Pediatric Fatty Liver Disease (PeFLD): all is not NAFLD - pathophysiological insights and

approach to management

Robert Hegarty1, Maesha Deheragoda2, Emer Fitzpatrick1, Anil Dhawan1

1 Pediatric Liver, GI and Nutrition Centre and Mowatlabs, King’s College London at King’s College Hospital, London2 Liver Histopathology, Institute of Liver Studies, King’s College Hospital, London

Corresponding author:

Professor Anil Dhawan MD FRCPCHPediatric Liver, GI and Nutrition Centre, King’s College Hospital, Denmark Hill, London, UKSE5 9RSanil.dhawan@nhs.net

Conflict of interest: The authors of this manuscript have no conflicts of interest to declare.

Keywords: pediatric fatty liver disease, non-alcoholic fatty liver disease, microvesicular steatosis

Word count: 5200

Number of figures and tables: 5

Financial disclosure: The authors received no financial support in relation to the production of the manuscript.

Authors contribution: AD came up with the idea of the manuscript. RH wrote the first draft of the manuscript. RH, MD, EF and AD contributed to the revised version.

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Abstract

The recognition of a pattern of steatotic liver injury where histology mimicked alcoholic liver

disease but alcohol consumption was denied, led to the identification of non-alcoholic fatty

liver disease (NAFLD). Non-alcoholic fatty liver disease has since become the most common

chronic liver disease in adults owing to the global epidemic of obesity. However, in

pediatrics, the term NAFLD seems incongruous: alcohol consumption is largely not a factor

and inherited metabolic disorders (IMD) can mimic or co-exist with a diagnosis of NAFLD.

The term pediatric fatty liver disease (PeFLD) may be more appropriate. In this article, we

summarise the known causes of steatosis in children according to their typical, clinical

presentation: 1. acute liver failure 2. neonatal or infantile jaundice 3. hepatomegaly,

splenomegaly or hepato-splenomegaly 4. developmental delay / psychomotor retardation

and perhaps most commonly; 5. the asymptomatic child with incidental discovery of

abnormal liver enzymes. We offer this model as a means to provide pathophysiological

insights and an approach to management of the ever more complex subject of fatty liver.

Lay summary

Fatty liver disease caused by sedentary lifestyle is the most common long-term liver disease

in adults today. Caution must be exercised when calling children with fatty liver disease as

there may be hidden problems with their metabolism.

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Introduction

The recognition of a pattern of steatotic liver injury where histology mimicked alcoholic liver

disease but alcohol consumption was denied, led to the identification of non-alcoholic fatty

liver disease (NAFLD) (1) - the most common chronic liver disease in adults today. The global

epidemic of obesity has been attributed to be a major factor in the pathogenesis of this

condition. The pediatric community also joined the bandwagon of adult hepatology and

embraced the term despite alcohol consumption, or certainly its contribution to liver

disease state, being minimal pre adolescence (2). The cause and effect relationship of

obesity to fatty liver appears to hold true in adults to a great extent but extrapolation of this

relationship to the pediatric age group, particularly to young children, should be with

caution as inherited metabolic disorders (IMD) can mimic or co-exist with a diagnosis of

NAFLD. This review is aimed at discussing the ever more complex subject of fatty liver

disease in children and young adults by describing its etiopathogenesis according to the

clinical phenotype. Furthermore, the nomenclature of NAFLD as a diagnostic entity needs to

be revisited in children: the term pediatric fatty liver disease (PeFLD) may be more

appropriate as alcohol consumption is usually not a factor, therefore, NAFLD is incongruous.

Steatosis is defined by the presence of fat in hepatocytes when examined under light

microscopy and can be classed as microvesicular or macrovesicular. In macrovesicular

steatosis there is accumulation of large fat droplets from excess delivery of free fatty acids

and the nucleus is displaced to the periphery of the cell. The fat droplets are usually large

and occupy much of the cell, but small droplet macrovesicular steatosis can also be seen, in

which one or more well defined small fat droplets are present and may not cause nuclear

displacement. In microvesicular steatosis hepatocytes have a “bubbly” cytoplasmic

appearance and contain small vesicles of fat (usually <1um in diameter) without

displacement of the nucleus (3). In practice, both macrovesicular and microvesicular

steatosis can co-exist (for example, in Wilson disease or in NAFLD). Whilst a liver

histopathologist is easily able to recognise the different types of steatosis present in a

biopsy sample, interpretation of the etiological or prognostic significance of the different

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types of steatosis present can be more challenging. The clinical associations of

microvesicular steatosis are different in adults and children; their significance attributed

disparately too in clinical practice whereby a link to an IMD is typically considered in

children. However, whether microvesicular steatosis is the histological bystander in an adult

with viral hepatitis or the diagnostic feature in an infant with an IMD, a common

mechanistic concept can be considered at the cellular level: increased delivery of lipids,

impaired efflux of lipids or increased intrinsic esterification of fatty acids due to organelle

dysfunction.

