Advertisement

Genetic and Epigenetic Culprits in the Pathogenesis of Nonalcoholic Fatty Liver Disease

  • Alexander J. Kovalic
    Affiliations
    Wake Forest Baptist Medical Center, Department of Internal Medicine, Winston-Salem, NC, United States
    Search for articles by this author
  • Pratik Banerjee
    Affiliations
    University of Memphis, School of Public Health, Division of Epidemiology, Biostatistics, and Environmental Health, Memphis, TN, United States
    Search for articles by this author
  • Quynh T. Tran
    Affiliations
    University of Tennessee Health Science Center, Department of Preventive Medicine, Memphis, TN, United States
    Search for articles by this author
  • Ashwani K. Singal
    Affiliations
    University of Alabama at Birmingham, Department of Medicine, Division of Gastroenterology and Hepatology, Birmingham, AL, United States
    Search for articles by this author
  • Sanjaya K. Satapathy
    Correspondence
    Address for correspondence: Sanjaya K. Satapathy, Associate Professor of Medicine, Division of Transplant Surgery, Department of Surgery, Methodist University Hospital Transplant Institute, University of Tennessee Health Science Center, 1211 Union Avenue, Suite #340, Memphis, TN 38104, United States. Tel.: +1 901 516 9179; fax: +1 901 516 8993.
    Affiliations
    University of Tennessee Health Science Center, Methodist University Hospital Transplant Institute, Memphis, TN, United States
    Search for articles by this author
      Nonalcoholic Fatty Liver Disease (NAFLD) constitutes a wide spectrum of liver pathology with hepatic steatosis at the core of this pathogenesis. Variations of certain genetic components have demonstrated increased susceptibility for hepatic steatosis. Therefore, these inciting variants must be further characterized in order to ultimately provide effective, targeted therapies for NAFLD and will be the focus of this review. Several genetic variants revealed an association with NAFLD through Genome-wide Association Study, meta-analyses, and retrospective case–control studies. PNPLA3 rs738409 and TM6SF2 rs58542926 are the two genetic variants providing the strongest evidence for association with NAFLD. However, it remains to be determined if these genetic variants serve as the primary culprit which induces the pathogenesis of NAFLD. Prospective and intervention studies are urgently needed to firmly establish a cause-and-effect relationship between the presence of certain genetic variants and risk of NAFLD development and progression.

      Abbreviations:

      ACC2 (Acetyl-CoA Carboxylase 2), ACLY (ATP Citrate Lyase), BMI (Body Mass Index), CK-18 (Cytokeratin 18), CT (Computed Tomography), FASN (Fatty Acid Synthase), GWAS (Genome-wide Association Study), 1H-MRS (Proton Magnetic Resonance Spectroscopy), HCC (Hepatocellular Carcinoma), LT (Liver Transplantation), miRNA (MicroRNA), NAFLD (Nonalcoholic Fatty Liver Disease), NASH (Nonalcoholic Steatohepatitis), SCD1 (Stearoyl-CoA Desaturase 1), SNP (Single Nucleotide Polymorphism), US (Ultrasonography)

      Keywords

      Nonalcoholic Fatty Liver Disease (NAFLD) encompasses a spectrum of liver disease that ranges from simple triglyceride (TG) accumulation, or steatosis, in the liver to necro-inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC).
      • Caldwell S.
      • Argo C.
      The natural history of non-alcoholic fatty liver disease.
      Hepatic steatosis may be accompanied by other pathologic changes, such as lobular inflammation or hepatocellular ballooning, that define Non-alcoholic Steatohepatitis (NASH). Although the progression from isolated hepatic steatosis to NASH is unclear, cirrhosis and HCC remain common long-term complications found among patients with NASH.
      • Matteoni C.A.
      • Younossi Z.M.
      • Gramlich T.
      • et al.
      Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity.
      • Adams L.A.
      • Lymp J.F.
      • St Sauver J.
      • et al.
      The natural history of nonalcoholic fatty liver disease: a population-based cohort study.
      In a longitudinal study of patients with NAFLD, fibrosis stage, but no other histologic features of steatohepatitis, were associated independently with long-term overall mortality, liver transplantation, and liver-related events.
      • Angulo P.
      • Kleiner D.E.
      • Dam-Larsen S.
      • et al.
      Liver fibrosis, but no other histologic features, associates with long-term outcomes of patients with nonalcoholic fatty liver disease.
      A complex interplay between cellular, genetic, and environmental factors is implicated in the pathogenesis of NAFLD (Figure 1). Hepatocytes play a central role in lipid metabolism, importing serum free fatty acids and manufacturing, storing and exporting lipids and lipoproteins. Accumulation of TG can occur in the liver as a result of abnormal fatty acid metabolism,
      • Alpers D.H.
      • Sabesin S.M.
      • White H.M.
      Fatty liver; biochemical and clinical aspects.
      with excessive delivery of free fatty acids to the liver compared to that which can be metabolized (obesity, rapid weight loss, TPN), an increased mitochondrial synthesis of fatty acids, or a failure of the synthesis or secretion of apolipoproteins or TGs.
      • Sanyal A.J.
      • Campbell-Sargent C.
      • Mirshahi F.
      • et al.
      Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities.
      The primary metabolic abnormality catalyzing the transformation of hepatic steatosis to NASH is still unknown. Insulin resistance plays a key role, since it may influence several intracellular metabolic pathways.
      • Marchesini G.
      • Pagotto U.
      • Bugianesi E.
      • et al.
      Low ghrelin concentrations in nonalcoholic fatty liver disease are related to insulin resistance.
      Higher levels of fasting serum insulin have been frequently noted in NASH patients.
      • Chitturi S.
      • Abeygunasekera S.
      • Farrell G.C.
      • et al.
      NASH and insulin resistance: insulin hypersecretion and specific association with the insulin resistance syndrome.
      Conditions associated with peripheral insulin resistance, such as type 2 diabetes mellitus and obesity (in particular visceral adiposity), are frequently observed in patients with NAFLD. Insulin resistance has also been observed in patients with NASH who are not obese and those who have normal glucose tolerance.
      • Chitturi S.
      • Abeygunasekera S.
      • Farrell G.C.
      • et al.
      NASH and insulin resistance: insulin hypersecretion and specific association with the insulin resistance syndrome.
      It is postulated that impaired capacity to adequately expand the peripheral adipose tissue compartments drives dyslipidemia and insulin resistance.
      • Lotta L.A.
      • Gulati P.
      • Day F.R.
      • et al.
      Integrative genomic analysis implicates limited peripheral adipose storage capacity in the pathogenesis of human insulin resistance.
      Insulin resistance decreases peripheral glucose uptake and promotes a lipogenic state within hepatocytes.
      • Sanyal A.J.
      • Campbell-Sargent C.
      • Mirshahi F.
      • et al.
      Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities.
      • Gastaldelli A.
      • Cusi K.
      • Pettiti M.
      • et al.
