Hepatic Steatosis in the Pediatric Population: An Overview of Pathophysiology, Genetics, and Diagnostic Workup

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Abbreviations

ALT

alanine aminotransferase

AST

aspartate aminotransferase

CoA

coenzyme A

FFA

free fatty acid

HAART

highly active antiretroviral therapy

H‐MRS

proton magnetic resonance spectroscopy

HSC

hepatic stellate cell

LAL‐D

lysosomal acid lipase deficiency

LPS

lipopolysaccharide

NAFLD

nonalcoholic fatty liver disease

NASH

nonalcoholic steatohepatitis

NASPGHAN

North American Society for Pediatric Gastroenterology, Hepatology & Nutrition

SNP

single‐nucleotide polymorphism

TG

triglyceride

TLR4

Toll‐like receptor 4

tRNA

transfer RNA

VLDL

very low density lipoprotein

Pathophysiology of Pediatric Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) has become a burgeoning health problem in the 21st century and today is the most common liver disease in children and adults worldwide. ¹ NAFLD is a disease spectrum that begins with simple steatosis (>5% of hepatocytes contain macrovesicular fat). Although 90% to 95% of the general population will remain with simple steatosis, approximately 5% to 10% will progress to nonalcoholic steatohepatitis (NASH). ² Those with NASH may subsequently experience development of fibrosis (38%), cirrhosis (30%), and hepatocellular carcinoma (1%‐2%). ³ , ⁴ The exact mechanism behind the progression from NAFLD to NASH is complex and has yet to be completely elucidated. In the pediatric population, some children have minimal steatosis with fibrosis at diagnosis, whereas others have only steatosis without progression to fibrosis. Longitudinal studies in pediatric NAFLD are limited, although combining existing data for children diagnosed with NAFLD, 25% to 50% have NASH and 10% to 25% have advanced fibrosis. ⁵

The “multiple hit” hypothesis addresses key factors in the occurrence of NAFLD and NASH: dietary intake, environmental factors, insulin resistance, adipose tissue dysfunction, changes in gut microbiota, and genetic predisposition. ² , ⁴ Once considered an adult disease, children and adolescents are now significantly affected. ⁴ The consumption of large amounts of dietary carbohydrates and fat coupled with lack of physical activity have a synergistic effect in the development of fatty liver. This lifestyle translates to metabolic derangements within the body: insulin resistance with hyperinsulinemia and elevated glucose production in the liver. ⁴

The initial stage begins with increased carbohydrate precursors resulting in upregulation of de novo lipogenesis. In addition, high fat intake leads to increase in uptake of fatty acids (FA) from chylomicron particles byt white adipose tissue via lipoprotein lipase. Furthermore, in the presence of hyperinsulinemia, there is an increase in lipolysis resulting in formation of FFAs and glycerol delivered to the liver (Fig. 1A). The hepatic FFAs are esterified to form triglycerides (TGs), which are stored within the liver or exported as very low density lipoprotein (VLDL) particles. ⁴ Decreased disposal of FFAs and increased production lead to hepatic accumulation of FFAs along with TGs. Lipotoxicity occurs with accumulation of saturated fatty acids, which serve as ligands for Toll‐like receptors (TLRs), and subsequently leads to inflammation. Moreover, an increase of Unesterified cholesterol within hepatocytes and Kupffer cells activates proinflammatory cytokines and chemokines. ⁶ Ceramides, adipokines, and by‐products from the gut microbiome all accumulate within the liver and contribute to hepatocellular injury (Fig. 1B). ² , ⁴ , ⁷ Secretion of these proinflammatory mediators serves as catalysts for further recruitment of Kupffer cells and activation of hepatic stellate cells (HSCs) resulting in hepatic fibrosis. ² , ⁸ Unless the source of injury is removed, fibrosis will ensue. Over time, fibrosis leads to cirrhosis, which can become decompensated and progress to end‐stage liver disease or hepatocellular carcinoma. Although these later stages are rare in children and adolescents, they have been reported in early adulthood. ⁹ , ¹⁰

FIG 1.