Liver biopsies are, therefore, still indispensable (4). Specifically, in the context of steatosis

and NAFLD, the European Society of Pediatric Gastroenterology Hepatology and Nutrition

(ESPGHAN) expert committee recommend liver biopsies, “to exclude other treatable

disease, in cases of clinically suspected advanced liver disease, before

pharmacological/surgical treatment, and as part of a structured intervention protocol or

clinical research trial” (5)(6). We argue that in the hands of an expert paediatric liver

pathologist working in an experienced liver centre, the biopsy assumes greater importance

for accurate diagnosis of rare IMDs, particularly in young children, and we would advise

referral of biopsy material to such centres when an IMD is suspected. Biopsies enable

investigation of tissue specific gene and protein expression patterns underlying the

progression of PeFLD and they facilitate study of the natural history of PeFLD progressing

into adulthood. Furthermore, a large scale study of PeFLD biopsy material in conjunction

with a comprehensive clinical database would support development of scoring systems

specific for PeFLD.

In this article, we summarise the known causes of steatosis in children according to their

typical, clinical presentation: 1. acute liver failure 2. neonatal or infantile jaundice 3.

hepatomegaly, splenomegaly or hepatosplenomegaly 4. developmental delay /

psychomotor retardation and perhaps most commonly; 5. the asymptomatic child with

incidental discovery of abnormal liver enzymes. A comprehensive diagnostic work-up of

metabolic and non-metabolic etiologies are required and we describe our investigational

approach in children (Figure 1) as well as offering pathophysiological insights that can be

applied for all patients (Figure 2). In doing so we highlight the importance of identifying

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steatosis as an independent entity with its own merits that require careful consideration

(Figure 3).

1. Steatosis in the context of acute liver failure

The number of children with acute liver failure of unknown origin has declined from about

half of cases (7) to one third over the last decade (8). This is largely to do with the more-

ready recognition of underlying IMD that were hitherto undiagnosed. Fat as a finding on

biopsy or explant, is strongly suggestive of an IMD as the etiology of ALF. This is thought to

be predominantly due to mitochondrial related pathology although non-mitochondrial

related accumulation of fat mainly in the form of microvesicular steatosis is also recognised.

Impaired mitochondrial respiratory chain function

The normal function of the mitochondria includes beta oxidation of fatty acids, production

of energy through the electron transport chain and the Kreb cycle. The first step in this

process is the mobilisation of triglycerides from fat stores under fasting conditions. Long

chain fatty acids (12-20 carbons) released by lipases from triglycerides are activated to acyl-

CoA esters in the cell cytoplasm. Whilst shorter chain fatty acids (10 carbons) can

independently enter the mitochondria, long chain fatty acids need to be transported by the

carnitine shuttle. The carnitine shuttle is composed of carnitine palmitoyltransferase I

(CPT1), carnitine translocase and carnitine palmitoyltransferase II (CPT2). Several length

specific enzymes such as long chain acyl-CoA dehydrogenase (LCHAD; 12-18 carbons) and

medium chain acyl-CoA dehydrogenase (MCAD; 6-12 carbons) then act to shorten acyl CoA

in subsequent beta-oxidation cycles. The protons generated by dehydrogenases enter the

electron transport chain whilst acetyl-CoA enters the Kreb cycle or undergo ketogenesis.

Primary or acquired events that impair any of these functions lead to fatty acids being

poorly oxidised by the mitochondria to be esterified into triglycerides (9).

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This is the case with mitochondrial hepatopathies in which patchy or diffuse microvesicular

steatosis is a consistent finding with light microscopy (Figure 4A). Impaired mitochondrial

function can result from tissue mitochondrial DNA depletion or due to a translational

disorder secondary to mutations in nuclear or mitochondrial DNA that encode for

mitochondrial enzymes and proteins (10). Polymerase gamma catalytic subunit (POLG),

deoxyguanosine kinase (DGUOK), MPV17, succinate-CoA ligase (SUCLG1), twinkle protein

(TWINKLE) and TRMU are amongst the more common genotypes that cause ALF; all

evidenced to cause microvesicular steatosis (11–15). Mutations in the leucyl-tRNA

synthetase (LARS) gene, the enzyme responsible for making leucine, should also be

considered as recently reported as a cause of infantile, recurrent ALF in a group of Irish

travellers (16).

Impaired mitochondrial carnitine transport and fatty acid oxidation

Beta oxidation of fatty acids is one of the primary functions of the mitochondria. As in

mitochondrial respiratory chain disorders it is, therefore, not surprising to find that

microvesicular steatosis is a consistent finding in fatty acid oxidation disorders (FAOD).

Amongst them deficiencies in MCAD, LCHAD, very long-chain acyl-CoA dehydrogenase

(VLCHAD) and acyl-CoA dehydrogenase 9 (ACAD 9) can present as acute liver failure (ALF)

with microvesicular steatosis (17–19). Similarly, liver failure and steatosis can result when

the transport of fatty acids are impaired due to defects in the carnitine transporter: severe

neonatal forms of carnitine-palmitoyltransferase and carnitine-acylcarnitine translocase

(CACT) deficiencies can give rise to this phenotype (20,21).