      Relationship between hepatic/visceral fat and hepatic insulin resistance in nondiabetic and type 2 diabetic subjects.
      • Lomonaco R.
      • Ortiz-Lopez C.
      • Orsak B.
      • et al.
      Effect of adipose tissue insulin resistance on metabolic parameters and liver histology in obese patients with nonalcoholic fatty liver disease.
      • Pagano G.
      • Pacini G.
      • Musso G.
      • et al.
      Nonalcoholic steatohepatitis, insulin resistance, and metabolic syndrome: further evidence for an etiologic association.
      After the introduction of excess hepatic TGs, increased lipid peroxidation and reactive oxidative species are generated which insidiously promotes dysfunction of the mitochondria and endoplasmic reticulum (ER).
      • Koek G.H.
      • Liedorp P.R.
      • Bast A.
      The role of oxidative stress in non-alcoholic steatohepatitis.
      • Kujoth G.C.
      • Hiona A.
      • Pugh T.D.
      • et al.
      Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging.
      • Lee J.
      • Ozcan U.
      Unfolded protein response signaling and metabolic diseases.
      • Ozcan U.
      • Cao Q.
      • Yilmaz E.
      • et al.
      Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes.
      This increased cellular stress defines the lipotoxicity which ultimately concludes with hepatocellular death, which further amplifies the inflammatory response within this milieu.
      • Fuchs M.
      • Sanyal A.J.
      Lipotoxicity in NASH.
      Hepatic steatosis with ensuing lipotoxicity and inflammatory response set the stage for NASH. Several adipocytokines derived primarily from the adipose tissue depot such as leptin, TNF-alpha, IL-6, adiponectin, resistin has been implicated in the pathogenesis of NASH, however a detailed discussion of their mechanism is beyond the scope of this review, and has been recently reviewed by Polyzos et al.
      • Polyzos S.A.
      • Kountouras J.
      • Mantzoros C.S.
      Adipokines in nonalcoholic fatty liver disease.
      Adipokines serve as auxiliary catalysts at numerous points along the pathway of NAFLD pathogenesis.
      • Flier J.S.
      • Maratos-Flier E.
      Biology of obesity.
      Finally, intestinal dysbiosis has been shown to exacerbate this entire process through diet-induced changes affecting the gut-liver axis.
      • Miele L.
      • Valenza V.
      • La Torre G.
      • et al.
      Increased intestinal permeability and tight junction alterations in nonalcoholic fatty liver disease.
      • Vijay-Kumar M.
      • Aitken J.D.
      • Carvalho F.A.
      • et al.
      Metabolic syndrome and altered gut microbiota in mice lacking Toll-like receptor 5.
      It is imperative to note the underlying pathophysiology because it is the genetic variations within this mechanism that dictate functionality of proteins within these cellular processes. Thus, minor epigenetic changes or alterations within the genetic sequence can clearly predispose individuals to both the development and progression of NAFLD.
      Figure thumbnail gr1
      Figure 1Pathophysiology of NAFLD. Visualized here are the influences of environmental factors, genetic components, and the intestinal microbiome on the development of hepatic steatosis and the progression of NAFLD. (A) NAFLD is characterized as >5% triglyceride accumulation in hepatocytes. This can be isolated hepatic steatosis, or accompanied by minimal inflammation within the lobules. (B) Histology showing classic features of NASH such as steatosis, inflammation, and hepatocellular ballooning degeneration. (C) Trichrome stain revealing progression of NASH to cirrhosis. (D) Histology illustrating the progression of cirrhosis to HCC. 11HSD-1, 11β-Hydroxysteroid Dehydrogenase Type 1; CCL2, C-C Motif Chemokine Ligand 2; EGF, Epidermal Growth Factor; FA, Fatty Acid; IL-6, Interleukin 6; HCC, Hepatocellular Carcinoma; HDL, High Density Lipoprotein; HGF, Hepatocyte Growth Factor; LDL, Low Density Lipoprotein; PAI-1, Plasminogen Activator Inhibitor-1; PDGF, Platelet-Derived Growth Factor; RBP4, Retinol Binding Protein 4; ROS, Reactive Oxygen Species; SNP, Single Nucleotide Polymorphism; TG, Triglyceride; TGF-β, Transforming Growth Factor Beta; TNF-α, Tumor Necrosis Factor Alpha.
      Although there are clearly numerous components at play, the individual genetic elements responsible for these cellular functions remain critical to the inner workings of this pathophysiology. Several genetic disorders that alter the lipoprotein levels such as, disorders of high density lipoprotein (familial hypoalphalipoproteinemia, Tangier disease, and LCAT deficiency); familial hypocholesterolemias (familial hypobetalipoproteinemia, abetalipoproteinemia, PCSK9 loss of function mutations, familial combined hypolipidemia, and chylomicron retention disease); β-sitosterolemia; cerebrotendinous xanthomatosis; and lysosomal acid lipase deficiency exists that potentially can predispose for development of NAFLD.
      • Shapiro M.D.
      Rare genetic disorders altering lipoproteins.
      Clear genetic association of NAFLD has been shown with LIPA gene mutation in lysosomal acid lipase deficiency, and a targeted therapies currently exists for this condition.
      • Erwin A.L.
      The role of sebelipase alfa in the treatment of lysosomal acid lipase deficiency.
      The relevance of genetic factor in the context of NAFLD has been recently and elegantly outlined by twin studies.
      • Loomba R.
      • Schork N.
      • Chen C.H.
      • et al.
      Heritability of hepatic fibrosis and steatosis based on a prospective twin study.
      Particular variants or Single Nucleotide Polymorphisms (SNPs) across multiple genetic loci have been demonstrated to work synergistically with adipose tissue in order to augment the risk of NAFLD.
      • Stender S.
      • Kozlitina J.
      • Nordestgaard B.G.
      • et al.
      Adiposity amplifies the genetic risk of fatty liver disease conferred by multiple loci.
      SNPs and epigenetic modifications affecting the genes within this process have been demonstrated to increase the susceptibility for hepatic steatosis, a central crux of this pathology which this review will analyze in further detail.

      Genetic Variants Implicated in NAFLD Pathogenesis

      Genome Wide Association Studies

      Genome-wide Association Studies (GWAS) have identified associations of several genetic variants with NAFLD
      • Romeo S.
      • Kozlitina J.
      • Xing C.
      • et al.
      Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.
      • Chalasani N.
      • Guo X.
      • Loomba R.
      • et al.
      Genome-wide association study identifies variants associated with histologic features of nonalcoholic Fatty liver disease.
      • Speliotes E.K.
      • Yerges-Armstrong L.M.
      • Wu J.
      • et al.
      Genome-wide association analysis identifies variants associated with nonalcoholic fatty liver disease that have distinct effects on metabolic traits.
      • Kawaguchi T.
      • Sumida Y.
      • Umemura A.
      • et al.
      Genetic polymorphisms of the human PNPLA3 gene are strongly associated with severity of non-alcoholic fatty liver disease in Japanese.
      • Adams L.A.
      • White S.W.
