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Diagnosis and management of secondary causes of steatohepatitis

  • Roman Liebe
    Affiliations
    Department of Gastroenterology, Hepatology and Infectious Diseases, University Hospital Düsseldorf, Medical Faculty of Heinrich Heine University Düsseldorf, Moorenstr. 5, 40225 Düsseldorf, Germany
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  • Irene Esposito
    Affiliations
    Institute of Pathology, University Hospital Düsseldorf, Medical Faculty of Heinrich Heine University Düsseldorf, Moorenstr. 5, 40225 Düsseldorf, Germany
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  • Hans H. Bock
    Affiliations
    Department of Gastroenterology, Hepatology and Infectious Diseases, University Hospital Düsseldorf, Medical Faculty of Heinrich Heine University Düsseldorf, Moorenstr. 5, 40225 Düsseldorf, Germany
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  • Stephan vom Dahl
    Affiliations
    Department of Gastroenterology, Hepatology and Infectious Diseases, University Hospital Düsseldorf, Medical Faculty of Heinrich Heine University Düsseldorf, Moorenstr. 5, 40225 Düsseldorf, Germany
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  • Jan Stindt
    Affiliations
    Department of Gastroenterology, Hepatology and Infectious Diseases, University Hospital Düsseldorf, Medical Faculty of Heinrich Heine University Düsseldorf, Moorenstr. 5, 40225 Düsseldorf, Germany
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  • Ulrich Baumann
    Affiliations
    Division of Paediatric Gastroenterology and Hepatology, Department of Paediatric Liver, Kidney and Metabolic Diseases, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany

    Institute of Immunology and Immunotherapy, University of Birmingham, UK
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  • Tom Luedde
    Affiliations
    Department of Gastroenterology, Hepatology and Infectious Diseases, University Hospital Düsseldorf, Medical Faculty of Heinrich Heine University Düsseldorf, Moorenstr. 5, 40225 Düsseldorf, Germany
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  • Verena Keitel
    Correspondence
    Corresponding author. Address: Clinic for Gastroenterology, Hepatology and Infectious Diseases, University Hospital Düsseldorf, Medical Faculty of the Heinrich Heine University Düsseldorf, Moorenstrasse 5, D-40225 Düsseldorf, Germany; Tel.: (49) 211 8116330, fax: (49) 211 8118752.
    Affiliations
    Department of Gastroenterology, Hepatology and Infectious Diseases, University Hospital Düsseldorf, Medical Faculty of Heinrich Heine University Düsseldorf, Moorenstr. 5, 40225 Düsseldorf, Germany
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Open AccessPublished:February 09, 2021DOI:https://doi.org/10.1016/j.jhep.2021.01.045

      Summary

      The term non-alcoholic fatty liver disease (NAFLD) was originally coined to describe hepatic fat deposition as part of the metabolic syndrome. However, a variety of rare hereditary liver and metabolic diseases, intestinal diseases, endocrine disorders and drugs may underlie, mimic, or aggravate NAFLD. In contrast to primary NAFLD, therapeutic interventions are available for many secondary causes of NAFLD. Accordingly, secondary causes of fatty liver disease should be considered during the diagnostic workup of patients with fatty liver disease, and treatment of the underlying disease should be started to halt disease progression. Common genetic variants in several genes involved in lipid handling and metabolism modulate the risk of progression from steatosis to fibrosis, cirrhosis and hepatocellular carcinoma development in NAFLD, alcohol-related liver disease and viral hepatitis. Hence, we speculate that genotyping of common risk variants for liver disease progression may be equally useful to gauge the likelihood of developing advanced liver disease in patients with secondary fatty liver disease.

      Keywords

      Linked Article

      Introduction

      Non-alcoholic fatty liver disease (NAFLD) has emerged as a leading cause of chronic liver disease worldwide with an estimated global prevalence of 25% in the adult population.
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      Attributable fractions of NAFLD for mortality in the United States: results from NHANES III with 27 Years of follow-up.
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      Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes.
      A further increase in NAFLD prevalence is expected as the prevalence of obesity, type 2 diabetes mellitus (T2DM), and other aspects of the metabolic syndrome continue to grow.
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      Non-alcoholic fatty liver disease–a global public health perspective.
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      In the United States the Third National Health and Nutrition Examination Survey (NHANES III) revealed that 8%, 36% and 38% of all-cause, liver disease-associated and diabetes-associated mortality, respectively, were attributable to NAFLD in 2015.
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      • Thistle J.E.
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      • McGlynn K.A.
      Attributable fractions of NAFLD for mortality in the United States: results from NHANES III with 27 Years of follow-up.
      Fatty liver disease due to overnutrition and the ensuing metabolic syndrome has emerged as the most common liver disease worldwide.
      Current EASL and AASLD practice recommendations require the exclusion of secondary/alternate causes of hepatic fat accumulation, including excessive alcohol consumption, prior to confirmation of a primary NAFLD diagnosis. While the vast majority of hepatic steatosis/steatohepatitis cases in adults are linked to excess nutrition and metabolic syndrome (primary NAFLD), recognition of less common, secondary/alternate causes underlying fatty liver disease is clinically highly relevant, since specific treatment options are both available and required for many of these secondary forms of fatty liver disease.
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      European Association for the Study of the L, European Association for the Study of D, European Association for the Study of O
      EASL-EASD-EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease.
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      Secondary causes of fatty liver disease–an update on pathogenesis, diagnosis and treatment strategies.
      In a minority of patients, fatty liver disease may result from or coexist with other conditions, termed secondary steatosis/steatohepatitis.
      Common secondary causes underlying, mimicking or worsening NAFLD/non-alcoholic steatohepatitis (NASH) in adults comprise chronic HCV infection, drugs and toxins, hypothyroidism, pregnancy-associated diseases and nutrition- or intestine-related disorders.
      European Association for the Study of the L, European Association for the Study of D, European Association for the Study of O
      EASL-EASD-EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease.
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      Distinguishing between a mere association and a causal relationship with secondary NAFLD is difficult for some of these conditions, especially since risk factors for primary NAFLD were not commonly reported in historical studies. In infants and children a number of rare, inherited metabolic disorders may underlie, aggravate or coexist with fatty liver disease.
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      Moreover, several frequent genetic variants are known to modulate the risk of development and progression of fatty liver, and thus act as "disease modifiers", i.e. protective or progression factors in both metabolic syndrome-associated and secondary fatty liver disease.
      • Keitel V.
      • Vom Dahl S.
      • Häussinger D.
      Secondary causes of fatty liver disease–an update on pathogenesis, diagnosis and treatment strategies.
      Risk factors for steatohepatitis can be grouped into 3 categories: i) conditions that trigger secondary NAFLD development, ii) conditions and factors that can aggravate primary NAFLD (co-factors) and iii) risk factors for primary NAFLD including risk factors for insulin resistance, obesity and metabolic syndrome. The latter are not discussed in this review.
      The aim of this review is to give an update on the secondary causes of fatty liver disease, to outline a diagnostic approach for patients with potential alternative causes of steatosis/steatohepatitis and to summarise available treatment options for these patients.
      Patients with secondary causes of fatty liver disease deserve particular attention since specific and efficacious treatment options may be available.

      Secondary causes of fatty liver disease

      Common secondary causes of fatty liver disease in adults include viral hepatitis, especially chronic HCV genotype 3 infection, various drugs and industrial chemicals.
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      Effect of antiviral treatment on evolution of liver steatosis in patients with chronic hepatitis C: indirect evidence of a role of hepatitis C virus genotype 3 in steatosis.
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      Relationship between steatosis, inflammation, and fibrosis in chronic hepatitis C: a meta-analysis of individual patient data.
      • Murata Y.
      • Ogawa Y.
      • Saibara T.
      • Nishioka A.
      • Fujiwara Y.
      • Fukumoto M.
      • et al.
      Unrecognized hepatic steatosis and non-alcoholic steatohepatitis in adjuvant tamoxifen for breast cancer patients.
      • Satapathy S.K.
      • Kuwajima V.
      • Nadelson J.
      • Atiq O.
      • Sanyal A.J.
      Drug-induced fatty liver disease: an overview of pathogenesis and management.
      • Bruno S.
      • Maisonneuve P.
      • Castellana P.
      • Rotmensz N.
      • Rossi S.
      • Maggioni M.
      • et al.
      Incidence and risk factors for non-alcoholic steatohepatitis: prospective study of 5408 women enrolled in Italian tamoxifen chemoprevention trial.
      • Schumacher J.D.
      • Guo G.L.
      Mechanistic review of drug-induced steatohepatitis.
      • Wahlang B.
      • Beier J.I.
      • Clair H.B.
      • Bellis-Jones H.J.
      • Falkner K.C.
      • McClain C.J.
      • et al.
      Toxicant-associated steatohepatitis.
      This review outlines the most frequent causes of secondary hepatic steatosis, as well as diagnostic strategies and treatment options.

      HCV-associated NAFLD

      NAFLD and chronic HCV are among the most common liver diseases. Therefore, it is not surprising that these conditions can coexist in the same individual. The presence of risk factors for primary NAFLD, such as insulin resistance and high BMI, were not commonly reported in historical studies on secondary causes of NAFLD. Thus, the distinction between mere associations and causal relationships may be difficult.
      However, transfection of the core protein of HCV genotype 3 into Huh7 cells led to an accumulation of lipids, resulting in the enlargement of lipid droplets.
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      Furthermore, overexpression of the HCV core protein in mouse livers inhibited the activity of the microsomal triglyceride transfer protein (MTTP) resulting in reduced VLDL assembly and secretion, and retention of triglycerides in hepatocytes.
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      Hepatitis C virus core protein inhibits microsomal triglyceride transfer protein activity and very low density lipoprotein secretion: a model of viral-related steatosis.
      MTTP mRNA levels were reduced in liver biopsy samples from HCV-infected individuals irrespective of HCV genotype, however MTTP activity was significantly reduced only in patients with HCV genotype 3.
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      • Gerotto M.
      • Dal Pero F.
      • Bortoletto G.
      • et al.
      Liver microsomal triglyceride transfer protein is involved in hepatitis C liver steatosis.
      Interestingly, successful antiviral therapy with sustained clearance of HCV genotype 3 was associated with an improvement in steatosis.
      • Castera L.
      • Hezode C.
      • Roudot-Thoraval F.
      • Lonjon I.
      • Zafrani E.S.
      • Pawlotsky J.M.
      • et al.
      Effect of antiviral treatment on evolution of liver steatosis in patients with chronic hepatitis C: indirect evidence of a role of hepatitis C virus genotype 3 in steatosis.
      While chronic HCV is associated with fatty liver disease irrespective of genotype, a causal relationship has been convincingly demonstrated for genotype 3.