Impaired pyruvate metabolism

The end product of glycolysis is pyruvate which enters the mitochondria to be metabolised

by pyruvate dehydrogenase complex or pyruvate carboxylase for the formation of acetyl-

CoA or oxaloacetate respectively. Dihydrolipoamide dehydrogenase forms part of the

pyruvate dehydrogenase complex and its dysfunction results in recurrent episodes of ALF. In

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between episodes of ALF, microvesicular steatosis is evidenced but is not a permanent

feature (22). Steatosis is also a feature in pyruvate carboxylase deficiency (23).

Urea cycle disorders

Microvesicular steatosis is a consistent finding on liver histological examination of patients

with urea cycle disorders (UCD). Ornithine transcarbamylase (OTC) deficiency and

carbamoylphosphate synthetase I (CPSI) deficiency are such examples that can present

acutely in the neonatal period with rapid development of hyperammonemia. Mitochondrial

dysfunction is implicated in disorders of the urea cycle which operates mainly in the liver

between the mitochondria and cytoplasm. At the molecular level the accumulation of

ammonia has been attributed to the cause of increased mitochondrial permeability leading

to defective oxidative phosphorylation and the production of reactive oxygen species,

eventually leading to cell death (24). However, it is not known what relation the histological

findings of the liver have with respect to the variable clinical expression (25). Microvesicular

steatosis is also seen in lysinuric protein intolerance, a rare autosomal recessive disorder

that can cause post prandial hyperammonemia due to functional deficiencies of urea cycle

intermediates (26).

Impaired sugar metabolism

Disorders of sugar metabolism such as hereditary fructose intolerance due to deficiency in

aldolase b or galactosemia due to deficiency in galactose-1-phosphate uridyl transferase can

result in acute liver failure. In galactosemia there is early, often diffuse, macrovesicular fatty

infiltration of the liver alongside cholestasis with minimal inflammation. The pathogenesis of

galactosemia in recent studies link galactose exposure to oxidative stress (27). Reactive

oxygen species, in turn, causes direct damage to cellular components including the

mitochondria. Hereditary fructose intolerance also manifests as diffuse micro- as well as

macro- vesicular steatosis (28).

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Impaired endoplasmic reticulum function

Wolcott Rallison syndrome is a disorder thought to be related to endoplasmic reticulum

dysfunction in response to cellular stress. It can cause recurrent ALF triggered by febrile

illnesses and microvesicular steatosis is a documented feature (29,30). In Walcott Rallison

syndrome, the defect lies in the PKR-like endoplasmic reticulum transmembrane protein

(PERK) which senses cellular stress by detecting misfolded proteins: part of the cell unfolded

protein response (UPR). PERK activates stress related proteins such as activation

transcription factor-4 (ATF4) that regulate a variety of cellular processes including oxidative

stress (29). Maintenance of endoplasmic reticulum integrity by the UPR is integral to the

metabolism of lipid and glucose metabolism in the liver: knock out mice in cytoplasmic

polyadenylation element-binding (CPEB) protein 4, a regulator of UPR upregulation,

accumulate fat in the liver due to a defect in mitochondrial fatty acid oxidation and

respiration (31). In neuroblastoma amplification sequence (NBAS) deficiency, recently

discovered to be a cause of recurrent, docking and fusion of transport vesicles between the

endoplasmic reticulum and Golgi is thought to be impaired (30). It causes recurrent ALF

from infancy triggered by febrile illnesses (32)(33) demonstrated, by in vitro studies, that

indeed a temperature shift from 37 to 40 degrees C results in reduced NBAS protein levels

(30). Steatosis has been described in children with this disorder (34).

2. Steatosis in the context of neonatal or infantile conjugated jaundice

Tyrosinemia type 1

Tyrosinemia classically presents in infancy with jaundice and progressive liver disease and

coagulopathy if it is not picked up by routine screening. The main findings on histology

depend on the timing in which the biopsy is carried out with progressive fibrosis and

ultimately cirrhosis featuring later in the disease course. Patchy macro- and micro- vesicular

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steatosis are commonly identified (Figure 4B) (35). Mice models designed to elucidate the

pathophysiology of tyrosinemia suggest that homogentisate, an intermediate metabolite of

the tyrosine catabolic pathway, induce apoptosis by releasing cytochrome c of the

respiratory chain into the cytosol of hepatocytes (36).

Niemann-Pick disease type C

Lysosomal storage disorders can present in the neonatal period with jaundice and hepato-

splenomegaly. In NPC, mild to moderate micro- and macro- vesicular steatosis can be

observed although the prevailing finding on light microscopy is that of abnormal

accumulation of glycolipids and cholesterol within Kupffer cell lysosomes. Increased

glycogen content can resemble steatosis (37)(38).

Cystic fibrosis

In the infantile period, cholestasis is the predominant finding on liver biopsy and we have

also encountered periportal macrovesicular steatosis. Steatosis observed in older age

groups is thought to be multifactorial in etiology, influenced by nutrition, infection, drugs,

pancreatic insufficiency as well as genetic modifiers.