      • Marsh J.A.
      • et al.
      Association between liver-specific gene polymorphisms and their expression levels with nonalcoholic fatty liver disease.
      • Kitamoto T.
      • Kitamoto A.
      • Yoneda M.
      • et al.
      Genome-wide scan revealed that polymorphisms in the PNPLA3, SAMM50, and PARVB genes are associated with development and progression of nonalcoholic fatty liver disease in Japan.
      • Feitosa M.F.
      • Wojczynski M.K.
      • North K.E.
      • et al.
      The ERLIN1-CHUK-CWF19L1 gene cluster influences liver fat deposition and hepatic inflammation in the NHLBI Family Heart Study.
      • Kozlitina J.
      • Smagris E.
      • Stender S.
      • et al.
      Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease.
      • Shang X.R.
      • Song J.Y.
      • Liu F.H.
      • et al.
      GWAS-identified common variants with nonalcoholic fatty liver disease in Chinese children.
      • DiStefano J.K.
      • Kingsley C.
      • Craig Wood G.
      • et al.
      Genome-wide analysis of hepatic lipid content in extreme obesity.
      (Table 1). GWAS have become a novel, powerful tool in first assessing which genes are affiliated with a particular disease. The majority of these findings have incriminated genetic variants, such as SNPs, as potential mechanisms predisposing for NAFLD pathogenesis.
      Table 1Genome-Wide Association (GWA) and Genome-Wide Exome Studies Identifying Genetic Variants Associated with NAFLD.
      GeneSNPStudy populationNotable cohort characteristicsDiagnostic modalityNAFLD sample size
      Romeo et al., 2008
      • Romeo S.
      • Kozlitina J.
      • Xing C.
      • et al.
      Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.
       PNPLA3rs738409American%52 African American, 29% Caucasian, 17% Hispanic1H-MRS2111 total (controls vs NAFLD not reported)
      Chalasani et al., 2010
      • Chalasani N.
      • Guo X.
      • Loomba R.
      • et al.
      Genome-wide association study identifies variants associated with histologic features of nonalcoholic Fatty liver disease.
       COL13A1rs1227756American100% Caucasian femalesLiver biopsy236
       FDFT1rs2645424
      Speliotes et al., 2011
      • Speliotes E.K.
      • Yerges-Armstrong L.M.
      • Wu J.
      • et al.
      Genome-wide association analysis identifies variants associated with nonalcoholic fatty liver disease that have distinct effects on metabolic traits.
       GCKRrs780094American & Icelandic100% Caucasian Family studies includedCT and/or liver biopsy592
       LYPLAL1rs12137855
       NCANrs2228603
       PNPLA3rs738408
      Kawaguchi et al., 2012
      • Kawaguchi T.
      • Sumida Y.
      • Umemura A.
      • et al.
      Genetic polymorphisms of the human PNPLA3 gene are strongly associated with severity of non-alcoholic fatty liver disease in Japanese.
       PNPLA3Several (but rs738409 not tested)JapaneseLiver biopsy529
      Adams et al., 2013
      • Adams L.A.
      • White S.W.
      • Marsh J.A.
      • et al.
      Association between liver-specific gene polymorphisms and their expression levels with nonalcoholic fatty liver disease.
       LPPR4rs12743824AustralianAdolescents at 17 years of ageUS126
       GCrs222054
       LCP1rs7324845
       SLC38A8rs11864146
      Kitamoto et al., 2013
      • Kitamoto T.
      • Kitamoto A.
      • Yoneda M.
      • et al.
      Genome-wide scan revealed that polymorphisms in the PNPLA3, SAMM50, and PARVB genes are associated with development and progression of nonalcoholic fatty liver disease in Japan.
       PARVBrs5764455, rs6006611JapaneseLiver biopsy392
       PNPLA3rs738409, rs2896019, rs3810622
       SAMM50rs738491, rs2073082, rs3761472, rs2143571
      Feitosa et al., 2013
      • Feitosa M.F.
      • Wojczynski M.K.
      • North K.E.
      • et al.
      The ERLIN1-CHUK-CWF19L1 gene cluster influences liver fat deposition and hepatic inflammation in the NHLBI Family Heart Study.
       ERLIN1rs2862954, rs1408579, rs10883451American100% Caucasian Family studyCT2767
       CHUKrs11597086, rs11591741
       CWF19L1rs17729876, rs17668255, rs17668357, rs12784396
       PNPLA3rs738409
       PPP1R3Brs2126259
      Kozlitina et al., 2014
      • Kozlitina J.
      • Smagris E.
      • Stender S.
      • et al.
      Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease.
       TM6SF2rs58542926American48% African American, 32% Caucasian, 17% Hispanic1H-MRS2735 total [controls vs NAFLD not reported]
      Shang et al., 2015
      • Shang X.R.
      • Song J.Y.
      • Liu F.H.
      • et al.
      GWAS-identified common variants with nonalcoholic fatty liver disease in Chinese children.
       COL13A1rs1227756ChineseChildren aged 7–18US162
       EHBP1L1rs6591182
       FDFT1rs2645424
       GCKRrs7800094
       NCANrs2228603
       PDGFArs343064
       PNPLA3rs738409
      DiStefano et al., 2015
      • DiStefano J.K.
      • Kingsley C.
      • Craig Wood G.
      • et al.
      Genome-wide analysis of hepatic lipid content in extreme obesity.
       PNPLA3rs4823173, rs2896019, rs2281135American100% Caucasian 81% female Subjects undergoing bariatric surgeryHistology from intraoperative liver biopsy1386
       SUGP1rs10401969
      CHUK, Conserved Helix-Loop Helix Ubiquitous Kinase; COL13A1, Collagen Type XIII, alpha1; CT, Computed Tomography; CWF19L1, CWF19-like Protein 1; EHBP1L1, EH Domain Binding Protein 1-like 1 Gene; ERLIN1, ER Lipid Raft Associated 1; FDFT1, Farnesyl Diphosphate Farnesyl Transferase 1; GC, Group-specific Component; GCKR, Glucokinase Regulator; 1H-MRS, Proton Magnetic Resonance Spectroscopy; LCP1, Lymphocyte Cytosolic Protein-1; LPPR4, Lipid Phosphate Phosphatase-Related Protein Type 4; LYPLAL1, Lysophospholipase-like 1; NCAN, Neurocan; PARVB, Parvin, Beta; PDGFA, Platelet-derived Growth Factor Alpha Polypeptide; PNPLA3, Patatin-like Phospholipase Domain Containing 3; PPP1R3B, Protein Phosphatase 1 Regulatory Subunit 3B; SAMM50, Sorting and Assembly Machinery Component 50; SLC38A8, Solute Carrier Family 38 Member 8; SUGP1, SURP and G Patch Domain Containing 1; TM6SF2, Transmembrane 6 Superfamily Member 2; US, Ultrasonography.
      Romero et al. for the first time reported in a GWAS a relationship of NAFLD with a SNP identified in patatin-like phospholipase domain-containing 3 (PNPLA3) on chromosome 22, PNPLA3 rs738409, which has founded this variant as a prime candidate of genetic-associated NAFLD.