      Environmental toxin- and drug-associated fatty liver disease

      Steatosis and steatohepatitis can be induced by a wide variety of environmental toxicants including metals (lead, arsenic, mercury, cadmium), herbicides (e.g. dioxine), pesticides/fungicides (e.g. triflumizole, fludioxonil), polychlorinated biphenyls and chloroalkenes (see Box 1 and Table 1).
      • Wahlang B.
      • Beier J.I.
      • Clair H.B.
      • Bellis-Jones H.J.
      • Falkner K.C.
      • McClain C.J.
      • et al.
      Toxicant-associated steatohepatitis.
      ,
      • Wahlang B.
      • Jin J.
      • Beier J.I.
      • Hardesty J.E.
      • Daly E.F.
      • Schnegelberger R.D.
      • et al.
      Mechanisms of environmental contributions to fatty liver disease.
      ,
      • Foulds C.E.
      • Trevino L.S.
      • York B.
      • Walker C.L.
      Endocrine-disrupting chemicals and fatty liver disease.
      The latter comprise perchloroethylene, trichloroethylene and vinyl chloride, which are widely used as degreasers and dry-cleaning fluids.
      • Wahlang B.
      • Beier J.I.
      • Clair H.B.
      • Bellis-Jones H.J.
      • Falkner K.C.
      • McClain C.J.
      • et al.
      Toxicant-associated steatohepatitis.
      ,
      • Wahlang B.
      • Jin J.
      • Beier J.I.
      • Hardesty J.E.
      • Daly E.F.
      • Schnegelberger R.D.
      • et al.
      Mechanisms of environmental contributions to fatty liver disease.
      These agents may act both as causative factors for secondary NAFLD development but may also worsen pre-existing primary NAFLD (e.g. as co-factors).
      Secondary causes of fatty liver disease.
      cART, combined antiretroviral therapy; HELLP, haemolysis, elevated liver enzymes, thrombocytopenia (low platelets); NAFLD, non-alcoholic fatty liver disease. ∗indicates reported progression of liver disease to fibrosis/cirrhosis; ∗∗ indicates reported progression to fibrosis/cirrhosis as well as hepatocellular carcinoma development. # indicates conditions which may cause secondary NAFLD, § indicates conditions which may aggravate primary NAFLD (act as co-factors for primary NAFLD). Adapted from.
      • Keitel V.
      • Vom Dahl S.
      • Häussinger D.
      Secondary causes of fatty liver disease–an update on pathogenesis, diagnosis and treatment strategies.
      Molecular mechanisms contributing to toxicant-associated steatosis or steatohepatitis (referred to as TASH) include disruption of endocrine metabolic regulation as well as chemical-nutrient interactions that may represent a "second hit" in patients with pre-existing metabolic abnormalities.
      • Wahlang B.
      • Jin J.
      • Beier J.I.
      • Hardesty J.E.
      • Daly E.F.
      • Schnegelberger R.D.
      • et al.
      Mechanisms of environmental contributions to fatty liver disease.
      Exogenous toxins and chemicals may interfere with any aspect of hormone action (so called “endocrine disrupting chemicals” or “metabolism disrupting chemicals”) resulting in the development of obesity, T2DM and fatty liver disease.
      • Heindel J.J.
      • Blumberg B.
      • Cave M.
      • Machtinger R.
      • Mantovani A.
      • Mendez M.A.
      • et al.
      Metabolism disrupting chemicals and metabolic disorders.
      ,
      • Gore A.C.
      • Chappell V.A.
      • Fenton S.E.
      • Flaws J.A.
      • Nadal A.
      • Prins G.S.
      • et al.
      EDC-2: the endocrine society's second scientific statement on endocrine-disrupting chemicals.
      They may also cause hormone-independent metabolic abnormalities, e.g. by altering hepatokine production (e.g. fibroblast-growth factor-21, insulin growth factor-1), or by directly interfering with lipid metabolism, e.g. through modulation of expression of adiponutrin (patatin-like phospholipase-containing A3 [PNPLA3]) or by interfering with nuclear hormone receptors involved in metabolic regulation (see Table 1).
      • Wahlang B.
      • Jin J.
      • Beier J.I.
      • Hardesty J.E.
      • Daly E.F.
      • Schnegelberger R.D.
      • et al.
      Mechanisms of environmental contributions to fatty liver disease.
      ,
      • Shi H.
      • Jan J.
      • Hardesty J.E.
      • Falkner K.C.
      • Prough R.A.
      • Balamurugan A.N.
      • et al.
      Polychlorinated biphenyl exposures differentially regulate hepatic metabolism and pancreatic function: implications for nonalcoholic steatohepatitis and diabetes.
      The latter include the peroxisome proliferator-activated receptors (PPARs), the constitutive androstane receptor (CAR), the pregnane X receptor (PXR), the farnesoid X receptor (FXR), the liver X receptor (LXR) as well as oestrogen (ER) and androgen receptors (AR).
      • Wahlang B.
      • Jin J.
      • Beier J.I.
      • Hardesty J.E.
      • Daly E.F.
      • Schnegelberger R.D.
      • et al.
      Mechanisms of environmental contributions to fatty liver disease.
      ,
      • Heindel J.J.
      • Blumberg B.
      • Cave M.
      • Machtinger R.
      • Mantovani A.
      • Mendez M.A.
      • et al.
      Metabolism disrupting chemicals and metabolic disorders.
      ,
      • Angrish M.M.
      • Kaiser J.P.
      • McQueen C.A.
      • Chorley B.N.
      Tipping the balance: hepatotoxicity and the 4 apical key events of hepatic steatosis.
      Interestingly, intrauterine or early childhood exposure to endocrine disrupting chemicals may favour obesity development and/or trigger alterations in the hepatic epigenome that predispose to NAFLD/NASH development.
      • Foulds C.E.
      • Trevino L.S.
      • York B.
      • Walker C.L.
      Endocrine-disrupting chemicals and fatty liver disease.
      Table 1Toxicant-associated fatty liver disease.
      Molecular targetMechanisms contributing to fatty liver diseaseExample of toxicant
      Hepatic nuclear hormone receptors:

      PPARs, PXR, CAR, LXR, FXR
      De novo lipogenesis

      Decreased fatty acid oxidation

      Decreased gluconeogenesis

      Hepatokine expression

      Immune-modulation
      Polyfluoroalkyl substances (PFOA, PFOS), PCBs, perchloroethylene, vinyl chloride
      Oestrogen and androgen receptors:

      ERα, ERβ, AR
      Endocrine metabolic disruptionPCBs
      Aryl hydrocarbon receptorIncreased expression of target genes

      Increased hepatic lipid uptake (CD36, FABP1)

      Inflammation and oxidative stress
      Dioxins, dioxin-like PCBs
      Hepatokines/enterokines:

      FGF21, GLP-1, IGF1
      Reduced FGF21 expression

      Reduced GLP-1

      Increased expression of IGF1
      PCBs
      Epidermal growth factor receptorReduced epidermal growth factor receptor signalling

      Reduced HNF4a expression
      PCBs, OCPs
      Energy metabolism, CREB, AMP, mTORGlycogen depletion, lipid accumulation

      Oxidative stress
      Dioxin
      Adiponutrin (PNPLA3)Decreased hepatic expression, but also altered by CAR (upregulation) and aryl hydrocarbon receptor (downregulation)PCBs
      Pancreatic atrophy and altered inflammation and gene expressionIncreased IL-6 expression

      Altered gene expression
      PCBs
      Adipose tissueIncreased differentiation of mesenchymal stem cells to adipocytes

      Increased adipocyte differentiation

      Increased adipocyte proliferation

      Increased fatty acid uptake
      Triflumizole, fludioxonil, tributyltin, dioxin, bisphenyl A, PCBs
      Different molecular targets as well as different mechanisms may contribute to toxicant-associated fatty liver disease. For details see text and also.
      • Wahlang B.
      • Jin J.
      • Beier J.I.
      • Hardesty J.E.
      • Daly E.F.
      • Schnegelberger R.D.
      • et al.
      Mechanisms of environmental contributions to fatty liver disease.
      • Foulds C.E.
      • Trevino L.S.
      • York B.
      • Walker C.L.
      Endocrine-disrupting chemicals and fatty liver disease.
      • Heindel J.J.
      • Blumberg B.
      • Cave M.
      • Machtinger R.
      • Mantovani A.
      • Mendez M.A.
      • et al.
      Metabolism disrupting chemicals and metabolic disorders.
      ,
      • Shi H.
      • Jan J.
      • Hardesty J.E.
      • Falkner K.C.
      • Prough R.A.
      • Balamurugan A.N.
      • et al.
      Polychlorinated biphenyl exposures differentially regulate hepatic metabolism and pancreatic function: implications for nonalcoholic steatohepatitis and diabetes.
      AR, androgen receptor; CAR, constitutive androstane receptor; CREB, cAMP response element-binding protein; ER, oestrogen receptor; FABP1, fatty acid binding protein 1; FGF21, fibroblast-growth factor-21; FXR, farnesoid X receptor; GLP-1, glucagon-like peptide 1; HNF4a, hepatocyte nuclear factor 4a; IGF1, insulin-like growth factor-1; LXR, liver X receptor; mTOR, mammalian target of rapamycin; OCPs, organochlorine pesticides; PCBs, polychlorinated biphenyls; PFOA, perfluorooctanoic acid; PFOS, perfluorooctane sulfonate; PPAR, peroxisome proliferator-activated receptor; PXR, pregnane X receptor.
      Drugs that have been linked to steatosis and steatohepatitis development include amiodarone, diltiazem, tamoxifen, methotrexate, corticosteroids, irinotecan, valproic acid, tetracycline, aspirin and different antiretrovirals (see Box 1).
      • Satapathy S.K.
      • Kuwajima V.
      • Nadelson J.
      • Atiq O.
      • Sanyal A.J.
      Drug-induced fatty liver disease: an overview of pathogenesis and management.
      ,
      • Schumacher J.D.
      • Guo G.L.
      Mechanistic review of drug-induced steatohepatitis.
      Like toxicants, drugs may cause steatohepatitis or aggravate pre-existing primary NAFLD.
      Cationic amphiphilic drugs (CADs) can induce hepatic triglyceride accumulation primarily through their toxicity on hepatocyte mitochondria, leading to inhibition of mitochondrial respiration, beta-oxidation and/or oxidative phosphorylation, or all of the above.
      • Satapathy S.K.
      • Kuwajima V.
      • Nadelson J.
      • Atiq O.
      • Sanyal A.J.
      Drug-induced fatty liver disease: an overview of pathogenesis and management.
      ,
      • Schumacher J.D.
      • Guo G.L.
      Mechanistic review of drug-induced steatohepatitis.
      This class of drugs comprises amiodarone, perhexiline, amitriptyline, propranolol, pirprofen, clozapine, irinotecan and tamoxifen.
      • Schumacher J.D.
      • Guo G.L.
      Mechanistic review of drug-induced steatohepatitis.
      Some of these drugs induce steatohepatitis through additional mechanisms beyond mitochondrial toxicity, e.g. disruption of phospholipid metabolism in lysosomes, prevention of lipid export from hepatocytes, reduced intestinal barrier function or increased fatty acid synthesis.
      • Schumacher J.D.
      • Guo G.L.
      Mechanistic review of drug-induced steatohepatitis.
      Two additional compounds, valproic acid and tetracycline, cause steatohepatitis via mechanisms that differ from those of CADs and chemotherapeutic agents.
      • Schumacher J.D.
      • Guo G.L.
      Mechanistic review of drug-induced steatohepatitis.
      Drugs may also contribute to metabolic changes resulting in obesity, steatosis or steatohepatitis through their impact on the gut microbiome,
      • Maier L.
      • Pruteanu M.
      • Kuhn M.
      • Zeller G.
      • Telzerow A.
      • Anderson E.E.
      • et al.
      Extensive impact of non-antibiotic drugs on human gut bacteria.
      but this complex and not yet completely understood interaction is beyond the scope of this review.
      The prevalence of drug-induced NAFLD varies; it is estimated that approximately 2% of all NAFLD cases are attributable to drug toxicity.
      • Satapathy S.K.
      • Kuwajima V.
      • Nadelson J.
      • Atiq O.
      • Sanyal A.J.
      Drug-induced fatty liver disease: an overview of pathogenesis and management.
      Adjuvant treatment with tamoxifen following resection of breast cancer induced NAFLD within 2 years in almost 40% of patients in a clinical trial setting,
      • Murata Y.
      • Ogawa Y.
      • Saibara T.
      • Nishioka A.
      • Fujiwara Y.
      • Fukumoto M.
      • et al.
      Unrecognized hepatic steatosis and non-alcoholic steatohepatitis in adjuvant tamoxifen for breast cancer patients.
      while another adjuvant trial for endometrial cancer reported that tamoxifen increased the risk of NAFLD in overweight patients (hazard ratio 2.0; 95% CI 1.1–3.5).
      • Bruno S.
      • Maisonneuve P.
      • Castellana P.
      • Rotmensz N.
      • Rossi S.
      • Maggioni M.
      • et al.
      Incidence and risk factors for non-alcoholic steatohepatitis: prospective study of 5408 women enrolled in Italian tamoxifen chemoprevention trial.
      Treatment with the chemotherapeutic agent irinotecan prior to resection of hepatic colorectal metastases increased the risk of steatohepatitis compared to no neoadjuvant chemotherapy (odds ratio [OR] 8.3; 95% CI 2.0–23.6).
      • Vauthey J.N.
      • Pawlik T.M.
      • Ribero D.
      • Wu T.T.
      • Zorzi D.
      • Hoff P.M.
      • et al.
      Chemotherapy regimen predicts steatohepatitis and an increase in 90-day mortality after surgery for hepatic colorectal metastases.
      For an overview of the molecular mechanisms by which individual drugs interfere with hepatocyte mitochondrial function and lipid metabolism see.
      • Satapathy S.K.
      • Kuwajima V.
      • Nadelson J.
      • Atiq O.
      • Sanyal A.J.
      Drug-induced fatty liver disease: an overview of pathogenesis and management.
      ,
      • Schumacher J.D.
      • Guo G.L.
      Mechanistic review of drug-induced steatohepatitis.
      While treatment with methotrexate, tamoxifen, corticosteroids and chemotherapeutic agents (such as irinotecan and oxaliplatin) may induce steatohepatitis, progression of steatosis/steatohepatitis towards fibrosis or cirrhosis is rarely observed in drug-induced fatty liver disease.
      • Satapathy S.K.
      • Kuwajima V.
      • Nadelson J.
      • Atiq O.
      • Sanyal A.J.
      Drug-induced fatty liver disease: an overview of pathogenesis and management.