Alpha 1-antitrypsin deficiency

Alpha 1-antitrypsin deficiency liver biopsy typically demonstrates giant cell hepatitis with

variable degrees of cell necrosis, inflammation, bile duct damage with PAS-positive diastase

resistant periportal globules (39). Predominantly macrovesicular steatosis is seen, classically

in a periportal distribution in the neonatal period. The mechanism of hepatocyte injury in ZZ

alpha 1-antitrypsin deficiency has come to be understood to intricately involve the

endoplasmic reticulum – mitochondria axis: mutant Z proteins are retained in the

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endoplasmic reticulum leading to autophagic response with caspase activation, redox injury

and mitochondrial changes (40)(41).

Inborn error of bile acid synthesis

This is a group of rare disorders that typically presents in infancy with cholestasis or

spasticity in adult life. Both micro- and macro- vesicular steatosis is evidenced on liver

biopsy along with giant cell hepatitis and extramedullary haematopoiesis (42). The hepatic

steatosis is likely to be due to disordered cholesterol homeostasis: tendon xanthoma and

atherosclerosis is observed in inborn errors of bile acid synthesis as bile acid intermediates

build up and are deposited around the body (43).

Citrin deficiency

Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) is recognised to be

accompanied by a diffusely fatty liver which is histologically similar to NAFLD (44)(45)(46).

NICCD may resolve by the age of 1 with the appropriate treatment. Older children may

present with failure to thrive and dyslipidemia caused by citrin deficiency (FTTDCD) which is

also characterized by a fatty liver.

Intestinal failure associated liver disease

Conjugated jaundice in the context of an infant born prematurely or with intestinal failure is

a common scenario in pediatric practice. The hepatic manifestations of intestinal failure is

multiple and include periportal inflammation and cholestasis, bile duct proliferation,

steatosis and perivenular fibrosis (47). Steatosis may commence in acinar zone 1 and

progress to a pan-lobular distribution. The steatosis can be attributed to delivery of lipid

emulsion from parenteral nutrition itself (48) excess energy (49) as well as the effects of

insulin on increasing mitochondrial fatty acid biosynthesis (50). Exogenous fat in the form of

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parenteral nutrition, as opposed to enterally delivered fat, lacks apolipoproteins and

interferes physiological processing of triglycerides; characteristically fat vacuolations are

found in Kupffer cells and hepatocyte lysosomes (51)(52).

3. Steatosis in the context of organomegaly

Glycogen storage disorders

Disorders that cause abnormal breakdown and synthesis of glycogen resulting in its excess

end organ accumulation is classed as glycogen storage disorders (GSD). Those that present

in their hepatic forms include types III, IV, VI, IX and XI. Dyslipidaemia is well described and

leads to mixed macro- and micro- vesicular in a patchy or diffuse distribution (53) and the

mechanism is two-fold. The first is exemplified by GSD III (glycogen debranching enzyme

deficiency), a ketotic form of GSD, whereby increased fatty acids and glycerol are released

from the adipose in order to provide an alternative energy source (39). Children with this

condition have higher serum triglyceride levels leading to fatty infiltration of the liver. The

second mechanism is due to the inherent physiological link of glycolysis to mitochondrial

function and beta oxidation of fatty acids. In GSD I (glucose-6-phosphatase deficiency), a

hypoketotic GSD, intracellular accumulation of glucose-6-phosphate leads to increased

glycolysis and their downstream products, acetyl CoA and malonyl CoA (54). This is

important as malonyl CoA is the primary regulator which determines the switch between

the reciprocal relationship of fatty acid synthesis and oxidation: when levels are elevated, as

in GSD I, malonyl CoA inhibits fatty acid beta-oxidation and promotes its synthesis (55). This

leaves more substrate to be esterified into triglycerides in the mitochondria (Figure 4C).

Mauriac syndrome

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Mauriac syndrome which is characterized by growth failure, cushingoid appearance and

hepatomegaly can be considered under the umbrella of disordered glycogen storage.

Glycogenic hepatopathy can exist in the context of a high HbA1C without the other

associated clinical features. The accumulation of glycogen and fat in the liver is observed

due to poor glycaemic control in patients with type I diabetes mellitus and the coexistence

of megamitochondria may indicate mitochondrial stress (56).

4. Steatosis in the context of developmental delay / psychomotor retardation

Disorders of protein metabolism

Disorders of amino acid metabolism and organic acid metabolism can present acutely in the

neonatal period with encephalopathy or later on in childhood with psychomotor

retardation. These patients have a variable degree of liver involvement from mild

abnormalities in their transaminases to frank hepatitis with hyperammonemia.

Microvesicular steatosis is consistent finding in organic acidemias and amino acidopathies.

Propionic and methylmalonic acidemias are such examples and reduced levels of complex III

and IV secondary to accumulation of toxic metabolites have been implicated in their

pathogenesis. In glutaric aciduria type II, an inherited disorder of amino acid and fatty acid

metabolism, there is defective electron transfer flavoprotein. The biochemical consequence

of which is deficient transfer of electrons to the mitochondrial respiratory chain. Micro- and

macro- vesicular steatosis is observed (57). Homocysteinuria, similarly, should form part of

the differential diagnosis (58).