      • Romeo S.
      • Kozlitina J.
      • Xing C.
      • et al.
      Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.
      The variant rs738409 (c.444 C>G, p.I148M), a non-synonymous cytosine to guanine mutation resulting in isoleucine to methionine conversion, correlates with increased hepatic lipid content and predisposes to fatty liver-associated liver disease, from simple steatosis to steatohepatitis, fibrosis and HCC.
      • Romeo S.
      • Kozlitina J.
      • Xing C.
      • et al.
      Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.
      • Dongiovanni P.
      • Donati B.
      • Fares R.
      • et al.
      PNPLA3 I148M polymorphism and progressive liver disease.
      Overexpression of the I148M variant in mouse liver promotes accumulation of triacylglycerol, increased synthesis of fatty acids and impaired hydrolysis of triacylglycerol.
      • Smagris E.
      • BasuRay S.
      • Li J.
      • et al.
      Pnpla3I148M knockin mice accumulate PNPLA3 on lipid droplets and develop hepatic steatosis.
      This genetic variant has been examined extensively in subsequent studies, and is analyzed in detail in this review. However, this study utilized an American cohort, of which African American subjects were predominant. This demographic composition may result in slightly skewed data because the prevalence of NAFLD is highest in Hispanics and Caucasians, while least common among African American individuals, respectively.
      • Kalia H.S.
      • Gaglio P.J.
      The prevalence and pathobiology of nonalcoholic fatty liver disease in patients of different races or ethnicities.
      The population with the highest prevalence was grossly underrepresented in this study, and therefore, may serve as a poor indicator of true genetic association. This GWAS analyzed cohort patients using Proton Magnetic Resonance Spectroscopy (1H-MRS) and fails to report the percentage of total patients with NAFLD as calculated via 1H-MRS, but only summarizes hepatic TG percentage within each ethnic group. Hepatic TG content was also stated as skewed, and a power transformation was applied to that trait prior to analysis. Nevertheless, this large-scale cohort was a landmark study that demonstrated a significant association between PNPLA3 rs738409 and NAFLD.
      Chalasani et al. in another GWAS retrospectively analyzed data from the NAFLD database Study.
      • Chalasani N.
      • Guo X.
      • Loomba R.
      • et al.
      Genome-wide association study identifies variants associated with histologic features of nonalcoholic Fatty liver disease.
      The entire genome of 236 American subjects confirmed by histology via liver biopsy was analyzed. PNPLA3 was not verified in this cohort, but SNPs in the COL13A1 and FDFT1 genes were found to be associated with NAFLD. Despite providing convincing evidence, there was one major drawback of this study in that this cohort was purely composed of Caucasian females. A highly homogenous group such as this may not represent the most indicative genetic associations in the general population.
      In the largest GWAS to date, Speliotes et al. not only was able to reproduce an association with PNPLA3 rs738409, but also established a new relationship between NAFLD and glucokinase regulatory protein GCKR rs780094 and NCAN rs2228603.
      • Speliotes E.K.
      • Yerges-Armstrong L.M.
      • Wu J.
      • et al.
      Genome-wide association analysis identifies variants associated with nonalcoholic fatty liver disease that have distinct effects on metabolic traits.
      This variant produces a GCKR with defective inhibitory function, leading to increased glucokinase activity and hepatic glucose uptake.
      • Santoro N.
      • Zhang C.K.
      • Zhao H.
      • et al.
      Variant in the glucokinase regulatory protein (GCKR) gene is associated with fatty liver in obese children and adolescents.
      The resultant unimpeded hepatic glycolysis reduces glucose levels, inducing malonyl-CoA synthesis, a substrate for lipogenesis that causes liver fat deposition and impairs mitochondrial β-oxidation. This cohort was comprised of populations in family-based studies from the Framingham Heart Study, Family Heart Study, Old Order Amish community, and Reykjavik Study from Iceland. All of the individuals in this cohort were Caucasian and from either America or Iceland. While the sheer number of individuals tested provides powerful data, this analysis will represent skewed information from within the same genetic pools, which could alter the validity of the GWAS as compared to the entire population. Also, the modality of NAFLD diagnosis was initially established with Computed Tomography (CT), and then if positive, followed up by liver biopsy. Utilizing this methodology, patients with lower levels of hepatic steatosis may have gone undetected in the initial screening process by CT, and thus, not confirmed on liver biopsy. Even though constraints certainly exist, this study was not only able to reproduce results on PNPLA3 rs738409, but also pioneered the association between NAFLD and GCKR rs780094 and NCAN rs2228603. Additionally, the multidimensional function of GCKR needs a mention here as a recent meta-analysis also suggests that the rs780094 polymorphism in GCKR is associated with elevated T2DM risk, which may indirectly influence the risk of developing NAFLD.
      • Li H.
      • Xu R.
      • Peng X.
      • et al.
      Association of glucokinase regulatory protein polymorphism with type 2 diabetes and fasting plasma glucose: a meta-analysis.
      Romero et al. in the first GWAS provided the framework for the remaining nine studies whose results are further detailed in Table 1. Three of the studies were strictly in Japanese and Chinese cohorts, the latter being primarily focused in a pediatric population.
      • Shang X.R.
      • Song J.Y.
      • Liu F.H.
      • et al.
      GWAS-identified common variants with nonalcoholic fatty liver disease in Chinese children.
      Of note, the individuals in the Chinese pediatric population were diagnosed with NAFLD based upon US. Another study was performed in Australia, which also used US for detection of hepatic steatosis.
      • Adams L.A.
      • White S.W.
      • Marsh J.A.
      • et al.
      Association between liver-specific gene polymorphisms and their expression levels with nonalcoholic fatty liver disease.
      Patients with limited hepatic steatosis would potentially go undetected utilizing this modality. The remaining GWAS were performed in American cohorts, which were able to confirm the prevalence of SNPs from previous work as well as identify new potential genetic variants associated with NAFLD.

      Candidate Gene Studies

      After the explosion of information from numerous GWAS, many candidate gene studies have been performed in order to examine the influence of particular SNPs on NAFLD pathogenesis. The most prominent genetic variants
      • Wang J.
      • Guo X.F.
      • Yu S.J.
      • et al.
      Adiponectin polymorphisms and non-alcoholic fatty liver disease risk: a meta-analysis.
      • Li M.R.
      • Zhang S.H.
      • Chao K.
      • et al.
      Apolipoprotein C3 (-455T>C) polymorphism confers susceptibility to nonalcoholic fatty liver disease in the Southern Han Chinese population.
      • Zhang H.
      • Chen L.
      • Xin Y.
      • et al.
      Apolipoprotein c3 gene polymorphisms are not a risk factor for developing non-alcoholic Fatty liver disease: a meta-analysis.
      • Zain S.M.
      • Mohamed Z.
      • Mohamed R.