      Gut-related causes of fatty liver disease

      Gut-related causes of fatty liver disease may result from malnutrition, increased intestinal permeability, small intestinal bacterial overgrowth (SIBO), changes in the gut microbiome, malabsorption and intestinal failure requiring parenteral nutrition (Box 1).
      • Miele L.
      • Valenza V.
      • La Torre G.
      • Montalto M.
      • Cammarota G.
      • Ricci R.
      • et al.
      Increased intestinal permeability and tight junction alterations in nonalcoholic fatty liver disease.
      • Albillos A.
      • de Gottardi A.
      • Rescigno M.
      The gut-liver axis in liver disease: pathophysiological basis for therapy.
      • Buchman A.L.
      • Naini B.V.
      • Spilker B.
      The differentiation of intestinal-failure-associated liver disease from nonalcoholic fatty liver and nonalcoholic steatohepatitis.
      These conditions are causative factors for secondary NAFLD since they may trigger steatosis development independently of insulin resistance and obesity.
      Celiac disease (CD), which is associated with SIBO and altered intestinal permeability, was detected in 3.4–7% of adult patients with NAFLD.
      • Bardella M.T.
      • Valenti L.
      • Pagliari C.
      • Peracchi M.
      • Fare M.
      • Fracanzani A.L.
      • et al.
      Searching for coeliac disease in patients with non-alcoholic fatty liver disease.
      ,
      • Kamal S.
      • Aldossari K.K.
      • Ghoraba D.
      • Abdelhakam S.M.
      • Kamal A.H.
      • Bedewi M.
      • et al.
      Clinicopathological and immunological characteristics and outcome of concomitant coeliac disease and non-alcoholic fatty liver disease in adults: a large prospective longitudinal study.
      In contrast, the hazard ratio (HR) for NAFLD within the first year of a CD diagnosis was 13.3 (95% CI 3.5–50.3), with an overall HR of 2.8 for the complete CD cohort (26,816 individuals), and an HR of 4.6 in children.
      • Reilly N.R.
      • Lebwohl B.
      • Hultcrantz R.
      • Green P.H.
      • Ludvigsson J.F.
      Increased risk of non-alcoholic fatty liver disease after diagnosis of celiac disease.
      Three common mechanisms link CD with SIBO: i) nutrient malabsorption, ii) intestinal barrier dysfunction with reduced tight junction integrity, and iii) bacterial overgrowth.
      • Freeman H.J.
      Hepatic manifestations of celiac disease.
      ,
      • Hoffmanova I.
      • Sanchez D.
      • Tuckova L.
      • Tlaskalova-Hogenova H.
      Celiac disease and liver disorders: from putative pathogenesis to clinical implications.
      Similar to patients with intestinal failure (see below). Patients with CD have lower serum levels of choline, which may predispose to steatosis development.
      • Buchman A.L.
      • Naini B.V.
      • Spilker B.
      The differentiation of intestinal-failure-associated liver disease from nonalcoholic fatty liver and nonalcoholic steatohepatitis.
      ,
      • Bertini I.
      • Calabro A.
      • De Carli V.
      • Luchinat C.
      • Nepi S.
      • Porfirio B.
      • et al.
      The metabonomic signature of celiac disease.
      Furthermore, increased intestinal permeability and dysbiosis with decreased bacterial beta diversity are not only observed in patients with CD but also commonly in those with NASH and ASH, where they may contribute to hepatic inflammation and steatosis development.
      • Hoffmanova I.
      • Sanchez D.
      • Tuckova L.
      • Tlaskalova-Hogenova H.
      Celiac disease and liver disorders: from putative pathogenesis to clinical implications.
      ,
      • Lang S.
      • Schnabl B.
      Microbiota and fatty liver disease-the known, the unknown, and the future.
      Thus, CD may predispose to fatty liver disease.
      Intestinal failure is characterised by an impaired ability to absorb sufficient nutrients and fluid, and the resulting requirement for intravenous supplementation.
      • Buchman A.L.
      • Naini B.V.
      • Spilker B.
      The differentiation of intestinal-failure-associated liver disease from nonalcoholic fatty liver and nonalcoholic steatohepatitis.
      Intestinal failure may occur in a variety of malabsorption disorders comprising short bowel syndrome, radiation enteritis, and refractory sprue.
      • Buchman A.L.
      • Naini B.V.
      • Spilker B.
      The differentiation of intestinal-failure-associated liver disease from nonalcoholic fatty liver and nonalcoholic steatohepatitis.
      Intestinal failure-associated liver disease (IFALD) is defined as an elevation of aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (GGT) and alkaline phosphatase (ALP) 1.5-fold above the upper limit of normal for more than 6 months in adults and 6 weeks in children.
      • Buchman A.L.
      • Naini B.V.
      • Spilker B.
      The differentiation of intestinal-failure-associated liver disease from nonalcoholic fatty liver and nonalcoholic steatohepatitis.
      ,
      • Beath S.
      • Pironi L.
      • Gabe S.
      • Horslen S.
      • Sudan D.
      • Mazeriegos G.
      • et al.
      Collaborative strategies to reduce mortality and morbidity in patients with chronic intestinal failure including those who are referred for small bowel transplantation.
      On liver histology, microvesicular or combined micro- and macrovesicular steatosis is found in about 65% of patients with IFALD.
      • Buchman A.L.
      • Naini B.V.
      • Spilker B.
      The differentiation of intestinal-failure-associated liver disease from nonalcoholic fatty liver and nonalcoholic steatohepatitis.
      Another hallmark of IFALD is the presence of biochemical and/or histological cholestasis.
      • Buchman A.L.
      • Naini B.V.
      • Spilker B.
      The differentiation of intestinal-failure-associated liver disease from nonalcoholic fatty liver and nonalcoholic steatohepatitis.
      Fibrosis and cirrhosis may also develop in IFALD. Patients with IFALD have reduced plasma-free choline concentrations, which may contribute to steatosis development by reducing phospholipid synthesis and transport.
      • Buchman A.L.
      • Naini B.V.
      • Spilker B.
      The differentiation of intestinal-failure-associated liver disease from nonalcoholic fatty liver and nonalcoholic steatohepatitis.
      This hypothesis is supported by the fact that steatosis and steatohepatitis are triggered in rodents by choline-deficient diets.
      • Buchman A.L.
      • Naini B.V.
      • Spilker B.
      The differentiation of intestinal-failure-associated liver disease from nonalcoholic fatty liver and nonalcoholic steatohepatitis.
      Thus, intestinal failure-associated fatty liver is related to long-term parenteral nutrition and is characterised by the presence of cholestasis, low plasma-free choline concentrations and either microvesicular or a combination of micro- and macrovesicular steatosis on histology.
      The historical bariatric surgery procedure of jejunoileal bypass conferred an increased risk of liver failure, severe steatohepatitis (6–26%) and elevated serum liver function tests (up to 40%).
      • Haines N.W.
      • Baker A.L.
      • Boyer J.L.
      • Glagov S.
      • Schneir H.
      • Jaspan J.
      • et al.
      Prognostic indicators of hepatic injury following jejunoileal bypass performed for refractory obesity: a prospective study.
      • Kneeman J.M.
      • Misdraji J.
      • Corey K.E.
      Secondary causes of nonalcoholic fatty liver disease.
      • Allard J.P.
      Other disease associations with Non-Alcoholic Fatty Liver Disease (NAFLD).
      In this procedure 25–30 cm of jejunum were anastomosed end-to-end to 25–30 cm of the terminal ileum, while the rest of the small intestine was anastomosed in an end-to-side procedure to the sigmoid colon.
      • Haines N.W.
      • Baker A.L.
      • Boyer J.L.
      • Glagov S.
      • Schneir H.
      • Jaspan J.
      • et al.
      Prognostic indicators of hepatic injury following jejunoileal bypass performed for refractory obesity: a prospective study.
      Therefore, this procedure mimicked intestinal failure and predisposed patients to fatty liver disease development.
      While bariatric surgical procedures such as gastric banding, sleeve gastrectomy, Roux-en-Y gastric bypass, and biliopancreatic diversion with duodenal switch successfully induce long-term weight loss and ameliorate NAFLD/NASH in the majority of cases,
      • Lassailly G.
      • Caiazzo R.
      • Buob D.
      • Pigeyre M.
      • Verkindt H.
      • Labreuche J.
      • et al.
      Bariatric surgery reduces features of nonalcoholic steatohepatitis in morbidly obese patients.
      • Mathurin P.
      • Hollebecque A.
      • Arnalsteen L.
      • Buob D.
      • Leteurtre E.
      • Caiazzo R.
      • et al.
      Prospective study of the long-term effects of bariatric surgery on liver injury in patients without advanced disease.
      • O'Brien P.E.
      • Hindle A.
      • Brennan L.
      • Skinner S.
      • Burton P.
      • Smith A.
      • et al.
      Long-term outcomes after bariatric surgery: a systematic review and meta-analysis of weight loss at 10 or more years for all bariatric procedures and a single-centre review of 20-year outcomes after adjustable gastric banding.
      severe hepatic dysfunction with features of aggressive NASH has been reported in rare cases.
      • Tsai J.H.
      • Ferrell L.D.
      • Tan V.
      • Yeh M.M.
      • Sarkar M.
      • Gill R.M.
      Aggressive non-alcoholic steatohepatitis following rapid weight loss and/or malnutrition.
      It was speculated that rapid fat mobilisation in obese patients may increase oxidative stress in hepatocytes and result in a NAFLD/NASH-like phenotype.
      • Tsai J.H.
      • Ferrell L.D.
      • Tan V.
      • Yeh M.M.
      • Sarkar M.
      • Gill R.M.
      Aggressive non-alcoholic steatohepatitis following rapid weight loss and/or malnutrition.
      Severe malnutrition in young children is characterised by hypoalbuminemia and the development of fatty liver disease. While the aetiology underlying malnutrition-associated fatty liver disease is not entirely clear, severe impairment of hepatic peroxisomal and mitochondrial function was observed in a low protein diet animal model.
      • van Zutphen T.
      • Ciapaite J.
      • Bloks V.W.
      • Ackereley C.
      • Gerding A.
      • Jurdzinski A.
      • et al.
      Malnutrition-associated liver steatosis and ATP depletion is caused by peroxisomal and mitochondrial dysfunction.
      Interestingly, administration of the PPAR-α agonist fenofibrate ameliorated hepatic steatosis and restored mitochondrial ATP synthesis.
      • van Zutphen T.
      • Ciapaite J.
      • Bloks V.W.
      • Ackereley C.
      • Gerding A.
      • Jurdzinski A.
      • et al.
      Malnutrition-associated liver steatosis and ATP depletion is caused by peroxisomal and mitochondrial dysfunction.
      Following complete pancreatectomy, approximately 37–50% of patients developed fatty liver disease.
      • Hashimoto D.
      • Chikamoto A.
      • Taki K.
      • Arima K.
      • Yamashita Y.
      • Ohmuraya M.
      • et al.
      Residual total pancreatectomy: short- and long-term outcomes.
      ,
      • Hata T.
      • Ishida M.
      • Motoi F.
      • Sakata N.
      • Yoshimatsu G.
      • Naitoh T.
      • et al.
      Clinical characteristics and risk factors for the development of postoperative hepatic steatosis after total pancreatectomy.
      The development of steatosis correlated with poor postoperative nutritional status, more severe weight loss and inadequate pancreatic enzyme replacement therapy.
      • Hata T.
      • Ishida M.
      • Motoi F.
      • Sakata N.
      • Yoshimatsu G.
      • Naitoh T.
      • et al.
      Clinical characteristics and risk factors for the development of postoperative hepatic steatosis after total pancreatectomy.
      Thus, malabsorption and malnutrition appear to be the main contributors to fatty liver disease development in these patients. While earlier case series of pancreatectomised patients reported a progression of fatty liver disease to fibrosis and cirrhosis, this has not been observed in more recent publications and systematic reviews, and steatosis seems to improve with adequate pancreatic enzyme replacement therapy.
      • Hata T.
      • Ishida M.
      • Motoi F.
      • Sakata N.
      • Yoshimatsu G.
      • Naitoh T.
      • et al.
      Clinical characteristics and risk factors for the development of postoperative hepatic steatosis after total pancreatectomy.
      • Barbier L.
      • Jamal W.
      • Dokmak S.
      • Aussilhou B.
      • Corcos O.
      • Ruszniewski P.
      • et al.
      Impact of total pancreatectomy: short- and long-term assessment.
      • Scholten L.
      • Stoop T.F.
      • Del Chiaro M.
      • Busch O.R.
      • van Eijck C.
      • Molenaar I.Q.
      • et al.
      Systematic review of functional outcome and quality of life after total pancreatectomy.
      • Suzuki S.
      • Miura J.
      • Shimizu K.
      • Tokushige K.
      • Uchigata Y.
      • Yamamoto M.
      Clinicophysiological outcomes after total pancreatectomy.
      Hepatic complications of anorexia nervosa are mainly due to severe malnutrition and present as mild to moderate elevations of serum ALT and AST (found in up to 50% of cases).
      • Rosen E.
      • Bakshi N.
      • Watters A.
      • Rosen H.R.
      • Mehler P.S.
      Hepatic complications of anorexia nervosa.
      A recent case series of 34 patients with anorexia nervosa identified mild steatosis on ultrasound in 16 patients, but significant steatosis was confirmed by MRI in only 1 patient, indicating that NAFLD is rare in this condition.
      • Fanin A.
      • Miele L.
      • Bertolini E.
      • Giorgini A.
      • Pontiroli A.E.
      • Benetti A.
      Liver alterations in anorexia nervosa are not caused by insulin resistance.
      In contrast, refeeding of these patients leads to an increased risk of steatosis development and needs to be carefully monitored.
      • Rosen E.
      • Bakshi N.
      • Watters A.
      • Rosen H.R.
      • Mehler P.S.
      Hepatic complications of anorexia nervosa.
      ,
      • Sakada M.
      • Tanaka A.
      • Ohta D.
      • Takayanagi M.
      • Kodama T.
      • Suzuki K.
      • et al.
      Severe steatosis resulted from anorexia nervosa leading to fatal hepatic failure.
      Psoriasis has been linked to an increased risk of metabolic syndrome (MetS) and NAFLD. A meta-analysis of 6 studies comprising 267,761 participants reported an increased OR for NAFLD development in psoriatic patients compared to non-psoriatic controls (OR 2.15; 95% CI 1.57–2.94).
      • Candia R.
      • Ruiz A.
      • Torres-Robles R.
      • Chavez-Tapia N.
      • Mendez-Sanchez N.
      • Arrese M.
      Risk of non-alcoholic fatty liver disease in patients with psoriasis: a systematic review and meta-analysis.
      The risk of concurrent NAFLD was significantly greater in patients with psoriatic arthritis (OR 2.25; 95% CI 1.37–3.71; 3 studies; 505 participants) and in patients with moderate to severe psoriasis (OR 2.07; 95% CI 1.59–2.71) compared to those with mild disease.
      • Candia R.
      • Ruiz A.
      • Torres-Robles R.
      • Chavez-Tapia N.
      • Mendez-Sanchez N.
      • Arrese M.
      Risk of non-alcoholic fatty liver disease in patients with psoriasis: a systematic review and meta-analysis.
      A small study in 76 patients with psoriasis and psoriatic arthritis revealed an increased prevalence of NAFLD in the psoriasis group compared to the psoriatic arthritis subgroup (33% vs. 15%, p = 0.044). MetS was present in up to 35%, and the prevalence of liver fibrosis, defined as liver stiffness ≥7.0 kPa on transient elastography, was up to 28% in both groups.
      • Ortolan A.
      • Lorenzin M.
      • Tadiotto G.
      • Russo F.P.
      • Oliviero F.
      • Felicetti M.
      • et al.
      Metabolic syndrome, non-alcoholic fatty liver disease and liver stiffness in psoriatic arthritis and psoriasis patients.
      Novel data indicate that chronic inflammation and elevated serum levels of high-sensitivity C-reactive protein in patients with psoriasis are associated with increased visceral adiposity.
      • Sajja A.
      • Abdelrahman K.M.
      • Reddy A.S.
      • Dey A.K.
      • Uceda D.E.
      • Lateef S.S.
      • et al.
      Chronic inflammation in psoriasis promotes visceral adiposity associated with noncalcified coronary burden over time.
      Furthermore, induction of a psoriasis-like skin condition in mice triggered skin and systemic inflammation, resulting in a pre-diabetic phenotype and metabolic dysfunction.
      • Evans E.A.
      • Sayers S.R.
      • Kodji X.
      • Xia Y.
      • Shaikh M.
      • Rizvi A.
      • et al.
      Psoriatic skin inflammation induces a pre-diabetic phenotype via the endocrine actions of skin secretome.
      These data suggest that psoriasis may act as a risk factor for primary NAFLD but may also represent a co-factor which may worsen primary NAFLD. As discussed, drug therapy (e.g. methotrexate) may also contribute to steatosis development in these patients.