(A) Development of steatosis. Glucose (from the carbohydrate) is converted into pyruvate, which enters the Krebs cycle and is subsequently converted into acetyl‐CoA, then into malonyl‐CoA, and eventually to FFAs and TGs. White adipose tissue (WAT) takes up fatty acids (FA) from chylomicron (CM) particles via lipoprotein lipase. Additional source of FFAs comes from lipolysis in WAT. These processes lead to accumulation of hepatic FFAs and TGs. ² , ⁴ (B) Progression of NAFLD. Accumulation of FFAs and TGs within the cytosol leads to impaired mitochondrial‐β‐oxidation and inadequate disposal of FFAs. Increased production of lipotoxic lipids, unesterified cholesterol, and ceramides causes damage to hepatocytes and increases inflammation. Further hepatocellular injury occurs with recruitment of inflammatory cytokines. Adipokines, oxidative stress, lipid peroxidation, and formation of aldehyde products contribute to further damage. ATP depletion also occurs. Lipopolysaccharides, by‐products from gut microbiome, induce TLR‐4 expression, which promotes hepatic inflammation. ⁷ With Kupffer cell and HSC activation, collagen production increases with a net matrix deposition and fibrosis ensues. ⁸ This process leads to cirrhosis, which can become decompensated cirrhosis or predispose to hepatocellular carcinoma.

Genetics of Pediatric NAFLD

The risk for NAFLD appears to be linked to pathogenic variants that lead to hepatic fat accumulation through various mechanisms. In addition, several variants have been identified that may confer protection against chronic liver disease. Although there is no robust evidence for the role of all identified genetic variants in pediatric NAFLD it is important to highlight a few that have been investigated (Table 1).

TABLE 1.

Genes and Their Proposed Mechanisms and Effect in Pediatric NAFLD

Gene	Protein Variant	SNP	Protective or Pathogenic	Function	Effect of Variant
PNPLA3, patatin‐like phospholipase domain containing 3	I148M	rs738409 >G	Pathogenic	Accumulation of mutated protein in lipid droplets in hepatocytes, which inhibits adipose TG lipase	Increased hepatocyte TG content
TM6SF2, transmembrane 6 superfamily member 2	E167K	rs58542926 C>T	Pathogenic	Decreases VLDL‐mediated lipid secretion	Increased hepatocyte TG content
GCKR, glucokinase regulatory protein	P446L	rs780094 C>T	Pathogenic	Lack of inhibition of glucokinase enzymatic activity and unrestrained lipogenesis	Increased hepatocyte TG content
MBOAT7, membrane‐bound O‐acyl transferase 7	TCM4	rs641738	Pathogenic	Altered remodeling of phospholipids	Increase in free polyunsaturated fatty acids and hepatic fibrosis
HSD17B13, 17‐β hydroxysteroid dehydrogenase 13	Splice variant	rs72613567 T>A	Protective	Unknown	Protection against chronic liver disease

The most widely studied variant is PNPLA3‐I148M, which was discovered in 2008 through a multiethnic, population‐based study. ¹¹ This gene is found more frequently in Hispanics followed by Europeans and then African Americans. ¹¹ The association between PNPLA3‐I148M and hepatic fat content on proton magnetic resonance spectroscopy (H‐MRS) was highly significant (P = 7.0 × 10⁻¹⁴) after adjusting for body mass index, diabetes status, ethanol use, and global and local ancestry. ¹¹ Carriage of this gene variant in children also increases the risk for liver disease and may interact with dietary factors. To date, PNPLA3‐I148M is the major common genetic variant in NAFLD. ¹² , ¹³ , ¹⁴

Other variants with moderate effect size include TM6SF2, GCKR, and MBOAT7. TM6SF2 variant has been associated with elevated hepatic fat fraction, alanine aminotransferase (ALT), and prevalence of fibrosis in a cohort of obese youth who carried the minor allele. ¹⁵ Similarly, GCKR was confirmed to predispose to fatty liver and dyslipidemia in obese children and adolescents independent of PNPLA3. ¹ A single‐nucleotide polymorphism in MBOAT7 was first discovered to increase risk in alcohol‐related cirrhosis, and more recently has been shown to increase risk for steatosis and histological liver damage in NAFLD independent of obesity. ¹⁶

On the contrary, there are genes that confer protection against chronic liver disease. Recently, one such variant in HSD17B13 was found to decrease risk for NASH in human liver samples and decrease levels of ALT and aspartate aminotransferase (AST). ¹⁷ This finding was replicated in a pediatric study in which carriers of the HSD17B13 rare allele showed lower rates of hepatic steatosis, lower serum transaminase levels than noncarriers, and lower Pediatric NAFLD Fibrosis Index scores even after adjustments for confounders. ¹⁸