      Common variant in the glucokinase regulatory gene rs780094 and risk of nonalcoholic fatty liver disease: a meta-analysis.
      • Sun M.Y.
      • Zhang L.
      • Shi S.L.
      • et al.
      Associations between methylenetetrahydrofolate reductase (MTHFR) polymorphisms and non-alcoholic fatty liver disease (NAFLD) risk: a meta-analysis.
      • Li L.
      • Wang S.J.
      • Shi K.
      • et al.
      Correlation between MTP -493G>T polymorphism and non-alcoholic fatty liver disease risk: a meta-analysis.
      • Tan H.L.
      • Mohamed R.
      • Mohamed Z.
      • et al.
      Phosphatidylethanolamine N-methyltransferase gene rs7946 polymorphism plays a role in risk of nonalcoholic fatty liver disease: evidence from meta-analysis.
      • Sookoian S.
      • Pirola C.J.
      Meta-analysis of the influence of I148M variant of patatin-like phospholipase domain containing 3 gene (PNPLA3) on the susceptibility and histological severity of nonalcoholic fatty liver disease.
      • Zhang L.
      • You W.
      • Zhang H.
      • et al.
      PNPLA3 polymorphisms (rs738409) and non-alcoholic fatty liver disease risk and related phenotypes: a meta-analysis.
      • Xu R.
      • Tao A.
      • Zhang S.
      • et al.
      Association between patatin-like phospholipase domain containing 3 gene (PNPLA3) polymorphisms and nonalcoholic fatty liver disease: a HuGE review and meta-analysis.
      • Wang J.
      • Guo X.
      • Wu P.
      • et al.
      Association between the Pro12Ala polymorphism of PPAR-gamma gene and the non-alcoholic fatty liver disease: a meta-analysis.
      • Lee Y.H.
      • Bae S.C.
      • Song G.G.
      Meta-analysis of associations between the peroxisome proliferator-activated receptor-gamma Pro12Ala polymorphism and susceptibility to nonalcoholic fatty liver disease, rheumatoid arthritis, and psoriatic arthritis.
      • Musso G.
      • Cassader M.
      • Bo S.
      • et al.
      Sterol regulatory element-binding factor 2 (SREBF-2) predicts 7-year NAFLD incidence and severity of liver disease and lipoprotein and glucose dysmetabolism.
      • Pirola C.J.
      • Sookoian S.
      The dual and opposite role of the TM6SF2-rs58542926 variant in protecting against cardiovascular disease and conferring risk for nonalcoholic fatty liver: a meta-analysis.
      • Goffredo M.
      • Caprio S.
      • Feldstein A.E.
      • et al.
      Role of TM6SF2 rs58542926 in the pathogenesis of nonalcoholic pediatric fatty liver disease: a multiethnic study.
      exhibiting a predilection or progression of NAFLD are displayed in Table 2.
      Table 2Meta-analyses and Prospective Evidence for the Association of Genetic Variants with NAFLD.
      GeneSNPStudy typeStudy populationNotable cohort characteristicsSample sizeDiagnostic modality
      ADIPOQrs266729
      • Wang J.
      • Guo X.F.
      • Yu S.J.
      • et al.
      Adiponectin polymorphisms and non-alcoholic fatty liver disease risk: a meta-analysis.
      Meta-analysisMultiethnic (primarily Asian)HB vs PB not reported9 retrospective case–control studies 1223 cases/1580 controlsLiver biopsy only in 3/9 studies
      APOC3rs2854116
      • Li M.R.
      • Zhang S.H.
      • Chao K.
      • et al.
      Apolipoprotein C3 (-455T>C) polymorphism confers susceptibility to nonalcoholic fatty liver disease in the Southern Han Chinese population.
      Prospective case–controlChinese67% male subjects300 cases/300 controlsUS
      rs2854116 and rs2854117
      • Zhang H.
      • Chen L.
      • Xin Y.
      • et al.
      Apolipoprotein c3 gene polymorphisms are not a risk factor for developing non-alcoholic Fatty liver disease: a meta-analysis.
      Meta-analysis
      This study demonstrated no association with NAFLD.
      Multiethnic5 HB vs 2 PB controls; 1 study included pediatric subjects; 2 studies did not report gender %7 prospective case–control studies 1745 cases/1437 controlsLiver biopsy only in 2/7 studies 1H-MRS or US in others
      GCKRrs780094
      • Zain S.M.
      • Mohamed Z.
      • Mohamed R.
      Common variant in the glucokinase regulatory gene rs780094 and risk of nonalcoholic fatty liver disease: a meta-analysis.
      Meta-analysisMultiethnic5 HB vs 2 PB controls; 1 of the Asian studies examines pediatric subjects5 retrospective case–control studies 2090 cases/3003 controlsLiver biopsy only in 3/5 studies
      MTHFRrs1801133
      • Sun M.Y.
      • Zhang L.
      • Shi S.L.
      • et al.
      Associations between methylenetetrahydrofolate reductase (MTHFR) polymorphisms and non-alcoholic fatty liver disease (NAFLD) risk: a meta-analysis.
      Meta-analysisMultiethnicAll 8 studies PB controls8 retrospective case–control studies 785 cases/1188 controlsLiver biopsy in 4/5 studies, not reported in Chinese and Ukrainian studies
      rs18101131
      • Sun M.Y.
      • Zhang L.
      • Shi S.L.
      • et al.
      Associations between methylenetetrahydrofolate reductase (MTHFR) polymorphisms and non-alcoholic fatty liver disease (NAFLD) risk: a meta-analysis.
      Meta-analysisMultiethnicAll 5 studies PB controls5 retrospective case–control studies 476 cases/679 controlsLiver biopsy in 3/4 studies, not reported in Ukrainian study
      MTTPrs1800591
      • Li L.
      • Wang S.J.
      • Shi K.
      • et al.
      Correlation between MTP -493G>T polymorphism and non-alcoholic fatty liver disease risk: a meta-analysis.
      Meta-analysisMultiethnic6 HB vs 5 PB controls11 retrospective case–control studies 636 cases/918 controlsNot reported
      PEMTrs7946
      • Tan H.L.
      • Mohamed R.
      • Mohamed Z.
      • et al.
      Phosphatidylethanolamine N-methyltransferase gene rs7946 polymorphism plays a role in risk of nonalcoholic fatty liver disease: evidence from meta-analysis.
      Meta-analysisMultiethnic (primarily Asian)4 HB vs 2 PB controls;

      1 study included pediatric subjects
      6 retrospective case–control studies 792 cases/2722 controlsLiver biopsy only in 2/6 studies
      PNPLA3rs738409
      • Sookoian S.
      • Pirola C.J.
      Meta-analysis of the influence of I148M variant of patatin-like phospholipase domain containing 3 gene (PNPLA3) on the susceptibility and histological severity of nonalcoholic fatty liver disease.
      Meta-analysisMultiethnic11 HB vs 5 PB controls; 5 studies included pediatric subjects16 retrospective case–control studies 12,677 totalLiver biopsy only in 6/16 studies 1H-MRS, US, or CT in others
      rs738409
      • Zhang L.