      Endocrine disorders that predispose individuals to fatty liver disease

      Endocrine disorders (other than MetS/T2DM) that predispose to NAFLD development include hypopituitarism, growth hormone (GH) deficiency as well as hypothyroidism.
      • Lonardo A.
      • Mantovani A.
      • Lugari S.
      • Targher G.
      NAFLD in some common endocrine diseases: prevalence, pathophysiology, and principles of diagnosis and management.
      • Cotter T.G.
      • Rinella M.
      Nonalcoholic fatty liver disease 2020: the state of the disease.
      • Ferrandino G.
      • Kaspari R.R.
      • Spadaro O.
      • Reyna-Neyra A.
      • Perry R.J.
      • Cardone R.
      • et al.
      Pathogenesis of hypothyroidism-induced NAFLD is driven by intra- and extrahepatic mechanisms.
      In a single centre analysis of 879 patients with panhypopituitarism, hypothalamic obesity or craniopharyngioma, 21 (2.3%) patients had liver histology or imaging findings of NAFLD in the absence of other liver diseases or alcohol consumption exceeding 14 units/week (=140 g/week).
      • Adams L.A.
      • Feldstein A.
      • Lindor K.D.
      • Angulo P.
      Nonalcoholic fatty liver disease among patients with hypothalamic and pituitary dysfunction.
      These 21 patients had hypopituitarism of mixed aetiology (mainly brain tumours but also idiopathic hypopituitarism), showed rapid development of NAFLD (on average after 6.4±7.5 years), which was highly progressive, with 60% of biopsied patients already having cirrhosis.
      • Adams L.A.
      • Feldstein A.
      • Lindor K.D.
      • Angulo P.
      Nonalcoholic fatty liver disease among patients with hypothalamic and pituitary dysfunction.
      Over the follow-up period, 2 patients received a liver transplantation and 2 died of liver-related complications.
      • Adams L.A.
      • Feldstein A.
      • Lindor K.D.
      • Angulo P.
      Nonalcoholic fatty liver disease among patients with hypothalamic and pituitary dysfunction.
      Following the diagnosis of pituitary/hypothalamic disease, most patients gained weight, 62% developed diabetes or glucose intolerance, 67% had hypertriglyceridaemia and 33% hypercholesterolaemia.
      • Adams L.A.
      • Feldstein A.
      • Lindor K.D.
      • Angulo P.
      Nonalcoholic fatty liver disease among patients with hypothalamic and pituitary dysfunction.
      The metabolic changes associated with hypopituitarism are most likely a consequence of GH deficiency.
      • Adams L.A.
      • Feldstein A.
      • Lindor K.D.
      • Angulo P.
      Nonalcoholic fatty liver disease among patients with hypothalamic and pituitary dysfunction.
      It is noteworthy that 19 of the 21 patients were on corticosteroid supplementation therapy, which may have contributed to NAFLD development.
      • Adams L.A.
      • Feldstein A.
      • Lindor K.D.
      • Angulo P.
      Nonalcoholic fatty liver disease among patients with hypothalamic and pituitary dysfunction.
      Similar cases of rapidly progressive NAFLD following hypothalamic and pituitary dysfunctions have been reported in both children and young adults.
      • Terawaki Y.
      • Murase K.
      • Motonaga R.
      • Tanabe M.
      • Nomiyama T.
      • Shakado S.
      • et al.
      A probable case of burn-out NASH caused by panhypopituitarism secondary to craniopharyngioma.
      ,
      • Altuntas B.
      • Ozcakar B.
      • Bideci A.
      • Cinaz P.
      Cirrhotic outcome in patients with craniopharyngioma.
      In a Japanese study of patients with hypothalamic/pituitary dysfunction and GH deficiency, 77% of patients had NAFLD compared to 12% in age-, sex- and BMI-matched controls.
      • Nishizawa H.
      • Iguchi G.
      • Murawaki A.
      • Fukuoka H.
      • Hayashi Y.
      • Kaji H.
      • et al.
      Nonalcoholic fatty liver disease in adult hypopituitary patients with GH deficiency and the impact of GH replacement therapy.
      GH replacement therapy over 6–12 months significantly improved AST, ALT and GGT serum values as well as histological steatosis and fibrosis scores.
      • Nishizawa H.
      • Iguchi G.
      • Murawaki A.
      • Fukuoka H.
      • Hayashi Y.
      • Kaji H.
      • et al.
      Nonalcoholic fatty liver disease in adult hypopituitary patients with GH deficiency and the impact of GH replacement therapy.
      Thus, GH deficiency may underlie secondary NAFLD. Subcutaneous administration of synthetic growth hormone-releasing hormone (tesamorelin, 2 mg) over 12 months significantly reduced hepatic fat content as measured by magnetic resonance spectroscopy in patients with HIV in a phase II trial (NCT NCT02196831).
      • Stanley T.L.
      • Fourman L.T.
      • Feldpausch M.N.
      • Purdy J.
      • Zheng I.
      • Pan C.S.
      • et al.
      Effects of tesamorelin on non-alcoholic fatty liver disease in HIV: a randomised, double-blind, multicentre trial.
      Overt and subclinical hypothyroidism have been linked to NAFLD in a number of epidemiological studies and are often associated with features of the metabolic syndrome including obesity, dyslipidaemia and insulin resistance, which are at least partly reversible by levothyroxine replacement therapy.
      • Lonardo A.
      • Ballestri S.
      • Mantovani A.
      • Nascimbeni F.
      • Lugari S.
      • Targher G.
      Pathogenesis of hypothyroidism-induced NAFLD: evidence for a distinct disease entity? Digestive and liver disease.
      • Liangpunsakul S.
      • Chalasani N.
      Is hypothyroidism a risk factor for non-alcoholic steatohepatitis?.
      • Chung G.E.
      • Kim D.
      • Kim W.
      • Yim J.Y.
      • Park M.J.
      • Kim Y.J.
      • et al.
      Non-alcoholic fatty liver disease across the spectrum of hypothyroidism.
      • Xu L.
      • Ma H.
      • Miao M.
      • Li Y.
      Impact of subclinical hypothyroidism on the development of non-alcoholic fatty liver disease: a prospective case-control study.
      Interestingly, selective agonists for the thyroid hormone receptor (THR)-β are currently being evaluated for the treatment of NASH (e.g. phase III trial NCT04197479 [resmetirom]; phase II trial NCT02927184 [VK2809]), and a THR-β agonist (resmetirom) effectively reduced hepatic fat content after 12 and 36 weeks of treatment in a phase II trial.
      • Harrison S.A.
      • Bashir M.R.
      • Guy C.D.
      • Zhou R.
      • Moylan C.A.
      • Frias J.P.
      • et al.
      Resmetirom (MGL-3196) for the treatment of non-alcoholic steatohepatitis: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial.
      Mechanisms contributing to NAFLD development in primary hypothyroidism comprise direct effects of thyroid stimulating hormone (TSH) on hepatic triglyceride metabolism, as well as impaired glucose sensing by pancreatic beta-cells in the presence of reduced levels of thyroid hormones (TH), resulting in reduced insulin secretion and de-repression of lipolysis in adipose tissue, which in turn increases shuttling of free fatty acids to the liver.
      • Ferrandino G.
      • Kaspari R.R.
      • Spadaro O.
      • Reyna-Neyra A.
      • Perry R.J.
      • Cardone R.
      • et al.
      Pathogenesis of hypothyroidism-induced NAFLD is driven by intra- and extrahepatic mechanisms.
      ,
      • Lonardo A.
      • Ballestri S.
      • Mantovani A.
      • Nascimbeni F.
      • Lugari S.
      • Targher G.
      Pathogenesis of hypothyroidism-induced NAFLD: evidence for a distinct disease entity? Digestive and liver disease.
      Administration of methimazole (MMI), an antithyroid drug used in Grave’s disease, induced a subclinical hypothyroidism with unaltered TH levels despite elevated TSH levels. MMI-treated mice developed hepatic steatosis despite unaltered physical activity, respiratory quotient (as a measure of energy consumption) and food intake.
      • Zhou L.
      • Ding S.
      • Li Y.
      • Wang L.
      • Chen W.
      • Bo T.
      • et al.
      Endoplasmic reticulum stress may play a pivotal role in lipid metabolic disorders in a novel mouse model of subclinical hypothyroidism.
      MMI therapy in humans may trigger acute hepatotoxicity, commonly presenting with cholestasis, but has also been associated with steatosis. A recent analysis of serum metabolic changes in patients with MMI-induced liver toxicity revealed elevated serum fatty acid levels. In contrast, intermediates of the tricarboxylic acid cycle, of the pentose phosphate pathway and several amino acids including methionine were reduced, suggesting that MMI and MMI-associated changes in TH and TSH levels may also trigger metabolic changes in humans.
      • Li X.
      • Yang J.
      • Jin S.
      • Dai Y.
      • Fan Y.
      • Fan X.
      • et al.
      Mechanistic examination of methimazole-induced hepatotoxicity in patients with Grave's disease: a metabolomic approach.
      Interestingly, a diet deficient in methionine and choline is used in rodents to induce NASH independently of obesity, insulin resistance and dyslipidaemia.
      • Larter C.Z.
      • Yeh M.M.
      Animal models of NASH: getting both pathology and metabolic context right.
      These data indicate that subclinical and overt hypothyroidism may cause secondary NAFLD but may also worsen primary NAFLD and even act as risk factor for primary NAFLD.
      We conclude that in the absence of metabolic risk factors, secondary causes of hepatic steatosis should be considered and excluded, particularly in paediatric and lean individuals.
      Polycystic ovary syndrome (PCOS) is strongly associated with insulin resistance and metabolic syndrome; thus, it is not surprising that NAFLD is a common finding in women with PCOS.
      • Cerda C.
      • Perez-Ayuso R.M.
      • Riquelme A.
      • Soza A.
      • Villaseca P.
      • Sir-Petermann T.
      • et al.
      Nonalcoholic fatty liver disease in women with polycystic ovary syndrome.
      A recent meta-analysis of 17 studies including 2,734 patients with PCOS and 2,561 controls of similar age and BMI, found an OR for NAFLD of 2.54 in patients with PCOS compared to controls.
      • Rocha A.L.L.
      • Faria L.C.
      • Guimaraes T.C.M.
      • Moreira G.V.
      • Candido A.L.
      • Couto C.A.
      • et al.
      Non-alcoholic fatty liver disease in women with polycystic ovary syndrome: systematic review and meta-analysis.
      The increased prevalence of NAFLD in young patients with PCOS, even in the absence of obesity and metabolic syndrome, underscores the relevance of further factors, such as hyperandrogenism in disease development.
      • Rocha A.L.L.
      • Faria L.C.
      • Guimaraes T.C.M.
      • Moreira G.V.
      • Candido A.L.
      • Couto C.A.
      • et al.
      Non-alcoholic fatty liver disease in women with polycystic ovary syndrome: systematic review and meta-analysis.
      ,
      • Jones H.
      • Sprung V.S.
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      • Aziz N.
      • et al.
      Polycystic ovary syndrome with hyperandrogenism is characterized by an increased risk of hepatic steatosis compared to nonhyperandrogenic PCOS phenotypes and healthy controls, independent of obesity and insulin resistance.
      Indeed, hyperandrogenism and higher serum testosterone values were significantly associated with NAFLD in patients with PCOS, independent of the presence of insulin resistance.
      • Rocha A.L.L.
      • Faria L.C.
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      • Moreira G.V.
      • Candido A.L.
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      • et al.
      Non-alcoholic fatty liver disease in women with polycystic ovary syndrome: systematic review and meta-analysis.
      While PCOS-associated insulin resistance may trigger primary NAFLD, hyperandrogenism represents an independent risk factor for steatosis development in females and may thus cause secondary NAFLD.
      In contrast, male hypogonadism (characterised by low testosterone levels) has been associated with an increased risk of NAFLD, independently of BMI, insulin resistance and T2DM in a number of epidemiological studies.
      • Kim S.
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      • Cho B.
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      • et al.
      A low level of serum total testosterone is independently associated with nonalcoholic fatty liver disease.
      ,
      • Yim J.Y.
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      • Kim D.
      • Ahmed A.
      Serum testosterone and non-alcoholic fatty liver disease in men and women in the US.
      This is in line with animal studies, where a high-fat diet (HFD) increased hepatic steatosis in castrated rats despite lower overall weight gain compared to HFD-fed controls.
      • Nikolaenko L.
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      • et al.
      Testosterone replacement ameliorates nonalcoholic fatty liver disease in castrated male rats.
      Supplementation of testosterone ameliorated HFD-induced steatosis in the castrated rodents.
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      • et al.
      Testosterone replacement ameliorates nonalcoholic fatty liver disease in castrated male rats.
      ,
      • Senmaru T.
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      • et al.
      Testosterone deficiency induces markedly decreased serum triglycerides, increased small dense LDL, and hepatic steatosis mediated by dysregulation of lipid assembly and secretion in mice fed a high-fat diet.
      Changes in the abundance and composition of the intestinal microbiome, as well as in hepatic lipid assembly and secretion, may contribute to steatosis in HFD-fed castrated rodents.
      • Senmaru T.
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      • et al.
      Testosterone deficiency induces markedly decreased serum triglycerides, increased small dense LDL, and hepatic steatosis mediated by dysregulation of lipid assembly and secretion in mice fed a high-fat diet.
      ,
      • Harada N.
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      • et al.
      Castration influences intestinal microflora and induces abdominal obesity in high-fat diet-fed mice.
      Thus, female androgen excess (e.g. PCOS) and male androgen deficiency (e.g. hypogonadal males) promote overlapping metabolic phenotypes including NAFLD.
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      • O'Reilly M.W.
      Mechanisms in endocrinology: the sexually dimorphic role of androgens in human metabolic disease.
      Both conditions may constitute secondary NAFLD but may also act as co-factors aggravating primary NAFLD.