Myopathic disorders

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Disorders that primarily affect the muscle are another class of disorders that must be taken

into consideration whereby the replacement of muscle by fat and connective tissue takes

place leading to pseudohypertrophy. Obesity and NAFLD are known associations with

muscular dystrophy. The altered composition of lipid in cells observed in muscular

dystrophy is likely due to decreased physical activity as well as decreased intracellular

carnitine and mitochondrial metabolism (59). One must keep an open mind as elevated

amino transferases, often due to muscle rather than hepatocyte release and usually in the

context of a high creatinine phosphokinase, rapid weight gain or microvesicular steatosis on

liver biopsy may be the presenting problem (28,60). Similar findings may be observed in

limb-girdle muscular dystrophy (61).

In patients with spinal muscular atrophy the SMN protein is deficient leading to defective

motor neurones. Secondary metabolic defects have been proposed to mimic that of acyl-

CoA dehydrogenase deficiency – perhaps part of the explanation for liver biopsy findings in

these patients (62,63). Mitochondrial cytopathies can also present under the heading of a

patient with microvesicular steatosis with developmental delay or neurological impairment.

GFM1 mutation, Leigh disease and MNGIE syndrome are such examples (64–66).

Hypothyroidism

The negative correlation between free T4 levels and presence of NAFLD has been previously

demonstrated (67)(68). Hepatic fat deposition may be secondary to the hormone’s role in

lipid metabolism (69)(70) as well as its association to metabolic syndrome (71). However,

even after the removal of these confounding factors the association between

hypothyroidism and NAFLD is significant suggesting that there may be a direct correlation

(72). It is not clear if NAFLD patients with hypothyroidism demonstrate a different steatotic

pattern histologically to those without hypothyroidism. Certainly in hypothyroid rats mild

microvesicular steatosis with random distribution was demonstrated (73). Further data from

animals show that hypothyroidism promotes hepatic enzymes of lipid metabolism (74),

oxidative stress (75) and farnesoid receptor (76) alluding to potential mechanistic pathways.

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Celiac disease and inflammatory bowel disease

Liver steatosis is well recognized in gastrointestinal disease. In celiac disease the histological

findings are frequently non-specific although steatosis, both micro and macro- vesicular, in a

focal or diffuse distribution, are seen . The mechanism of liver injury may be related to the

increase in permeability of the gut or related autoimmunity (77); steatosis related to

malabsorption, altered intestinal microbiota and nutritional deficiency states as seen in

starvation-associated kwashiorkor (78)(79). Hepatic steatosis in inflammatory bowel

disease, similarly, is multifactorial in etiology and its prevalence has been estimated to be

around 23% (range, 1.5%-55%)(80). The distribution is usually zone 1 and the causes have

been attributed to co-existing metabolic syndrome, chronic inflammation, steroid exposure,

drug-induced and alteration in the gut microbiota (81)(82)(83)(84).

Lysosomal acid lipase deficiency

Lysosomal acid lipase deficiency (LAL-D) was originally described in infants with failure to

thrive, diarrhoea and hepato-splenomegaly. Recent understanding has evolved to a very

heterogeneous condition that can present in older children or adults (85). Abnormal

accumulation of cholesteryl esters within lysosomes due to deficient LAL results in the

appearance of microvesicular steatosis on liver biopsy associated with vacuolated Kupffer

cells (Figure 4D). Whilst immunohistochemistry with lysosomal markers can confirm the

lysosomal nature of the lipid deposits (86), detection of vacuolated Kupffer cells, that would

support the diagnosis, is possible using Diastase Periodic Acid-Schiff (DPAS) stain. It has been

observed in the context of children with NAFLD who failed to respond to weight loss and the

diagnosis brought to light following liver biopsy (87). Importantly LAL-D patients

demonstrate substantially higher low-density lipoprotein-cholesterol levels (88).

Abetalipoproteinemia / hypobetaliproproteinemia

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Defective processing and packaging of apolipoprotein (apo) B as a result of mutations in the

MTP and APOB genes results in impaired lipid absorption and transport (89)(90). It is

characterized clinically by fat malabsorption and failure to thrive in the first year of life

followed by cerebellar dysfunction and retinal degeneration that is amenable to high dose

oral vitamin E supplementation (91). Hypobetalipoproteinemia may be asymptomatic or

associated with less severe diarrhoea. Hepatic manifestation includes steatosis (92) but has

been reported to lead to chronic liver disease and transplantation (93). Very-low density

lipoprotein retained in the cell has been postulated as the mechanism (89)(93).

Lipodystrophies

Lipodystrophy is an umbrella term describing patients with selective loss of body fat (94).