      • You W.
      • Zhang H.
      • et al.
      PNPLA3 polymorphisms (rs738409) and non-alcoholic fatty liver disease risk and related phenotypes: a meta-analysis.
      Meta-analysisAsian4 HB vs 8 PB controls12 retrospective case–control studies 4495 cases/7431 controlsLiver biopsy only in 5/12 studies
      rs738409
      • Xu R.
      • Tao A.
      • Zhang S.
      • et al.
      Association between patatin-like phospholipase domain containing 3 gene (PNPLA3) polymorphisms and nonalcoholic fatty liver disease: a HuGE review and meta-analysis.
      Meta-analysisMultiethnic15 HB vs 8 PB controls23 retrospective case–control studies 5826 cases/10,796 controlsLiver biopsy only in10/23 studies
      PPARγrs1805192 and rs1801282
      • Wang J.
      • Guo X.
      • Wu P.
      • et al.
      Association between the Pro12Ala polymorphism of PPAR-gamma gene and the non-alcoholic fatty liver disease: a meta-analysis.
      Meta-analysis
      This study demonstrated no association with NAFLD.
      Caucasian and Asian4 HB vs 4 PB controls8 retrospective case–control studies 1697 cases/2427 controlsLiver biopsy in 5/9 studies
      rs1805192 and rs1801282
      • Lee Y.H.
      • Bae S.C.
      • Song G.G.
      Meta-analysis of associations between the peroxisome proliferator-activated receptor-gamma Pro12Ala polymorphism and susceptibility to nonalcoholic fatty liver disease, rheumatoid arthritis, and psoriatic arthritis.
      Meta-analysis
      This study suggested association with NAFLD in the East Asian population, however not in the European population. ADIPOQ, Adiponectin; APOC3, Apolipoprotein C-III; CT, Computed Tomography; GCKR, Glucokinase Regulator; 1H-MRS, Proton Magnetic Resonance Spectroscopy; HB, Hospital-Based Controls; MTHFR, Methylenetetrahydrofolate REDUCTASE; MTTP, Microsomal Triglyceride Transfer Protein; PB, Population-Based Controls; PEMT, Phosphatidylethanolamine N-methyltransferase; PNPLA3, Patatin-like Phospholipase Domain Containing 3; PPARγ, Peroxisome Proliferator-Activated Receptor Gamma; SREBF-2, Sterol Regulatory Element-binding Factor 2; TM6SF2, Transmembrane 6 Superfamily Member 2; US, Ultrasonography.
      Caucasian and AsianHB vs PB not reported5 retrospective case–control studies 1225 cases/2016 controlsNot reported
      SREBF-2rs133291
      • Musso G.
      • Cassader M.
      • Bo S.
      • et al.
      Sterol regulatory element-binding factor 2 (SREBF-2) predicts 7-year NAFLD incidence and severity of liver disease and lipoprotein and glucose dysmetabolism.
      Prospective cohortItalian65% male subjects48 cases/127 controlsLiver biopsy
      TM6SF2rs58542926
      • Pirola C.J.
      • Sookoian S.
      The dual and opposite role of the TM6SF2-rs58542926 variant in protecting against cardiovascular disease and conferring risk for nonalcoholic fatty liver: a meta-analysis.
      Meta-analysisMultiethnic4 HB vs 3 PB vs 1 mixed controls;

      1 of the 2 Italian studies in pediatric
      8 retrospective case–control studies 5537 totalLiver biopsy only in 3/8 studies
      rs58542926
      • Goffredo M.
      • Caprio S.
      • Feldstein A.E.
      • et al.
      Role of TM6SF2 rs58542926 in the pathogenesis of nonalcoholic pediatric fatty liver disease: a multiethnic study.
      Prospective cohortMultiethnicPediatric cohort957 totalLiver biopsy only in 11 patients
      a This study demonstrated no association with NAFLD.
      b This study suggested association with NAFLD in the East Asian population, however not in the European population. ADIPOQ, Adiponectin; APOC3, Apolipoprotein C-III; CT, Computed Tomography; GCKR, Glucokinase Regulator; 1H-MRS, Proton Magnetic Resonance Spectroscopy; HB, Hospital-Based Controls; MTHFR, Methylenetetrahydrofolate REDUCTASE; MTTP, Microsomal Triglyceride Transfer Protein; PB, Population-Based Controls; PEMT, Phosphatidylethanolamine N-methyltransferase; PNPLA3, Patatin-like Phospholipase Domain Containing 3; PPARγ, Peroxisome Proliferator-Activated Receptor Gamma; SREBF-2, Sterol Regulatory Element-binding Factor 2; TM6SF2, Transmembrane 6 Superfamily Member 2; US, Ultrasonography.
      PNPLA3 rs738409 remains the most highly proclaimed genetic variant incriminate in the pathogenesis of NAFLD. To date, three large-scale meta-analyses
      • Sookoian S.
      • Pirola C.J.
      Meta-analysis of the influence of I148M variant of patatin-like phospholipase domain containing 3 gene (PNPLA3) on the susceptibility and histological severity of nonalcoholic fatty liver disease.
      • Zhang L.
      • You W.
      • Zhang H.
      • et al.
      PNPLA3 polymorphisms (rs738409) and non-alcoholic fatty liver disease risk and related phenotypes: a meta-analysis.
      • Xu R.
      • Tao A.
      • Zhang S.
      • et al.
      Association between patatin-like phospholipase domain containing 3 gene (PNPLA3) polymorphisms and nonalcoholic fatty liver disease: a HuGE review and meta-analysis.
      (Table 2) have been performed across a vast array of ethnicities and large sample populations, confirming the evidence for this relationship. Support from this data aligns with the intracellular actions of PNPLA3 as a lipase in both hepatocytes and hepatic stellate cells. Aberrant function of PNPLA3 rs738409 results in an absence of lipase activity, thus leading to intracellular TG or retinol accumulation in hepatocytes and hepatic stellate cells, respectively.
      • Chamoun Z.
      • Vacca F.
      • Parton R.G.
      • et al.
      PNPLA3/adiponutrin functions in lipid droplet formation.
      • He S.
      • McPhaul C.
      • Li J.Z.
      • et al.
      A sequence variation (I148M) in PNPLA3 associated with nonalcoholic fatty liver disease disrupts triglyceride hydrolysis.
      • Huang Y.
      • Cohen J.C.
      • Hobbs H.H.
      Expression and characterization of a PNPLA3 protein isoform (I148M) associated with nonalcoholic fatty liver disease.
      However a recent study has argued against this mechanism as knockout mice fail to develop steatosis and it is more likely to be a gain-of-function mutation that modulates the composition of hepatic lipid, resulting in lipotoxicity.
      • Basantani M.K.
      • Sitnick M.T.
      • Cai L.
      • et al.
      Pnpla3/adiponutrin deficiency in mice does not contribute to fatty liver disease or metabolic syndrome.