      Hereditary diseases associated with fatty liver disease

      A heterogeneous group of genetic diseases may predispose individuals, especially children and young adults, to the development of fatty liver disease (Box 1, Fig. 2, Table 2). A more extensive overview of the secondary causes of fatty liver disease in infants and children is provided in.
      • Hegarty R.
      • Deheragoda M.
      • Fitzpatrick E.
      • Dhawan A.
      Paediatric Fatty Liver Disease (PeFLD): all is not NAFLD–pathophysiological insights and approach to management.
      Figure thumbnail gr1
      Fig. 1Histopathology of metabolic syndrome-associated NAFLD and secondary causes of NAFLD.
      H&E staining of liver tissue obtained by percutaneous ultrasound-guided biopsy. ASH, alcoholic steatohepatitis; A1AT, alpha-1 antitrypsin deficiency; GSD, glycogen storage disease type III; GT, genotype; LAL-D, lysosomal acid lipase deficiency; NAFLD, non-alcoholic fatty liver disease. Images are shown in 20x magnification.
      Figure thumbnail gr2
      Fig. 2Pathways affected by toxicants, congenital diseases, and NAFLD-associated genetic risk variants and their contribution to hepatic triglyceride accumulation.
      Variants in the PNPLA3, MBOAT7, GCKR and TM6SF2 genes promote hepatic lipid accumulation. In contrast, variants in HSD17B13 and MARC1 protect from steatosis development. Variants in ApoB and MTTP underlie abetalipoproteinaemia and familial hypobetalipoproteinaemia. ACAD9, acyl-CoA dehydrogenase 9; AhR, aryl hydrocarbon receptor; ApoB, apolipoprotein B; AR, androgen receptor; BPA, bisphenol A; CACT, carnitine-acylcarnitine translocase; CAR, constitutive androstane receptor; CPS1, carbamoyl phosphate synthetase-1; CPT1, carnitine palmitoyltransferase 1; EGFR, epidermal growth factor receptor; ER, oestrogen receptor; FA, fatty acid; FABP1, fatty acid binding protein 1; FGF21, fibroblast-growth factor-21; FXR, farnesoid X receptor; GCKR, glucokinase regulatory protein; GLP-1, glucagon-like peptide-1; HNF4a, hepatocyte nuclear factor 4a; HSD17B13, hydroxysteroid 17-beta dehydrogenase-13; IGF1, insulin-like growth factor-1; IMM, inner mitochondrial membrane; LDLR, LDL receptor; LXR, liver X receptor; MARC1, mitochondrial amidoxime reducing component-1; MBOAT7, membrane-bound O-acyltransferase domain-containing-7; MCAD, medium chain acyl-coA dehydrogenase; mTOR, mammalian target of rapamycin; MTTP, microsomal triglyceride transfer protein; NAFLD, non-alcoholic fatty liver disease; OCPs, organochlorine pesticides; OMM, outer mitochondrial membrane; OTC, ornithine transcarbamylase; PCBs, polychlorinated biphenyls; PCE, percloroethene; PFOA, perfluorooctanoic acid; PFOS, perfluorooctane sulfonate; PNPLA3, patatin-like phospholipase domain-containing A3; TCA, tricarboxylic acid; TM6SF2, transmembrane 6 superfamily member-2; VC, vinyl chloride. Modified from
      • Liebe Roman
      • Keitel-Anselmino Verena
      Genetisches Risiko bei metabolischer Fettlebererkrankung.
      10.1007/s11428-020-00647-2 (Liebe & Keitel, Diabetologe).
      Table 2Prevalence of congenital diseases associated with secondary NAFLD (#) and/or acting as risk factor aggravating primary NAFLD (§).
      Disease

      Main causative variant; rs-number;
      Prevalence/GnomAD risk allele frequency in Europeans
      Haemochromatosis (§)

      HFE p.C282Y; rs1800562
      MAF: 0.0577

      = 6% of the population
      Alpha-1 antitrypsin deficiency (#, §)

      SERPINA1 p.E366K; rs28929474
      MAF: 0.0184

      = 2% of the population
      Wilsons disease (#)

      ATP7B p.H1069Q; rs76151636 (>300 variants known)
      Prevalence 1:30,000

      MAF= 0.001524
      Congenital lipodystrophy (#) various genes (AGPAT2, BSCL2, LMNA, PPARγ)Prevalence 1:106
      Abetalipoproteinaemia (#)

      MTTP p.G865ter; rs146064714
      Prevalence <1:106

      MAF Europeans: 0.00002638

      MAF Ashkenazi: 0.002679
      Hypobetalipoproteinaemia (#)

      APOB, PCSK9, ANGPTL3
      FHBL is estimated to occur in 1 in 1,000 to 3,000 individuals
      Familial hyperlipidaemia (#)

      LDLR, PCSK9, APOB
      Familial hypercholesterinaemia

      1:160,000-300,000 for homozygous familial hyperlipidaemia
      Lysosomal acid lipase deficiency (#)

      (Wolman disease or cholesteryl ester storage disease)

      LIPA c.894G>A, p.N298N, splice defect causing p.S275_Y298del; rs116928232)
      1 in 40,000 to 1 in 300,000 depending on ethnicity and geographical location

      MAF Europeans: 0.001295
      Glycogen storage diseases (#)Currently 15 subtypes

      Prevalence varies by population: British Columbia 1 in 43,000; US 1 in 20,000–25,000 births; Netherlands 1 in 40,000 births
      Hereditary fructose intolerance (#)

      ALDOB p.A150P; rs1800546 (historically denoted as p.A149P)
      Approximately 1.3% of neonates harbour one copy of the prevalent p.A149P disease allele

      MAF Europeans: 0.004866
      Urea cycle disorders (#)

      Ornithine transcarbamylase deficiency

      Carbamoylphosphate synthetase I deficiency

      Lysinuric protein intolerance
      The incidence of the urea cycle disorders is estimated at 1 in 8,000 to 1 in 44,000 births.
      Citrin deficiency (#)

      Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD)

      Adult onset: citrullinaemia type 2 (CTLN2)
      Prevalence:

      NICCD 1:17,000

      CTLN2 1:100,000-1:200,000 higher disease allele frequencies, but probably incomplete penetrance
      For references refer to text and for prevalence data to.
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      FHBL, familial hypobetalipoproteinaemia; MAF, minor allele frequency.
      Inborn errors of lipid metabolism, such as congenital lipodystrophy, abetalipoproteinaemia (ABL), familial hypobetalipoproteinaemia (FHBL), familial hyperlipidaemia/hypercholesterolaemia as well as lysosomal acid lipase deficiency (LAL-D, previously termed cholesteryl ester storage disease in adults/Wolman’s disease in infancy) may underlie fatty liver disease.
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      Hepatic steatosis was diagnosed using ultrasound in all individuals with ABL due to genetic variants in the MTTP gene, which encodes an apolipoprotein B lipid-loading and chaperone protein, resulting in defective chylomicron and VLDL secretion. In FHBL, steatosis was found in more than 50% of affected individuals, while its prevalence reached 100% in patients with LAL-D.
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      On liver histology, LAL-D presents predominately as microvesicular steatosis (Fig. 1), which is uncommon in other metabolic causes of liver disease except fatty acid oxidation disorders and urea cycle disorders (UCDs).
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      LAL-D results from pathogenic variants in the lipase A (LIPA) gene, which have an estimated minor allele frequency (MAF) of 0.0018–0.0024 and are characterised by a residual enzyme activity of <1% or 1–5% in the paediatric and adult phenotype, respectively.
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      The most common pathogenic LIPA variant, accounting for approximately 60% of LAL-D variants, is the splice junction variant c.894G>A, resulting in an in-frame deletion of 72 base pairs (p.S275_Y298del; rs116928232, MAF = 0.0013).
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      A splice junction mutation causes deletion of a 72-base exon from the mRNA for lysosomal acid lipase in a patient with cholesteryl ester storage disease.
      UCDs (cumulative incidence about 1:8,000) have been described as both cause and consequence of fatty liver disease, with microvesicular steatosis being a common finding on liver histology.
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      Presentation in the neonatal period with acute liver failure has been described for the most common, X-chromosome linked ornithine transcarbamylase (OTC) deficiency as well as the autosomal recessive carbamoylphosphate synthetase I (CPSI) deficiency.
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      Due to the X-chromosomal transmission, the clinical presentation of OTC deficiency is highly variable; in patients who present in adulthood, liver histology can be characterised by signs of portal-to-portal bridging fibrosis and enlarged glycogen-positive hepatocytes, resembling glycogen storage disorders (GSDs).
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      Hepatocellular carcinoma (HCC) development, even in the absence of cirrhosis, has been described for various UCDs.
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      While citrin deficiency is an uncommon UCD in Europe and North America, the prevalence of NICCD in Japan is about 1:17,000, whereas that of CTLN2 is 1:100,000–1:200,000. Fatty liver disease is a common finding in patients with CTLN2 (prevalence 89%), despite a low BMI (median BMI was only 18.3 kg/m2).
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      Similar to reports in other UCDs, HCC development has been described in patients with citrin deficiency.
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      Malfunction in mitochondrial beta-oxidation contributes to lipid accumulation in hepatocyte-like cells derived from citrin deficiency-induced pluripotent stem cells.
      In vitro analysis of pluripotent stem cells derived from the fibroblasts of a citrin-deficient patient suggested that the observed lipid accumulation is due to aberrant mitochondrial β-oxidation, which could be partially reversed by a PPAR-α agonist in this model.
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      Interestingly, metabolic NAFLD/NASH may cause urea cycle disturbances via hypermethylation of OTC and CPSI promoter regions, subsequent suppression of OTC and CPSI mRNA and protein expression, and thus enzyme function, leading to increased serum ammonia levels.
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      Other genetic diseases associated with hepatic triglyceride accumulation include Wilson’s disease, haemochromatosis, alpha-1 antitrypsin (A1AT) deficiency, hereditary fructose intolerance, GSDs (especially types I and III, but also types IV, VI, IX, XI) and fatty acid oxidation disorders (Table 2).
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      • Ellervik C.
      • Birgens H.
      • Tybjaerg-Hansen A.
      • Nordestgaard B.G.
      Hemochromatosis genotypes and risk of 31 disease endpoints: meta-analyses including 66,000 cases and 226,000 controls.
      ,
      • Krawczyk M.
      • Liebe R.
      • Lammert F.
      Toward genetic prediction of nonalcoholic fatty liver disease trajectories: PNPLA3 and beyond.
      In earlier studies, heterozygosity for p.C282Y was associated with advanced fibrosis in patients with NASH, indicating that the presence of p.C282Y may predispose to disease progression rather than the development of fatty liver disease per se.
      • Nelson J.E.
      • Bhattacharya R.
      • Lindor K.D.
      • Chalasani N.
      • Raaka S.
      • Heathcote E.J.
      • et al.
      HFE C282Y mutations are associated with advanced hepatic fibrosis in Caucasians with nonalcoholic steatohepatitis.
      Interestingly, the NAFLD-associated genetic variants in the PNPLA3 (rs738409) and transmembrane 6 superfamily member-2 (TM6SF2, rs58542926) genes significantly increased the risk of cirrhosis development in homozygous carriers of p.C282Y, with a population attributable fraction of 13.8% for PNPLA3 and 6.8% for TM6SF2, respectively.
      • Buch S.
      • Sharma A.
      • Ryan E.
      • Datz C.
      • Griffiths W.
      • Way M.
      • et al.
      Variants in PCSK7, PNPLA3and TM6SF2 are risk factors for the development of cirrhosis in people with hereditary haemochromatosis.
      This finding suggests that all 3 common variants are determinants of liver disease progression and act as co-factors for primary NAFLD.
      Pathogenic variants in the SERPINA1 gene underlie A1AT deficiency, characterised by lung emphysema (due to loss of enzymatic activity) and liver disease (following hepatic accumulation of the misfolded mutant protein).
      • Gooptu B.
      • Dickens J.A.
      • Lomas D.A.
      The molecular and cellular pathology of alpha(1)-antitrypsin deficiency.
      ,
      • Greene C.M.
      • Marciniak S.J.
      • Teckman J.
      • Ferrarotti I.
      • Brantly M.L.
      • Lomas D.A.
      • et al.
      alpha1-Antitrypsin deficiency.
      Presence of the c.1096G>A variant (p.E366K = p.E342K, rs28929474, MAF = 0.0184), which encodes the Pi∗Z protein, is found in 2–4% of Europeans, but in up to 20% of patients with NAFLD.
      • Cacciottolo T.M.
      • Gelson W.T.
      • Maguire G.
      • Davies S.E.
      • Griffiths W.J.
      Pi∗Z heterozygous alpha-1 antitrypsin states accelerate parenchymal but not biliary cirrhosis.
      ,
      • El-Rayah E.A.
      • Twomey P.J.
      • Wallace E.M.
      • McCormick P.A.
      Both alpha-1-antitrypsin Z phenotypes and low caeruloplasmin levels are over-represented in alcohol and nonalcoholic fatty liver disease cirrhotic patients undergoing liver transplant in Ireland.
      ,
      • Greene C.M.
      • Marciniak S.J.
      • Teckman J.
      • Ferrarotti I.
      • Brantly M.L.
      • Lomas D.A.
      • et al.
      alpha1-Antitrypsin deficiency.
      Homozygous carriers of the Pi∗Z variant have significantly higher controlled attenuation parameter values, a non-invasive measure of hepatic fat content.
      • Hamesch K.
      • Mandorfer M.
      • Pereira V.M.
      • Moeller L.S.
      • Pons M.
      • Dolman G.E.
      • et al.
      Liver fibrosis and metabolic alterations in adults with alpha-1-antitrypsin deficiency caused by the Pi∗ZZ mutation.
      ,
      • Dawwas M.F.
      • Davies S.E.
      • Griffiths W.J.
      • Lomas D.A.
      • Alexander G.J.
      Prevalence and risk factors for liver involvement in individuals with PiZZ-related lung disease.
      Furthermore, Pi∗Z homozygotes had significantly higher liver stiffness measurements compared to non-carriers of the variant.
      • Hamesch K.
      • Mandorfer M.
      • Pereira V.M.
      • Moeller L.S.
      • Pons M.
      • Dolman G.E.
      • et al.
      Liver fibrosis and metabolic alterations in adults with alpha-1-antitrypsin deficiency caused by the Pi∗ZZ mutation.
      Mice overexpressing the Pi∗Z variant develop pronounced hepatic steatosis, underscoring that the accumulation of the mutated protein in hepatocytes triggers triglyceride accumulation and indicating that the Pi∗Z variant may cause secondary NAFLD.
      • Hamesch K.
      • Mandorfer M.
      • Pereira V.M.
      • Moeller L.S.
      • Pons M.
      • Dolman G.E.
      • et al.
      Liver fibrosis and metabolic alterations in adults with alpha-1-antitrypsin deficiency caused by the Pi∗ZZ mutation.
      In contrast to the HFE p.C282Y variant, the risk associated with the SERPINA1 p.E366K/p.E342K variant was also found in heterozygous carriers, in whom the presence of the PI∗Z variant constituted a risk factor for the progression towards NAFLD-related cirrhosis (OR 6.7; 95% CI 1.5–31).
      • Strnad P.
      • Buch S.
      • Hamesch K.
      • Fischer J.
      • Rosendahl J.
      • Schmelz R.
      • et al.
      Heterozygous carriage of the alpha1-antitrypsin Pi∗Z variant increases the risk to develop liver cirrhosis.
      HCC risk is also increased in heterozygous carriers of the Pi∗Z variant (OR 1.86; 95% CI 0.74–4.67).
      • Krawczyk M.
      • Liebe R.
      • Lammert F.
      Toward genetic prediction of nonalcoholic fatty liver disease trajectories: PNPLA3 and beyond.
      ,
      • Zhou H.
      • Ortiz-Pallardo M.E.
      • Ko Y.
      • Fischer H.P.
      Is heterozygous alpha-1-antitrypsin deficiency type PIZ a risk factor for primary liver carcinoma?.
      These reports indicate that the Pi∗Z variant may act as a co-factor aggravating primary NAFLD.
      Even in late-onset NAFLD, assessment of family history might be helpful. A rare autosomal dominant form of NAFLD and/or dyslipidaemia with clinical presentation after the fourth decade of life may be caused by variants in the ABHD5 gene, which encodes comparative gene identification-58 (CGI-58), a regulatory co-factor of adipose triglyceride lipase (ATGL).
      • Youssefian L.
      • Vahidnezhad H.
      • Saeidian A.H.
      • Pajouhanfar S.
      • Sotoudeh S.
      • Mansouri P.
      • et al.
      Inherited non-alcoholic fatty liver disease and dyslipidemia due to monoallelic ABHD5 mutations.
      Interestingly, one of the mechanisms by which the common NAFLD-associated PNPLA3 variant p.I148M prevents triglyceride hydrolysis is through sequestration of CGI-58, which prevents ATGL from accessing lipid droplets.
      • Wang Y.
      • Kory N.
      • BasuRay S.
      • Cohen J.C.
      • Hobbs H.H.
      PNPLA3, CGI-58, and inhibition of hepatic triglyceride hydrolysis in mice.
      The prevalence of ABHD5-associated NAFLD was estimated to be 1 in 1,137 individuals in the general population. With the increasing availability and affordability of whole-exome sequencing, the discovery of more rare or private variants with higher penetrance is anticipated.
      A comprehensive list of genetic diseases resulting in paediatric NAFLD is beyond the scope of this review, but has recently been summarised in.
      • Yildiz Y.
      • Sivri H.S.
      Inborn errors of metabolism in the differential diagnosis of fatty liver disease.