Infants with the congenital forms fail to gain weight despite adequate calorie intake. Later

on patients exhibit paucity of adipose tissue, insulin resistance, hypertriglyceridemia and

macrovesicular hepatic steatosis (95)(96). The problem lies in the defective lipid formation

in the adipocytes (97). This results in excess circulating free fatty acids and eventual

accumulation of triglycerides in the liver (94). The effects are that of hepatic steatosis and

metabolic syndrome (98) and progressive insulin resistance with loss of the body fat

compartment (97).

Congenital disorders of glycosylation

Congenital disorders of glycosylation (CDG) is a group of disorders that result in defective

synthesis of glycoproteins or glycolipids (99). Since its first description in 1980 it has grown

to be a heterogeneous condition: mono- or multi-organ disease; presentation in children as

well as young adults; mild to severe phenotype and nearly 100 subtypes have been

discovered (99) (100). Liver involvement is seen in about 22% of patients and can manifest

as steatosis, cirrhosis and failure (101)(100)(102). The etiology of the steatosis remains

unknown but CDG that give rise to liver disease tend to be grouped at the early steps in the

glycosylation pathway involving the endoplasmic reticulum (100). The authors recommend

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CDG testing in patients with Wilsonian patients with hepatocerebral manifestation when

typical features are not met (100).

Ataxia telangiectasia

Ataxia telangiectasia (AT) is disorder that presents in childhood with ocular telangiectasia,

progressive cerebellar dysfunction, variable immunodeficiency, sensitivity to radiation and

increased cancer susceptibility (103). It falls under the umbrella of chromosome breakage

syndromes characterized by chromosome instability due to defective DNA repair

mechanisms. Fatty liver resembling NAFLD is common in patients with AT and may progress

to advanced liver disease at a young age (104). Patients with AT are vulnerable to DNA

damage from oxidative stress which leads to mitochondrial dysfunction (105); together with

impaired insulin secretion and development of diabetes this can lead to liver steatosis (106)

(107).

5. Steatosis in the context of an asymptomatic child with elevated aminotransferases

Steatosis as the hepatic manifestation of the metabolic syndrome (NAFLD)

NAFLD is present in 9% of children and young people between the ages of 2 and 19 (108),

3% of whom with steatohepatitic / fibrotic disease. In obese children, fatty liver may be

present in 55 - 80% (109)(110). Clearly this is a very heterogeneous group. Similar to adult

NAFLD, the disease may be progressive or not and prediction of those who have a

propensity to progressive fibrotic disease versus those who remain with simple steatosis is

not yet possible. Undoubtedly there are several modifiers involved whether these are

genetic or epigenetic (111). No risk factors for progressive disease in terms of nutrient

intake, BMI nor other clinical features have been identified which reliably distinguish those

children with simple non-progressive steatosis from those who develop fibrotic change. In

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children certainly, a higher body mass index in the morbidly obese range is not a useful

correlate of more advanced disease (112). Histologically, both pediatric and adult NAFLD is

characterized by predominantly macrovesicular steatosis affecting at least 5% of the

parenchyma (113). In adults, NAFLD typically features acinar zone 3 fat accumulation. If

fibrosis is seen, it usually commences in acinar zone 3. The progressive form of non-alcoholic

steatohepatitis (NASH) in adults features steatotic hepatocytes in a diffuse acinar zone 3

distribution, lobular inflammation and cell injury in the form of hepatocyte ballooning and

inflammation whereby ballooning denotes a pattern of liver cell injury of cytoplasmic

swelling and rounding (114). In pediatric NAFLD, inflammation is often portal based,

steatosis may be periportal in distribution, located in acinar zone 3 or panacinar and

ballooning is uncommon (115). The morphological features of pediatric NASH fall into three

types. The centrilobular pattern of steatosis and inflammation, hepatocellular ballooning

and perisinusoidal pattern of fibrosis characteristic of adult NASH is termed type 1 NASH.

This pattern is the least common form of paediatric NASH. Type 2 NASH is the predominant

form in the paediatric population characterised by the presence of zone 1 or panacinar

steatosis, portal inflammation, infrequent ballooning and portal based fibrosis. An overlap

between types 1 and 2 NASH can be seen in up to 75% (116). It is associated with more

advanced disease (117) although puberty seem to exert protective factors (118).

Furthermore, the presence of microvesicular steatosis in both 10% of adults and 19% of

children (119) more strongly correlates with severity of disease, both in terms of fibrosis and

presence of inflammation as well as ballooning and the presence of megamitochondria.

Again, the coexistence of micro- and macro- vesicular steatosis is not fully understood.

Certainly, accumulation of excess free fatty acids in a milieu of elevated plasma glucose and

insulin levels where packaging and transport of triglycerides is overwhelmed or impaired

leads to macrovesicular steatosis. In turn mitochondrial dysfunction may be precipitated by

tumour necrosis factor-alpha, reactive oxygen species, peroxynitrite and lipid peroxidation

products which disrupt respiratory chain polypeptides and mitochondrial DNA (120). The

downstream consequence of a blocked electron transport chain is a further increase in

mitochondrial reactive oxygen species. This may be a contributary mechanism for

microvesicular steatosis is this context. The heterogeneity of the disease needs much

further explanation, and in particular whether the term NAFLD is a mixed bag of different

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susceptibilities and in essence different diseases which manifest in the context of a

precipitant.