      The genetic variant in TM6SF2 rs58542926 on chromosome 19 (19p13.11) has been reported to correlate with steatosis and increased risk of advanced fibrosis in NAFLD patients,
      • Kozlitina J.
      • Smagris E.
      • Stender S.
      • et al.
      Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease.
      • Liu Y.L.
      • Reeves H.L.
      • Burt A.D.
      • et al.
      TM6SF2 rs58542926 influences hepatic fibrosis progression in patients with non-alcoholic fatty liver disease.
      independently of other factors, including diabetes, obesity, or PNPLA3 genotype. TM6SF2, is a transmembrane protein localized in ER and ER–Golgi compartments and functions as a lipid transporter.
      • Mahdessian H.
      • Taxiarchis A.
      • Popov S.
      • et al.
      TM6SF2 is a regulator of liver fat metabolism influencing triglyceride secretion and hepatic lipid droplet content.
      The amino acid change E167K causes loss of function of TM6SF2 protein, and downregulation of TM6SF2 reduces lipoproteins and apolipoprotein B (APOB) levels, and increases hepatic deposition of TGs and the amount and size of lipid droplets. In contrast, the size and number of lipid droplets diminishes when TM6SF2 is overexpressed, indicating that TM6SF2 plays a role in regulating hepatic lipid efflux.
      • Kozlitina J.
      • Smagris E.
      • Stender S.
      • et al.
      Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease.
      • Mahdessian H.
      • Taxiarchis A.
      • Popov S.
      • et al.
      TM6SF2 is a regulator of liver fat metabolism influencing triglyceride secretion and hepatic lipid droplet content.
      This genetic variant was first recognized after emerging as a leading candidate from the genome-wide exome study performed by Kozlitina et al.
      • Kozlitina J.
      • Smagris E.
      • Stender S.
      • et al.
      Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease.
      A myriad of studies have since validated its association with NAFLD, including one meta-analysis.
      • Pirola C.J.
      • Sookoian S.
      The dual and opposite role of the TM6SF2-rs58542926 variant in protecting against cardiovascular disease and conferring risk for nonalcoholic fatty liver: a meta-analysis.
      Despite a large sample size only three out of the eight studies confirmed NAFLD on liver biopsy, and one study from Italy included pediatric subjects.
      • Goffredo M.
      • Caprio S.
      • Feldstein A.E.
      • et al.
      Role of TM6SF2 rs58542926 in the pathogenesis of nonalcoholic pediatric fatty liver disease: a multiethnic study.
      Although the presence of several other genetic variants listed in Table 2 have proven a high propensity for NAFLD through retrospective evidence, only APOC3 rs2854116 and rs2854117 have been studied through meta-analysis of prospective data.
      • Zhang H.
      • Chen L.
      • Xin Y.
      • et al.
      Apolipoprotein c3 gene polymorphisms are not a risk factor for developing non-alcoholic Fatty liver disease: a meta-analysis.
      PPARγ rs1805192 and 1801282 have been examined in two meta-analyses. In the first meta-analysis, these genetic variants were found to have no association with NAFLD.
      • Wang J.
      • Guo X.
      • Wu P.
      • et al.
      Association between the Pro12Ala polymorphism of PPAR-gamma gene and the non-alcoholic fatty liver disease: a meta-analysis.
      The second meta-analysis provides somewhat weaker methodology, in that the incidence of hospital-based and population-based controls, as well as the diagnostic modality for NAFLD, was not reported.
      • Lee Y.H.
      • Bae S.C.
      • Song G.G.
      Meta-analysis of associations between the peroxisome proliferator-activated receptor-gamma Pro12Ala polymorphism and susceptibility to nonalcoholic fatty liver disease, rheumatoid arthritis, and psoriatic arthritis.
      Amidst these deficiencies, it was concluded that these genetic variants were associated with NAFLD in the East Asian population that was studied, but not the European population. Clearly, there is inconsistent data concerning the PPARγ rs1805192 and rs1801282 and further analysis is required regarding its role in NAFLD pathogenesis.
      Meta-analysis provides the highest level of evidence to date, however there is a multitude of cross-sectional studies analyzing the role of specific genetic variants in their association to NAFLD. Selected retrospective studies
      • Sookoian S.
      • Castano G.
      • Gianotti T.F.
      • et al.
      Polymorphisms of MRP2 (ABCC2) are associated with susceptibility to nonalcoholic fatty liver disease.
      • Kotronen A.
      • Yki-Jarvinen H.
      • Aminoff A.
      • et al.
      Genetic variation in the ADIPOR2 gene is associated with liver fat content and its surrogate markers in three independent cohorts.
      • Iwamoto N.
      • Ogawa Y.
      • Kajihara S.
      • et al.
      Gln27Glu beta2-adrenergic receptor variant is associated with hypertriglyceridemia and the development of fatty liver.
      • Nozaki Y.
      • Saibara T.
      • Nemoto Y.
      • et al.
      Polymorphisms of interleukin-1 beta and beta 3-adrenergic receptor in Japanese patients with nonalcoholic steatohepatitis.
      • Yoneda M.
      • Hotta K.
      • Nozaki Y.
      • et al.
      Association between angiotensin II type 1 receptor polymorphisms and the occurrence of nonalcoholic fatty liver disease.
      • Sazci A.
      • Akpinar G.
      • Aygun C.
      • et al.
      Association of apolipoprotein E polymorphisms in patients with non-alcoholic steatohepatitis.
      • Wang B.
      • Wang B.
      • Wang Y.
      • et al.
      Association of APPL1 gene polymorphism with non-alcoholic fatty liver disease susceptibility in a Chinese Han population.
      • Wang L.
      • Athinarayanan S.
      • Jiang G.
      • et al.
      Fatty acid desaturase 1 gene polymorphisms control human hepatic lipid composition.
      • Oliveira C.P.
      • Stefano J.T.
      • Cavaleiro A.M.
      • et al.
      Association of polymorphisms of glutamate-cystein ligase and microsomal triglyceride transfer protein genes in non-alcoholic fatty liver disease.
      • Carulli L.
      • Canedi I.
      • Rondinella S.
      • et al.
      Genetic polymorphisms in non-alcoholic fatty liver disease: interleukin-6-174G/C polymorphism is associated with non-alcoholic steatohepatitis.
      • Swellam M.
      • Hamdy N.
      Association of nonalcoholic fatty liver disease with a single nucleotide polymorphism on the gene encoding leptin receptor.
      • Zain S.M.
      • Mohamed Z.
      • Mahadeva S.
      • et al.
      Impact of leptin receptor gene variants on risk of non-alcoholic fatty liver disease and its interaction with adiponutrin gene.
      • Mancina R.M.
      • Dongiovanni P.
      • Petta S.
      • et al.
      The MBOAT7-TMC4 variant rs641738 increases risk of nonalcoholic fatty liver disease in individuals of European descent.
      • Gorden A.
      • Yang R.
      • Yerges-Armstrong L.M.
      • et al.