      Common genetic variants associated with steatosis/steatohepatitis susceptibility and disease progression

      It is estimated that approximately 50% of the interindividual differences in liver fat content are influenced by genetic variants.
      • Anstee Q.M.
      • Seth D.
      • Day C.P.
      Genetic factors that affect risk of alcoholic and nonalcoholic fatty liver disease.
      ,
      • Eslam M.
      • Valenti L.
      • Romeo S.
      Genetics and epigenetics of NAFLD and NASH: clinical impact.
      Furthermore, genetic risk variants may not only contribute to the development of steatosis and steatohepatitis but also to the progression towards liver fibrosis/cirrhosis and HCC.
      • Krawczyk M.
      • Liebe R.
      • Lammert F.
      Toward genetic prediction of nonalcoholic fatty liver disease trajectories: PNPLA3 and beyond.
      ,
      • Anstee Q.M.
      • Seth D.
      • Day C.P.
      Genetic factors that affect risk of alcoholic and nonalcoholic fatty liver disease.
      • Eslam M.
      • Valenti L.
      • Romeo S.
      Genetics and epigenetics of NAFLD and NASH: clinical impact.
      • Gellert-Kristensen H.
      • Richardson T.G.
      • Davey Smith G.
      • Nordestgaard B.G.
      • Tybjaerg-Hansen A.
      • Stender S.
      Combined effect of PNPLA3, TM6SF2, and HSD17B13 variants on risk of cirrhosis and hepatocellular carcinoma in the general population.
      • Krawczyk M.
      • Rau M.
      • Schattenberg J.M.
      • Bantel H.
      • Pathil A.
      • Demir M.
      • et al.
      Combined effects of the PNPLA3 rs738409, TM6SF2 rs58542926, and MBOAT7 rs641738 variants on NAFLD severity: a multicenter biopsy-based study.
      Variants in genes involved in hepatic lipid metabolism, such as PNPLA3, TM6SF2, membrane-bound O-acyltransferase domain-containing-7 (MBOAT7), glucokinase regulatory protein (GCKR), hydroxysteroid 17-beta dehydrogenase-13 (HSD17B13) and mitochondrial amidoxime reducing component-1 (MARC1), show a robust association with the development and/or progression of NAFLD.
      • Eslam M.
      • Valenti L.
      • Romeo S.
      Genetics and epigenetics of NAFLD and NASH: clinical impact.
      It is safe to assume that these genetic factors not only modulate hepatic triglyceride accumulation and progression towards cirrhosis in metabolic NAFLD (co-factor for primary NAFLD) but also in patients with secondary causes of fatty liver disease. At present, sufficiently powered genome-wide association studies to quantify their impact on the progression of secondary steatohepatitis have not been performed. However, there is evidence that the presence of the PNPLA3 variant p.I148M promotes progression of fatty liver disease to cirrhosis in patients with alcohol-related liver disease (ALD) and HCV.
      • Stickel F.
      • Buch S.
      • Lau K.
      • Meyer zu Schwabedissen H.
      • Berg T.
      • Ridinger M.
      • et al.
      Genetic variation in the PNPLA3 gene is associated with alcoholic liver injury in caucasians.
      • Valenti L.
      • Rumi M.
      • Galmozzi E.
      • Aghemo A.
      • Del Menico B.
      • De Nicola S.
      • et al.
      Patatin-like phospholipase domain-containing 3 I148M polymorphism, steatosis, and liver damage in chronic hepatitis C.
      • Cai T.
      • Dufour J.F.
      • Muellhaupt B.
      • Gerlach T.
      • Heim M.
      • Moradpour D.
      • et al.
      Viral genotype-specific role of PNPLA3, PPARG, MTTP, and IL28B in hepatitis C virus-associated steatosis.
      • Yasui K.
      • Kawaguchi T.
      • Shima T.
      • Mitsuyoshi H.
      • Seki K.
      • Sendo R.
      • et al.
      Effect of PNPLA3 rs738409 variant (I148 M) on hepatic steatosis, necroinflammation, and fibrosis in Japanese patients with chronic hepatitis C.
      • De Nicola S.
      • Dongiovanni P.
      • Aghemo A.
      • Cheroni C.
      • D'Ambrosio R.
      • Pedrazzini M.
      • et al.
      Interaction between PNPLA3 I148M variant and age at infection in determining fibrosis progression in chronic hepatitis C.

