygen species (ROS), which then further fuel an already upregulated obesity-related inflammatory environment. The role of cell death in the progression of disease has been demonstrated in a mouse model of NASH in which administration of a pan-caspase inhibitor, which mitigates hepatocyte death, resulted in decreased markers of liver fibrosis. Hepatocyte injury and death directly and indirectly activate the hepatic stellate cells, which are the primary collagen-forming cells in the liver. This activation can result in the development of fibrosis and, if activation is chronic, cirrhosis.
Genetics
Evidence for a genetic contribution to NAFLD is supported by increased prevalence in boys, certain ethnicities and races, and family clustering. In children with biopsy-proven NAFLD, 59% of their siblings and 78% of their parents were found to have evidence of fatty liver on MRI, significantly more than in relatives of age and body mass index (BMI)-matched children without NAFLD. There are many potential genetic contributors to NAFLD but those affecting lipid metabolism, insulin sensitivity, inflammation, oxidative stress, and fibrosis show particular promise as contributers.
One of the most exciting recent developments has been the association between NAFLD and variants of the adiponutrin gene, otherwise known as patatinlike phospholipase 3 (PNPLA3). GWAS conducted by Romeo and colleagues found that the PNPLA3 rs738409 SNP was seen more commonly in Hispanics and was associated with increased liver fat and hepatic inflammation, whereas the rs6006460 was seen more commonly in African Americans and correlated with less fat accumulation. A study of 475 obese and overweight children with a mean age of 10 years noted that homozygosity for the rs738409 variant was associated with a 52% increase in mean ALT levels compared with homozygous controls. Another study of 83 obese children using MRI to quantify hepatic lipid content found that the rs738409 variant was associated with both increased steatosis and Hispanic ethnicity. In a large adult cohort of 894 patients with biopsy-proven NAFLD, rs738409 correlated with worse hepatic inflammation and fibrosis. The same study also had a sizable pediatric cohort of 224 children, and although the investigators could not link the rs738409 SNP with histologic NAFLD severity, they found that this variant was associated with an earlier presentation of disease. A recent meta-analysis that reviewed 11 studies, including 2651 patients with biopsy-proven NAFLD confirmed that PNPLA3 polymorphisms could be correlated with increased fat deposition and worse histologic injury. In addition to its role in NAFLD, rs348409 seems to worsen disease severity in other liver diseases associated with hepatic fat accumulation, such as hepatitis C and alcohol-induced fatty liver disease, adding further proof to the importance of this gene variant.
The mechanism by which the PNPLA3 rs738409 SNP affects liver disease is still under investigation. In humans, PNPLA3 is most robustly expressed in liver but it is also found in muscle and adipose tissue. Expression seems to be directly related to nutritional intake, being downregulated in the fasting state and upregulated during feeding. In vitro and mouse work have shown that SREBP-1, which is activated by insulin, induces PNPLA3, which then promotes hepatic lipogenesis and modulates glucose homeostasis. Interestingly, the role of PNPLA3 on insulin resistance in humans remains controversial, with 2 studies in children demonstrating no association. PNPLA3 is the most widely studied gene affecting the development of NAFLD but the role of many other genes is being investigated.
A recent study by Speliotes and colleagues performed GWAS on subjects pooled from 3 large adult studies (Family Heart Study, Framingham Study, and Amish) to identify candidate genes in patients with evidence on NAFLD on computed tomography scan. Candidate genes were then correlated with histologic and metabolic liver data gathered in patients enrolled in the NASH Clinical research Network. This study demonstrated an association not only between PNPLA3 but also 3 other candidate genes in the development of histologically confirmed NAFLD. PNPLA3 rs738409 conferred the highest odds ratio of developing NAFLD at 3.26, confirming the significant role of PNPLA3. The other candidate genes included neurocan, lysophospholipaselike 1, and glucokinase regulatory protein and were associated with odds ratios of developing NAFLD of 1.6, 1.37, and 1.45, respectively. The function and mechanism of these candidate genes remain to be fully characterized.
Variants of apolipoprotein C3 (APOC3) have also been implicated in the development of NAFLD. Petersen and colleagues conducted a study on 95 healthy Indian Asian men and found that 2 variants in APOC3 (rs2854116 and rs28541) were associated with a 30% increase in the fasting plasma APOC3 concentration and an approximately 60% increase in fasting plasma triglycerides. A total of 30% of those patients possessing one of these variants of APOC3 were found to have fatty liver disease on MR spectroscopy. No patients with wild-type APOC3 had evidence of fatty liver disease, and these findings were reproduced in a cohort of 163 non–Indian Asian men. However, several recent studies have brought into question the role of APOC3 and NAFLD. In a study of 2239 individuals who underwent MR spectroscopy to diagnose NAFLD as a part of the Dallas heart study, no association could be found between APOC3 and hepatic triglyceride content or insulin resistance. A second study using a cohort of southern Europeans with biopsy-proven NAFLD also could not correlate APOC3 with liver disease severity. A possible explanation for the discrepancy in the findings of these studies on APOC3 may relate to the fact that healthy patients with simple steatosis were used in the Petersen study, whereas patients in the other studies were more likely to have more severe liver disease. Nevertheless, at this point the role of APOC3 in the development of NAFLD is in question and remains to be fully determined.
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