Inherited conditions associated with NAFLD that should be considered include Prader-Willi

(121), Alstrom (122) and Bardet Biedel syndromes (123). Prader-Willi syndrome patients

exhibit unique body composition in comparison to obese controls (124)(121) with higher

insulin sensitivity alluding to alternative pathogenic pathways (125)(126).

Wilson disease

A wide spectrum of histological findings can be found in Wilson disease, ranging from a

chronic porto-lobular hepatitis with no steatosis to a steatohepatitis-like picture, similar to

that seen in alcoholic liver disease. It has been known to masquerade NASH and

histologically portal inflammation as well as special stains for copper may point towards the

diagnosis but the distribution of copper can be patchy and may not be encountered in a

liver biopsy. Abundant hepatocyte ballooning and formation of Mallory’s hyaline may also

favor Wilson disease over steatohepatitis in a child. Both microvesicular and macrovesicular

steatosis can be encountered and work has been carried out to verify the link between

intracellular copper accumulation and, in its early stages, the findings of micro- and macro-

vesicular steatosis (127). In vivo studies demonstrated that excess copper catalyse

peroxisomal lipid peroxidation and mitochondrial copper accumulation lead to its structural

and functional disruption (128)(129)(130). Furthermore, in clinical practice, the degree of

steatosis correlated with hepatic parenchymal copper concentration (131). However, it is

likely that steatosis in Wilson is multifactorial in etiology as demonstrated by a recent study

which identified PNPLA G allele and pediatric age independent variables for steatosis in

Wilson disease (132).

Following recovery from sepsis

Steatosis is also seen in the setting of biopsies carried out following sepsis when

abnormalities in their liver function tests persist. In these patients, the etiology of liver

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damage may be multifactorial including hypoxia, bacterial toxin-mediated causes as well as

parenteral nutrition. At the molecular level, there is a recognised association between the

sepsis and mitochondrial dysfunction. Systemic inflammation causes: 1) impaired organ

perfusion and tissue hypoxia compromising oxidative phosphorylation and energy

production; 2) reactive oxygen species, products of inflammation, directly damage cell

constituents including the mitochondria; 3) genes transcribing mitochondrial proteins are

down regulated.

Viral hepatitis

Evidence of hepatotropic as well as non hepatotropic viral hepatitis should be sought.

Hepatitis B, delta and C are associated with hepatic micro- and macro- vesicular steatosis

(133)(134)(135). In a meta-analysis of hepatic steatosis in 4100 chronic hepatitis B virus

(HBV) infected patients the overall steatosis prevalence was 29.6% (133) similar to that of

the general population. However, the prevalence of steatosis in chronic hepatitis C virus

(HCV) infected adults is double (136) although this figure is lower in children (137)(135). The

mechanism is down to the increased risk of metabolic syndrome (138)(139) as well as HCV

specific factors. The virus has been shown to increase de novo lipogenesis (140), interfere

with mitochondrial fatty acid oxidation (141)(142) and decrease hepatocellular export of

fatty substrates (143). In particular, the prevalence and severity of steatosis in HCV

genotype 3 is greatest (ref) and the virus core protein has been shown to directly associate

with lipids acting as intracellular storage sites (144)(145)(146); the effects of which are

increased risk of fibrosis (147)(148) and carcinogenesis (149). Readers are referred to

reviews dedicated to the subject for further details (139). (cross tj, Lonardo A)

Drugs

Microvesicular steatosis in the context of pharmacotherapy has been well documented.

Corticosteroids are probably the most commonly associated drug with macrovesicular

19

steatosis. This is due to the direct effects that glucocorticoids incur on nuclear receptors and

inducing lipogenic enzymes as well as inhibiting fatty acid beta-oxidation (150). The

Conventionally, sodium valproate, aspirin, tetracycline, amiodarone and antiretrovirals have

been implicated. In sodium valproate therapy idiosyncratic hepatotoxicity is well known.

Valproate competitively inhibits beta oxidation of fatty acids in the mitochondria, impairing

lipid metabolism resulting microvesicular steatosis (151). Similarly, interference with beta

oxidation and oxidative phosphorylation has been proposed as the mechanism behind

microvesicular steatosis induced by tetracycline, amiodarone as well as nucleoside

analogues (152).

Congenital porto-systemic shunts

A congenital porto-systemic shunts result from failed involution of foetal vessels either

inside or outside of the liver; the ductus venosus being the most important (153). If left

untreated, it can lead to the development of chronic liver disease, liver tumours and

encephalopathy. Under these circumstances hepatocytes are deprived of nutrients and

oxygen perfusion leading to cell dysfunction (154). Microvesicular steatosis with

mitochondrial enlargement at the ultrastructural level has been observed in this setting

(155).

Conclusion

Steatosis is an important histological finding with diagnostic and prognostic implications.