Differential Diagnosis and Workup

The North American Society for Pediatric Gastroenterology, Hepatology & Nutrition (NASPGHAN) published clinical practice guidelines for diagnosis of NAFLD. The algorithm recommends workup for NAFLD and other causes of liver disease in children with persistently (>3 months) elevated ALT more than two times the upper limit of normal (normal: 22 U/L for girls and 26 U/L for boys). ³

Various conditions may be mistaken for or even coexist with NAFLD. Furthermore, NAFLD is a diagnosis of exclusion; therefore, other causes of hepatic steatosis should be considered (Table 2). For evaluation of hepatic steatosis, clinical history, physical examination, laboratory tests, and imaging will guide further workup. Use of certain medications and diet may predispose to fatty changes within the liver and can be ascertained by clinical history. Infections, autoimmune liver disease, and genetic/metabolic disorders can cause elevated ALT and should be tested for via appropriate serum studies. In children with persistently elevated ALT and/or imaging studies suggestive of steatosis with other clinical signs, such as organomegaly (liver and/or spleen), metabolic and genetic disorders should be included in the differential (Table 3). ¹⁹ Diagnostic workup for blood and urine tests depend on the condition being evaluated. ¹⁸ Abdominal ultrasound is helpful to assess for gallstones, biliary disease, and other anatomical abnormalities (including kidney involvement), but does not diagnose NAFLD because of inadequate detection and quantification of hepatic steatosis. Transient elastography by FibroScan has shown promise in predicting fibrosis, although studies for sensitivity in hepatic fat quantification in children are limited. ³ Hepatic H‐MRS has been used in research, is noninvasive, and is a highly sensitive radiological imaging modality for fat quantification and detection of fibrosis; however, H‐MRS is not widely available. Liver biopsy remains the gold standard for diagnosis of NAFLD and NASH and allows for evaluation of other liver diseases. Optimal timing of liver biopsy in children with presumed NAFLD has not been established. NASPGHAN guidelines recommend considering a liver biopsy in children with a high risk for NASH and fibrosis. ³

TABLE 2.

Clinical Conditions That Can Cause Hepatic Steatosis in Children

Medications

Amiodarone

Carbamazepine

Corticosteroids

Valproic acid

Antipsychotics

Antidepressants

HAART

Infections

Hepatitis A

Hepatitis B

Hepatitis C

Specific diets

Ketogenic diet

Parenteral nutrition

Severe malnutrition

Rapid weight loss

Autoimmune liver disease

Metabolic/Genetic disorders

TABLE 3.

Metabolic and Genetic Disorders Manifesting With Hepatic Steatosis

alpha‐1‐Antitrypsin deficiency

Wilson’s disease

Celiac disease

Hypothyroidism

Cystic fibrosis

Hypopituitarism/Septo‐optic dysplasia

Uncontrolled diabetes

Mauriac syndrome

Glycogen storage disease

Lipodystrophies

LAL‐D

Abetalipoproteinemia/Hypobetalipoproteinemia

Fatty acid oxidation disorders

Medium‐chain acyl‐CoA dehydrogenase deficiency

Long‐chain acyl‐CoA dehydrogenase deficiency

Very long‐chain acyl CoA dehydrogenase deficiency

Acyl‐CoA dehydrogenase 9 deficiency

Mitochondrial hepatopathies

Polymerase gamma catalytic subunit–related mitochondrial disease

Deoxyguanosine kinase deficiency

MPV17‐related hepatocerebral mitochondrial DNA depletion syndrome

tRNA‐specific 2‐thiouridylase deficiency

Urea cycle disorders

Ornithine transcarbamylase deficiency

Carbamoyl phosphate synthetase I deficiency

Disorders of carbohydrate metabolism

Galactosemia

Hereditary fructose intolerance

Disorders of endoplasmic reticulum function

Tyrosinemia type 1

Niemann‐Pick disease type C

Ataxia telangiectasia

Disorders of bile acid synthesis

Citrin deficiency

Disorders of protein metabolism

Methylmalonic acidemia

Propionic acidemia

Congenital disorders of glycosylation

In summary, NAFLD is a growing health problem in the pediatric population. It is pivotal to identify patients in the earlier, benign stage of the spectrum before irreversible liver damage occurs. Understanding of the disease mechanism, predisposing genetic variants, and appropriate diagnostic workup will refine disease management and positively influence the natural history of NAFLD.