      PNPLA3

      PNPLA3 encodes a lipase with hydrolase activity towards triglycerides and retinyl esters, which is expressed in hepatocytes and hepatic stellate cells.
      • Eslam M.
      • Valenti L.
      • Romeo S.
      Genetics and epigenetics of NAFLD and NASH: clinical impact.
      ,
      • Pirazzi C.
      • Valenti L.
      • Motta B.M.
      • Pingitore P.
      • Hedfalk K.
      • Mancina R.M.
      • et al.
      PNPLA3 has retinyl-palmitate lipase activity in human hepatic stellate cells.
      • Huang Y.
      • Cohen J.C.
      • Hobbs H.H.
      Expression and characterization of a PNPLA3 protein isoform (I148M) associated with nonalcoholic fatty liver disease.
      • Pingitore P.
      • Pirazzi C.
      • Mancina R.M.
      • Motta B.M.
      • Indiveri C.
      • Pujia A.
      • et al.
      Recombinant PNPLA3 protein shows triglyceride hydrolase activity and its I148M mutation results in loss of function.
      • Linden D.
      • Ahnmark A.
      • Pingitore P.
      • Ciociola E.
      • Ahlstedt I.
      • Andreasson A.C.
      • et al.
      Pnpla3 silencing with antisense oligonucleotides ameliorates nonalcoholic steatohepatitis and fibrosis in Pnpla3 I148M knock-in mice.
      The association of the PNPLA3 rs738409 variant (p.I148M, C>G, MAF = 0.2281) with the development and progression of NAFLD has been observed in a large number of different adult and paediatric cohorts (combined OR 3.41).
      • Anstee Q.M.
      • Darlay R.
      • Cockell S.
      • Meroni M.
      • Govaere O.
      • Tiniakos D.
      • et al.
      Genome-wide association study of non-alcoholic fatty liver and steatohepatitis in a histologically-characterised cohort.
      • Parisinos C.A.
      • Wilman H.R.
      • Thomas E.L.
      • Kelly M.
      • Nicholls R.C.
      • McGonigle J.
      • et al.
      Genome-wide and Mendelian randomisation studies of liver MRI yield insights into the pathogenesis of steatohepatitis.
      • Romeo S.
      • Kozlitina J.
      • Xing C.
      • Pertsemlidis A.
      • Cox D.
      • Pennacchio L.A.
      • et al.
      Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.
      • Singal A.G.
      • Manjunath H.
      • Yopp A.C.
      • Beg M.S.
      • Marrero J.A.
      • Gopal P.
      • et al.
      The effect of PNPLA3 on fibrosis progression and development of hepatocellular carcinoma: a meta-analysis.
      • Speliotes E.K.
      • Yerges-Armstrong L.M.
      • Wu J.
      • Hernaez R.
      • Kim L.J.
      • Palmer C.D.
      • et al.
      Genome-wide association analysis identifies variants associated with nonalcoholic fatty liver disease that have distinct effects on metabolic traits.
      • Walker R.W.
      • Belbin G.M.
      • Sorokin E.P.
      • Van Vleck T.
      • Wojcik G.L.
      • Moscati A.
      • et al.
      A common variant in PNPLA3 is associated with age at diagnosis of NAFLD in patients from a multi-ethnic biobank.
      • Kawaguchi T.
      • Sumida Y.
      • Umemura A.
      • Matsuo K.
      • Takahashi M.
      • Takamura T.
      • et al.
      Genetic polymorphisms of the human PNPLA3 gene are strongly associated with severity of non-alcoholic fatty liver disease in Japanese.
      • Xu R.
      • Tao A.
      • Zhang S.
      • Deng Y.
      • Chen G.
      Association between patatin-like phospholipase domain containing 3 gene (PNPLA3) polymorphisms and nonalcoholic fatty liver disease: a HuGE review and meta-analysis.
      • Hudert C.A.
      • Selinski S.
      • Rudolph B.
      • Blaker H.
      • Loddenkemper C.
      • Thielhorn R.
      • et al.
      Genetic determinants of steatosis and fibrosis progression in paediatric non-alcoholic fatty liver disease.
      • Namjou B.
      • Lingren T.
      • Huang Y.
      • Parameswaran S.
      • Cobb B.L.
      • Stanaway I.B.
      • et al.
      GWAS and enrichment analyses of non-alcoholic fatty liver disease identify new trait-associated genes and pathways across eMERGE Network.
      A similarly robust association of PNPLA3 p.I148M with the development and progression of ALD has also been observed.
      • Stickel F.
      • Buch S.
      • Lau K.
      • Meyer zu Schwabedissen H.
      • Berg T.
      • Ridinger M.
      • et al.
      Genetic variation in the PNPLA3 gene is associated with alcoholic liver injury in caucasians.
      ,
      • Buch S.
      • Stickel F.
      • Trepo E.
      • Way M.
      • Herrmann A.
      • Nischalke H.D.
      • et al.
      A genome-wide association study confirms PNPLA3 and identifies TM6SF2 and MBOAT7 as risk loci for alcohol-related cirrhosis.
      While expression levels and the biochemical function of the PNPLA3 p.I148M variant are not altered, ubiquitin-mediated degradation is impaired, resulting in the accumulation of mutated PNPLA3 in the membrane of hepatic lipid droplets and the sequestration of CGI-58 (Fig. 2).
      • Wang Y.
      • Kory N.
      • BasuRay S.
      • Cohen J.C.
      • Hobbs H.H.
      PNPLA3, CGI-58, and inhibition of hepatic triglyceride hydrolysis in mice.
      ,
      • BasuRay S.
      • Smagris E.
      • Cohen J.C.
      • Hobbs H.H.
      The PNPLA3 variant associated with fatty liver disease (I148M) accumulates on lipid droplets by evading ubiquitylation.
      ,
      • BasuRay S.
      • Wang Y.
      • Smagris E.
      • Cohen J.C.
      • Hobbs H.H.
      Accumulation of PNPLA3 on lipid droplets is the basis of associated hepatic steatosis.
      The presence of PNPLA3 p.I148M was associated with increased disease activity and severity (as measured by the NAFLD activity score – approximately 1 unit increase per risk allele), liver fibrosis and elevation of liver enzymes.
      • Anstee Q.M.
      • Darlay R.
      • Cockell S.
      • Meroni M.
      • Govaere O.
      • Tiniakos D.
      • et al.
      Genome-wide association study of non-alcoholic fatty liver and steatohepatitis in a histologically-characterised cohort.
      ,
      • Walker R.W.
      • Belbin G.M.
      • Sorokin E.P.
      • Van Vleck T.
      • Wojcik G.L.
      • Moscati A.
      • et al.
      A common variant in PNPLA3 is associated with age at diagnosis of NAFLD in patients from a multi-ethnic biobank.
      ,
      • Namjou B.
      • Lingren T.
      • Huang Y.
      • Parameswaran S.
      • Cobb B.L.
      • Stanaway I.B.
      • et al.
      GWAS and enrichment analyses of non-alcoholic fatty liver disease identify new trait-associated genes and pathways across eMERGE Network.
      ,
      • Chalasani N.
      • Guo X.
      • Loomba R.
      • Goodarzi M.O.
      • Haritunians T.
      • Kwon S.
      • et al.
      Genome-wide association study identifies variants associated with histologic features of nonalcoholic Fatty liver disease.
      ,
      • Koo B.K.
      • Joo S.K.
      • Kim D.
      • Bae J.M.
      • Park J.H.
      • Kim J.H.
      • et al.
      Additive effects of PNPLA3 and TM6SF2 on the histological severity of non-alcoholic fatty liver disease.
      Furthermore, the PNPLA3 p.I148M variant acts as a risk factor for HCC development in both NAFLD and ALD, independently of liver fibrosis.
      • Yang J.
      • Trepo E.
      • Nahon P.
      • Cao Q.
      • Moreno C.
      • Letouze E.
      • et al.
      PNPLA3 and TM6SF2 variants as risk factors of hepatocellular carcinoma across various etiologies and severity of underlying liver diseases.
      • Liu Y.L.
      • Patman G.L.
      • Leathart J.B.
      • Piguet A.C.
      • Burt A.D.
      • Dufour J.F.
      • et al.
      Carriage of the PNPLA3 rs738409 C >G polymorphism confers an increased risk of non-alcoholic fatty liver disease associated hepatocellular carcinoma.
      • Krawczyk M.
      • Stokes C.S.
      • Romeo S.
      • Lammert F.
      HCC and liver disease risks in homozygous PNPLA3 p.I148M carriers approach monogenic inheritance.
      Silencing of PNPLA3 in mice overexpressing PNPLA3 p.I148M (PNPLA3 148M/M knock-in mice) using antisense oligonucleotides (ASOs) or small hairpin RNA ameliorated steatosis, inflammation and fibrosis.
      • Linden D.
      • Ahnmark A.
      • Pingitore P.
      • Ciociola E.
      • Ahlstedt I.
      • Andreasson A.C.
      • et al.
      Pnpla3 silencing with antisense oligonucleotides ameliorates nonalcoholic steatohepatitis and fibrosis in Pnpla3 I148M knock-in mice.
      ,
      • BasuRay S.
      • Wang Y.
      • Smagris E.
      • Cohen J.C.
      • Hobbs H.H.
      Accumulation of PNPLA3 on lipid droplets is the basis of associated hepatic steatosis.
      Thus, targeting hepatic PNPLA3 expression using specific ASOs may offer a precision medicine approach for a subpopulation of patients with NAFLD.
      • Linden D.
      • Ahnmark A.
      • Pingitore P.
      • Ciociola E.
      • Ahlstedt I.
      • Andreasson A.C.
      • et al.
      Pnpla3 silencing with antisense oligonucleotides ameliorates nonalcoholic steatohepatitis and fibrosis in Pnpla3 I148M knock-in mice.

      Diagnostic approach – when to test for secondary causes of fatty liver disease

      Adult patients:

      Common causes of secondary NAFLD should be excluded in all patients (Fig. 3).
      • Keitel V.
      • Vom Dahl S.
      • Häussinger D.
      Secondary causes of fatty liver disease–an update on pathogenesis, diagnosis and treatment strategies.
      Therefore, hepatitis C serology (anti-HCV IgG) and serum TSH levels (to exclude hypothyroidism) should be determined in all patients. Nutrition- or gut-associated secondary NAFLD can often be suspected based on medical history and physical examination (malnutrition?, surgical scars?, parenteral nutrition?, diarrhoea?). CD should be considered in adult patients with normal/low BMI, abdominal symptoms (diarrhoea, steatorrhea) and anaemia, as well as in all paediatric patients with elevated liver enzymes.
      • Valvano M.
      • Longo S.
      • Stefanelli G.
      • Frieri G.
      • Viscido A.
      • Latella G.
      Celiac disease, gluten-free diet, and metabolic and liver disorders.
      • Rubio-Tapia A.
      • Hill I.D.
      • Kelly C.P.
      • Calderwood A.H.
      • Murray J.A.
      American College of G
      ACG clinical guidelines: diagnosis and management of celiac disease.
      • Husby S.
      • Koletzko S.
      • Korponay-Szabo I.
      • Kurppa K.
      • Mearin M.L.
      • Ribes-Koninckx C.
      • et al.
      European society paediatric gastroenterology, hepatology and nutrition guidelines for diagnosing coeliac disease 2020.
      For initial screening of CD in adults and children, total serum IgA and IgA anti-tissue transglutaminase (TTG) should be measured; upper gastrointestinal endoscopy with biopsies of the small intestine should be considered in adults with high suspicion of CD, even in the absence of anti-TTG antibodies.
      European Association for the Study of the L, European Association for the Study of D, European Association for the Study of O
      EASL-EASD-EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease.
      ,
      • Husby S.
      • Koletzko S.
      • Korponay-Szabo I.
      • Kurppa K.
      • Mearin M.L.
      • Ribes-Koninckx C.
      • et al.
      European society paediatric gastroenterology, hepatology and nutrition guidelines for diagnosing coeliac disease 2020.
      If IgA deficiency is suspected (affects approximately 2–3% of patients with CD), IgG anti-TTG and IgG-deamidated gliadin peptides should be determined (Fig. 3).
      • Rubio-Tapia A.
      • Hill I.D.
      • Kelly C.P.
      • Calderwood A.H.
      • Murray J.A.
      American College of G
      ACG clinical guidelines: diagnosis and management of celiac disease.
      Figure thumbnail gr3
      Fig. 3Extended diagnostic algorithm for secondary NAFLD in adults.
      Always consider liver biopsy. Also consider analysis of PNPLA3, TM6SF2, GCKR, MBOAT7 and HSD17B13, MARC1 risk variants in adult patients with steatohepatitis. A1AT, alpha-1 antitrypsin deficiency; ABL, abetalipoproteinaemia; ALD, alcohol-related liver disease; CAD, coronary artery disease; CAP, controlled attenuation parameter; CD, celiac disease; EGD, esophagogastroduodenoscopy; FH, familial hyperlipidaemia; FHBL, familial hypobetalipoproteinaemia; GSD, glycogen storage disease; KF, Kayser-Fleischer corneal ring; LAL-D, lysosomal acid lipase deficiency; LSM, liver stiffness measurement; SIBO, small intestinal bacterial overgrowth; SPE, serum protein electrophoresis; TPN, total parenteral nutrition; TSH, thyroid stimulating hormone; TTG, tissue transglutaminase; TG, triglyceride levels; US, ultrasound. Modified and adapted from
      • Keitel V.
      • Vom Dahl S.
      • Häussinger D.
      Secondary causes of fatty liver disease–an update on pathogenesis, diagnosis and treatment strategies.
      In women, signs and symptoms of PCOS (oligo- or anovulation, signs of hyperandrogenism, polycystic ovaries) should be searched for.
      • Lonardo A.
      • Mantovani A.
      • Lugari S.
      • Targher G.
      NAFLD in some common endocrine diseases: prevalence, pathophysiology, and principles of diagnosis and management.
      ,
      • EA-SPcwg Rotterdam
      Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS).
      When fatty liver occurs for the first time during pregnancy, acute fatty liver of pregnancy (AFLP) and HELLP (haemolysis, elevated liver enzymes, and low platelets) syndrome must be excluded.
      • Joshi D.
      • James A.
      • Quaglia A.
      • Westbrook R.H.
      • Heneghan M.A.
      Liver disease in pregnancy.
      AFLP often presents as acute liver failure, with a high maternal and foetal mortality rate, ranging from 1% to 20%.
      • Joshi D.
      • James A.
      • Quaglia A.
      • Westbrook R.H.
      • Heneghan M.A.
      Liver disease in pregnancy.
      Exclusion of haemochromatosis (ferritin, transferrin saturation, testing for the HFE p.C282Y gene variant) and analysis of the A1AT deficiency-associated SERPINA1 p.E366K/p.E342K (Pi∗Z) variant should be considered. Patients with Wilson’s disease may present with a history of jaundice, signs of Coombs-negative haemolytic anaemia, a plethora of neuro-psychiatric symptoms and acute liver failure.
      • European Association for Study of L.
      EASL clinical practice guidelines: Wilson's disease.
      If Wilson’s disease is suspected, the presence of a Kayser-Fleischer corneal ring and reduced serum ceruloplasmin levels are sufficient to confirm the diagnosis.
      • European Association for Study of L.
      EASL clinical practice guidelines: Wilson's disease.
      In the absence of Kayser-Fleischer rings, urinary copper excretion should be determined and depending on the results, copper quantification in a liver biopsy sample and sequencing of the ATP7B gene should be performed.
      • European Association for Study of L.
      EASL clinical practice guidelines: Wilson's disease.
      For the diagnostic workup of Wilson’s disease please refer to the current EASL clinical practice guideline.
      • European Association for Study of L.
      EASL clinical practice guidelines: Wilson's disease.
      Hypopituitarism resulting from tumours, cerebral toxoplasmosis or other infections is a rare cause of secondary NAFLD.
      • Lonardo A.
      • Mantovani A.
      • Lugari S.
      • Targher G.
      NAFLD in some common endocrine diseases: prevalence, pathophysiology, and principles of diagnosis and management.
      In patients with elevated serum cholesterol and triglyceride levels, various lipid metabolism disorders should be excluded. A dry blood spot assay is available for the diagnosis of LAL-D.
      • Keitel V.
      • Vom Dahl S.
      • Häussinger D.
      Secondary causes of fatty liver disease–an update on pathogenesis, diagnosis and treatment strategies.
      With an estimated prevalence of 1:177,000, LAL-D is a very rare cause of fatty liver disease.
      • Carter A.
      • Brackley S.M.
      • Gao J.
      • Mann J.P.
      The global prevalence and genetic spectrum of lysosomal acid lipase deficiency: a rare condition that mimics NAFLD.
      Liver biopsy may help identify alternative causes of liver disease and detect overlap syndromes (Fig. 3).
      Once the underlying aetiology of secondary NAFLD has been elucidated, the obvious next step is the removal of the damaging agent if possible. In case of non-modifiable causes, additional genotyping of frequent risk variants is advised to gauge the risk of rapid NAFLD progression, and thus determine the frequency of future surveillance.