Traditionally, in pediatrics, steatosis has been considered a finding that is linked to an

underlying IMD. Today with the global epidemic of obesity the subject of fatty liver is

becoming ever more complex. This review was aimed at describing the etiopathogenesis of

fatty liver in children and young adults according to different clinical presentations. We offer

20

this model as a means to the diagnostic approach in relation to clinical presentation. We

also request to shy away from the diagnostic entity of NAFLD, particularly in young children

and suggest that in this population the term PeFLD may be a more appropriate umbrella

term.

Acknowledgements: None

21

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Fig 1.

Microvesicular steatosis: disorders to consider and investigational approach according to

patient phenotype.

Fig 2.

A schematic representation of the mitochondria and other organelle dysfunction leading

to the accumulation of fat droplets.

A1AT-D, alpha 1-antitrypsin deficiency; ACAD9, acyl-CoA dehydrogenase 9; AT, ataxia telangiectasia; CACT, carnitine-acylcarnitine translocase; CPS1, carbamoylphosphate synthetase I; CDG, congenital disorder of glycosylation; CPT1, carnitine palmitoyltransferase 1; CPT2, carnitine palmitoyltransferase 2; DLD, dihydrolipoamide dehydrogenase deficiency; GSD, glycogen storage disease; HFI, hereditary fructose intolerance; LCHAD, long chain acyl-CoA dehydrogenase; LPI, lysinuric protein intolerance; LAL-D, lysosomal acid lipase deficiency; MCAD, medium chain acyl-CoA dehydrogenase; MD, muscular dystrophy; NAFLD, non-alcoholic fatty liver disease; NBAS, neuroblastoma amplification sequence; NPC, Niemann-Pick C; OA, organic acidemia; OTC, ornithine transcarbamylase deficiency; VLCHAD, very long chain acyl-CoA dehydrogenase; WRS, Wolcott Rallison syndrome.

Fig 3.

Balancing the contributions of fat: risk factors to consider when trying to understand the

etiologies of steatosis.

ALF, acute liver failure; BMI, body mass index; DM, diabetes mellitus; IR, insulin resistance; NAFLD, non-alcoholic fatty liver disease.

Fig. 4.

Patterns of steatosis in mitochondrial hepatopathy, tyrosinemia type 1, glycogen storage

disorder type 1a and lysosomal acid lipase deficiency.

Mitochondrial disorders can demonstrate varying degrees of steatosis (A, microvesicular

steatosis indicated by the short arrows, macrovesicular steatosis demonstrated by the long

arrows, H&E x200 magnification). Tyrosinemia type I in a 6-month child with mixed macro-

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and microvesicular steatosis (B, microvesicular steatosis indicated by the short arrows, H&E

x200 magnification). Liver biopsy from an 18-month old child with glycogen storage disease

type 1a showing severe predominantly microvesicular steatosis admixed with glycogen (C,

H&E x400 magnification. Inset shows microvesicular steatosis, short arrows, within

hepatocytes at ultrastructural level). A liver biopsy from a 9-year old child with lysosomal

acid lipase deficiency, demonstrating severe microvesicular steatosis (D, H&E x400

magnification). The inset shows cholesteryl ester storage material within Kupffer cells (Inset

D, short arrows, Diastase Periodic Acid Schiff x400 magnification).

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Fig. 1.

Presentation First line tests Second line tests Disorders to consider

Acute liver failure Blood

GlucoseKetonesLactate

Uric acidCreatine kinase

Acylcarnitine profileCarnitine (free and total)

Alpha 1-antitrypsin phenotype

Galactose-1-uridyl transferase enzymology

Amino acid profileAmmonia

Lipid profileBlood film

Hepatitis serologyOther viral serology and

PCRGlucose tolerance test

Copper studiesSweat test

Lysosomal acid lipase activity

GSD enzymologyTransferrin electrophoresis

UrineOrganic acids

Bile acids

ImagingLiver USS

Liver biopsy

MRI brain

Muscle biopsy

Skin biopsy

Bone marrow aspirate

Targeted next generation sequencing

Mitochondrial hepatopathiesCarnitine transporter defectsFatty acid oxidation disorders

Pyruvate metabolism disordersUrea cycle disorders

Disorders of sugar metabolismImpaired endoplasmic reticulum

function

Neonatal / infantile

conjugated jaundice

Tyrosinemia type 1Niemann-Pick disease type C

Cystic fibrosisAlpha 1-antitrypsin deficiency

Bile acid synthesis defectsCitrin deficiency

Intestinal failure associated liver disease

OrganomegalyGlycogen storage disorders

Mauriac syndrome

Developmental delay /

psychomotor retardation

Disorders of protein metabolismMyopathic disorders

Celiac diseaseLysosomal acid lipase deficiency

AbetalipoproteinemiaLipodystrophies

Congenital disorder of glycosylation

Asymptomatic child

Consider all of the aboveNon-alcoholic fatty liver disease

Wilson diseaseRecovery following sepsis

Drugs

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Porto-systemic shunt

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Fig. 2.

39

Fig. 3.

Fig. 4.

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