      Paediatric patients <18 years of age:

      The prevalence of NAFLD depends on the diagnostic tool used. If elevated AST and ALT levels are used as criterion, the prevalence is estimated at 3–7% for the general paediatric population, however, this increases to 28% in obese children.
      • Mann J.P.
      • Valenti L.
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      • Nobili V.
      Nonalcoholic fatty liver disease in children.
      • Nobili V.
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      • Alisi A.
      • Miele L.
      • Valenti L.
      • Vajro P.
      A 360-degree overview of paediatric NAFLD: recent insights.
      • Yu E.L.
      • Golshan S.
      • Harlow K.E.
      • Angeles J.E.
      • Durelle J.
      • Goyal N.P.
      • et al.
      Prevalence of nonalcoholic fatty liver disease in children with obesity.
      Secondary causes of NAFLD must be excluded not only in lean but also in overweight and obese children.
      • Hegarty R.
      • Deheragoda M.
      • Fitzpatrick E.
      • Dhawan A.
      Paediatric Fatty Liver Disease (PeFLD): all is not NAFLD–pathophysiological insights and approach to management.
      ,
      • Vos M.B.
      • Abrams S.H.
      • Barlow S.E.
      • Caprio S.
      • Daniels S.R.
      • Kohli R.
      • et al.
      NASPGHAN clinical practice guideline for the diagnosis and treatment of nonalcoholic fatty liver disease in children: recommendations from the expert committee on NAFLD (ECON) and the North American Society of Pediatric Gastroenterology, Hepatology And Nutrition (NASPGHAN).
      For an extensive overview of secondary causes of fatty liver disease in infants and children see Hegarty et al.
      • Hegarty R.
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      • Fitzpatrick E.
      • Dhawan A.
      Paediatric Fatty Liver Disease (PeFLD): all is not NAFLD–pathophysiological insights and approach to management.
      While in adults the percentage of patients with secondary causes of NAFLD/NASH is unknown, a recent study found that a secondary cause could be identified in 18% of obese children referred for NAFLD.
      • Di Sessa A.
      • Marzuillo P.
      • Guarino S.
      • Cirillo G.
      • Miraglia Del Giudice E.
      When a secondary form of pediatric non-alcoholic fatty liver disease should be suspected?.
      ,
      • Schwimmer J.B.
      • Newton K.P.
      • Awai H.I.
      • Choi L.J.
      • Garcia M.A.
      • Ellis L.L.
      • et al.
      Paediatric gastroenterology evaluation of overweight and obese children referred from primary care for suspected non-alcoholic fatty liver disease.
      Thus, in children presenting with NAFLD who do not normalise AST and ALT levels following weight reduction, treatable causes for liver disease should be formally excluded. In the absence of red flags (cholestasis, evidence of portal hypertension) this extended assessment should be staged over a year and should include testing for viral infections (acute HAV, chronic HBV and HCV, cytomegalovirus and Epstein-Barr virus), hypothyroidism, autoimmune hepatitis, CD, metabolic conditions including Wilsons’s disease, LAL-D, A1AT deficiency, and GSDs.
      • Hegarty R.
      • Deheragoda M.
      • Fitzpatrick E.
      • Dhawan A.
      Paediatric Fatty Liver Disease (PeFLD): all is not NAFLD–pathophysiological insights and approach to management.
      ,
      • Mann J.P.
      • Valenti L.
      • Scorletti E.
      • Byrne C.D.
      • Nobili V.
      Nonalcoholic fatty liver disease in children.
      ,
      • Vos M.B.
      • Abrams S.H.
      • Barlow S.E.
      • Caprio S.
      • Daniels S.R.
      • Kohli R.
      • et al.
      NASPGHAN clinical practice guideline for the diagnosis and treatment of nonalcoholic fatty liver disease in children: recommendations from the expert committee on NAFLD (ECON) and the North American Society of Pediatric Gastroenterology, Hepatology And Nutrition (NASPGHAN).
      ,
      • Vajro P.
      • Lenta S.
      • Socha P.
      • Dhawan A.
      • McKiernan P.
      • Baumann U.
      • et al.
      Diagnosis of nonalcoholic fatty liver disease in children and adolescents: position paper of the ESPGHAN Hepatology Committee.
      Very lean children with fatty liver on imaging should be screened for lipodystrophy.
      • Di Sessa A.
      • Marzuillo P.
      • Guarino S.
      • Cirillo G.
      • Miraglia Del Giudice E.
      When a secondary form of pediatric non-alcoholic fatty liver disease should be suspected?.
      An abdominal ultrasound should be performed to exclude anatomical abnormalities and to detect signs of portal hypertension. A liver biopsy should always be considered.
      • Vos M.B.
      • Abrams S.H.
      • Barlow S.E.
      • Caprio S.
      • Daniels S.R.
      • Kohli R.
      • et al.
      NASPGHAN clinical practice guideline for the diagnosis and treatment of nonalcoholic fatty liver disease in children: recommendations from the expert committee on NAFLD (ECON) and the North American Society of Pediatric Gastroenterology, Hepatology And Nutrition (NASPGHAN).
      A presentation-based approach to basic and advanced diagnostic procedures for secondary causes of NAFLD in children is described in.
      • Hegarty R.
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      • Fitzpatrick E.
      • Dhawan A.
      Paediatric Fatty Liver Disease (PeFLD): all is not NAFLD–pathophysiological insights and approach to management.

      Therapeutic interventions

      While no drug therapy has been approved for NAFLD yet, a reduction of steatosis can be achieved through structured lifestyle changes with weight reduction, a change in dietary habits with reduction of alcohol consumption, avoidance of high fructose drinks/foods and an increase in physical activity. In contrast to metabolic syndrome-associated NAFLD/NASH, specific therapies exist for a number of secondary causes of fatty liver disease.
      • Keitel V.
      • Vom Dahl S.
      • Häussinger D.
      Secondary causes of fatty liver disease–an update on pathogenesis, diagnosis and treatment strategies.
      These therapeutic approaches are listed in Table 3.
      • Keitel V.
      • Vom Dahl S.
      • Häussinger D.
      Secondary causes of fatty liver disease–an update on pathogenesis, diagnosis and treatment strategies.
      In secondary NAFLD/NASH, treatment of the underlying disease can halt disease progression and improve NAFLD/NASH. For example, almost 80% of patients with CD and abnormal liver function tests at the time of diagnosis normalised these values after a mean duration of 1.5 years on a gluten-free diet.
      • Castillo N.E.
      • Vanga R.R.
      • Theethira T.G.
      • Rubio-Tapia A.
      • Murray J.A.
      • Villafuerte J.
      • et al.
      Prevalence of abnormal liver function tests in celiac disease and the effect of a gluten-free diet in the US population.
      Similar effects are observed when patients with haemochromatosis receive early and regular phlebotomy.
      • Niederau C.
      • Fischer R.
      • Sonnenberg A.
      • Stremmel W.
      • Trampisch H.J.
      • Strohmeyer G.
      Survival and causes of death in cirrhotic and in noncirrhotic patients with primary hemochromatosis.
      These therapeutic options may justify early application of the extended diagnostic approach.
      Table 3Therapeutic approaches for secondary causes of NAFLD/NASH.
      Secondary NAFLD/NASHTherapy
      Chronic HCV infectionDirect antiviral therapy with high sustained virological response rates
      Nutritional/intestinal-related causes
      Coeliac diseaseGluten-free diet
      Small intestinal bacterial overgrowthAntibiotic treatment
      Endocrine disorders
      Polycystic ovary syndromeLifestyle change, weight reduction, metformin for insulin resistance/diabetes (consider GLP1 agonists for T2DM)
      HypothyroidismHormone substitution
      HypopituitarismDepending on the cause (tumour, infection), hormone replacement as necessary, consider GH substitution
      Growth hormone deficiencyDetermine underlying cause, if necessary hormone substitution
      Genetic diseases
      A1AT deficiencyNo causative therapy for liver phenotype, refrain from alcohol
      Wilson's diseaseCopper chelating agents, zinc acetate
      HaemochromatosisPhlebotomy, deferasirox, refrain from alcohol
      Congenital lipodystrophyStatins, fibrates, metformin, leptin replacement therapy
      Abeta-, HypobetalipoproteinaemiaLow-fat diet, high-dose vitamin E, vitamin suppl. (A, D, K)
      Familial hypercholesterolemiaStatins, ezetimib (possibly in combination), PCSK9 antibodies
      Glycogen storage diseasesDietary, ERT (type II)
      Cholesterol ester storage diseaseERT (sebelipase alfa)
      Hereditary fructose intoleranceReduce fructose (<2-2.5 g/day), sucrose or sorbitol in diet; refrain from fruit, replace fructose by glucose, maltose and starch; supplementary vitamin C/folic acid substitution
      Pregnancy-associated
      Acute fatty liver during pregnancyIntensive care, early delivery
      Steatosis-associated risk gene variantsNo current causative therapy
      PNPLA3 p.I148MPreclinical:
      PNPLA3 ASOs - reduction in PNPLA3 expression; inhibition of HSD17B13 enzymatic activity
      Adapted from.
      • Keitel V.
      • Vom Dahl S.
      • Häussinger D.
      Secondary causes of fatty liver disease–an update on pathogenesis, diagnosis and treatment strategies.
      ASOs, antisense oligonucleotides; ERT, enzyme replacement therapy; GH, growth hormone; GLP-1, glucagon-like peptide-1; HSD17B13, hydroxysteroid 17-beta dehydrogenase-13; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; PCSK9, proprotein convertase subtilisin kexin 9; PNPLA3, patatin-like phospholipase domain-containing A3; T2DM, type 2 diabetes mellitus.

      Summary and perspectives

      The majority of NAFLD cases are observed as a constituent of the metabolic syndrome, caused by excess nutrition. The absence of symptoms and signs of the metabolic syndrome on clinical assessment should warrant assessment of alternative causes of NAFLD. Drug treatment and environmental exposure to endocrine disrupting chemicals need to be considered. Several congenital metabolic defects may result in hepatic fat deposition and should be investigated, particularly in lean and paediatric patients. Identification of potential alternative causes of steatosis is vital since treatment options exist for many of these diseases.
      Where no specific treatment option is currently available, the risk of liver disease progression may be estimated by analysis of known genetic risk factors (PNPLA3, TM6SF2, GCKR, MBOAT7, HFE, A1AT; protective: HSD17B13, MARC1), with surveillance adjusted accordingly. Novel drugs in clinical trials for nutrition-induced NAFLD and its sequelae target central regulators and effectors of lipid deposition and metabolism. They may also prove useful for future treatment of congenital metabolic diseases, i.e. "secondary NAFLD", if the underlying gene defect impinges the same pathway that is targeted by the respective drug. A thyroid receptor agonist might in the future be used to treat patients with NAFLD due to hypothyroidism, while PPAR agonists may serve those with fatty acid oxidation disorders. An extended diagnostic approach aimed at identifying patients with NAFLD may not only prove cost-effective in paediatric patients, but may also help identify subgroups amenable to specific treatment for secondary causes of NAFLD, as well as subgroups at high risk of disease progression, leading to improved patient care.

      Abbreviations

      A1AT, alpha-1 antitrypsin, ABL, abetalipoproteinemia; AFLP, acute fatty liver of pregnancy; ALD, alcohol-related liver disease; ASO, antisense oligonucleotides; ATGL, adipose triglyceride lipase; CAD, cationic amphiphilic drug; CAR, constitutive androstane receptor; CD, celiac disease; CGI-58, comparative gene identification-58; CPSI, carbamoylphosphate synthetase I; CTLN2, citrullinemia type II; FHBL, familial hypobetalipoproteinemia; GCKR, glucokinase regulatory protein; GH, growth hormone; GSD, glycogen storage disorders; HCC, hepatocellular carcinoma; HR, hazard ratio; HSD17B13, hydroxysteroid 17-beta dehydrogenase-13; IFALD, intestinal failure-associated liver disease; LAL-D, lysosomal acid lipase deficiency; MAF, minor allele frequency (all taken from GnomAD, data are shown for individuals of European, non-Finnish ancestry); MARC1, mitochondrial amidoxime reducing component-1; MBOAT7, membrane-bound O-acyltransferase domain-containing-7; MMI, methamizole; MR, magnetic resonance; MRT/I , magnetic resonance tomography/imaging; MTTP, microsomal triglyceride transfer protein; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; NICCD, neonatal intrahepatic cholestasis caused by citrin deficiency; OGTT, oral glucose tolerance test; OR, odds ratio; OTC, ornithine transcarbamylase; PCOS, polycystic ovary syndrome; PNPLA3, patatin-like phospholipase domain-containing A3 (adiponutrin); PPAR, peroxisome proliferator-activated receptor; SIBO, small intestinal bacterial overgrowth; T2DM, type 2 diabetes mellitus; TH, thyroid hormone; TM6SF2, transmembrane 6 superfamily member-2; THR, thyroid hormone receptor; TSH, thyroid stimulating hormone; TTG, tissue transglutaminase; UCDs, urea cycle disorders.

      Financial support

      VK, HHB received funding from the German Research Foundation (DFG) through the Collaborative Research Centre SFB 974 and VK and UB from BMBF through HiChol ( 01GM1904A ), UB from Horizon 2020 through “Adeno-Associated Virus Vector-Mediated Liver Gene Therapy for Crigler-Najjar Syndrome” (755225).

      Authors’ contributions

      VK and RL initiated this work and drafted the manuscript; IE, HB, SVD, VK selected exemplary cases of secondary steatosis, JS designed figures; IE contributed pathological expertise; UB contributed paediatric expertise; TL helped to revise the manuscript. All authors critically revised the manuscript. All authors approved the final version of the manuscript.

      Conflict of interest

      The authors declare that there is no potential conflict of interest.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following is the supplementary data to this article:

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