      Genetic variation at NCAN locus is associated with inflammation and fibrosis in non-alcoholic fatty liver disease in morbid obesity.
      • Wu G.
      • Wang K.
      • Xue Y.
      • et al.
      Association of rs5764455 and rs6006473 polymorphisms in PARVB with liver damage of nonalcoholic fatty liver disease in Han Chinese population.
      • Hernaez R.
      • McLean J.
      • Lazo M.
      • et al.
      Association between variants in or near PNPLA3, GCKR, and PPP1R3B with ultrasound-defined steatosis based on data from the third National Health and Nutrition Examination Survey.
      • Vazquez-Chantada M.
      • Gonzalez-Lahera A.
      • Martinez-Arranz I.
      • et al.
      Solute carrier family 2 member 1 is involved in the development of nonalcoholic fatty liver disease.
      • Huang Y.S.
      • Chang C.H.
      • Lin T.L.
      • et al.
      Genetic variations of superoxide dismutase 2 and cytochrome P450 2E1 in non-alcoholic steatohepatitis.
      • Musso G.
      • Gambino R.
      • Pacini G.
      • et al.
      Transcription factor 7-like 2 polymorphism modulates glucose and lipid homeostasis, adipokine profile, and hepatocyte apoptosis in NASH.
      • Kiziltas S.
      • Ata P.
      • Colak Y.
      • et al.
      TLR4 gene polymorphism in patients with nonalcoholic fatty liver disease in comparison to healthy controls.
      • Yan X.
      • Xu L.
      • Qi J.
      • et al.
      sTRAIL levels and TRAIL gene polymorphisms in Chinese patients with fatty liver disease.
      • Aller R.
      • De Luis D.A.
      • Izaola O.
      • et al.
      Role of -55CT polymorphism of UCP3 gene on non alcoholic fatty liver disease and insulin resistance in patients with obesity.
      • Yuan C.
      • Lu L.
      • An B.
      • et al.
      Association between LYPLAL1 rs12137855 polymorphism with ultrasound-defined non-alcoholic fatty liver disease in a Chinese Han population.
      • Chen L.
      • Lin Z.
      • Jiang M.
      • et al.
      Genetic variants in the SAMM50 gene create susceptibility to nonalcoholic fatty liver disease in a Chinese Han population.
      from each genetic variant are detailed here in Table 3, which have been performed over a variety of populations and ethnicities. Certainly, there are many genetic variants embroiled in this disease process, and many of which, warrant further investigation. However, LYPLAL1 rs12137855 provides an interesting context to NAFLD pathogenesis. After being implicated in the landmark GWAS by Speliotes et al.,
      • Speliotes E.K.
      • Yerges-Armstrong L.M.
      • Wu J.
      • et al.
      Genome-wide association analysis identifies variants associated with nonalcoholic fatty liver disease that have distinct effects on metabolic traits.
      this genetic variant was shown to have no association with NAFLD in a Chinese study population through retrospective case–control analysis.
      • Yuan C.
      • Lu L.
      • An B.
      • et al.
      Association between LYPLAL1 rs12137855 polymorphism with ultrasound-defined non-alcoholic fatty liver disease in a Chinese Han population.
      Results of these studies potentially could be biased related to power of these studies and could have been confounded by the quality of the study design.
      Table 3Retrospective Candidate Gene Studies Linking Genetic Variants to NAFLD.
      GeneSNPStudy populationNotable cohort characteristicsSample sizeDiagnostic modality
      ABCC2rs17222723, rs8187710
      • Sookoian S.
      • Castano G.
      • Gianotti T.F.
      • et al.
      Polymorphisms of MRP2 (ABCC2) are associated with susceptibility to nonalcoholic fatty liver disease.
      ArgentinePoor selection of controls109 cases/58 controlsUS
      ACSL4rs7887981
      • Kotronen A.
      • Yki-Jarvinen H.
      • Aminoff A.
      • et al.
      Genetic variation in the ADIPOR2 gene is associated with liver fat content and its surrogate markers in three independent cohorts.
      FinnishValidated in 2 replication cohorts302 total with replication cohorts1H-MRS
      ADIPOR2rs767870
      • Kotronen A.
      • Yki-Jarvinen H.
      • Aminoff A.
      • et al.
      Genetic variation in the ADIPOR2 gene is associated with liver fat content and its surrogate markers in three independent cohorts.
      FinnishValidated in 2 replication cohorts302 total with replication cohorts1H-MRS
      ADRB2rs1042714
      • Iwamoto N.
      • Ogawa Y.
      • Kajihara S.
      • et al.
      Gln27Glu beta2-adrenergic receptor variant is associated with hypertriglyceridemia and the development of fatty liver.
      Japanese132 cases/119 controlsUS
      ADRB3rs4994
      • Nozaki Y.
      • Saibara T.
      • Nemoto Y.
      • et al.
      Polymorphisms of interleukin-1 beta and beta 3-adrenergic receptor in Japanese patients with nonalcoholic steatohepatitis.
      JapaneseExclusively NASH cases63 cases/100 controlsCT
      AGTR1rs3772630, rs3772622
      • Yoneda M.
      • Hotta K.
      • Nozaki Y.
      • et al.
      Association between angiotensin II type 1 receptor polymorphisms and the occurrence of nonalcoholic fatty liver disease.
      Japanese80% males in NAFLD cases167 cases/435 controlsLiver biopsy
      APOErs7412, rs429358
      • Sazci A.
      • Akpinar G.
      • Aygun C.
      • et al.
      Association of apolipoprotein E polymorphisms in patients with non-alcoholic steatohepatitis.
      Turkish57 cases/245 controlsLiver biopsy
      APPL1rs4640525
      • Wang B.
      • Wang B.
      • Wang Y.
      • et al.
      Association of APPL1 gene polymorphism with non-alcoholic fatty liver disease susceptibility in a Chinese Han population.
      Chinese280 cases/281 controlsUnreported
      FADS1rs174556
      • Wang L.
      • Athinarayanan S.
      • Jiang G.
      • et al.
      Fatty acid desaturase 1 gene polymorphisms control human hepatic lipid composition.
      American and ChineseAnalysis of explanted liver samples206 transplantsHistology of donor livers
      GCLC/GCLMrs17883901
      • Oliveira C.P.
      • Stefano J.T.
      • Cavaleiro A.M.
      • et al.
      Association of polymorphisms of glutamate-cystein ligase and microsomal triglyceride transfer protein genes in non-alcoholic fatty liver disease.
      Brazilian131 cases/141 controlsLiver biopsy
      IL-1rs16944
      • Nozaki Y.
      • Saibara T.
      • Nemoto Y.
      • et al.
      Polymorphisms of interleukin-1 beta and beta 3-adrenergic receptor in Japanese patients with nonalcoholic steatohepatitis.
      JapaneseExclusively NASH cases63 cases/100 controlsCT
      IL-6rs1800795
      • Carulli L.
      • Canedi I.
      • Rondinella S.
      • et al.
      Genetic polymorphisms in non-alcoholic fatty liver disease: interleukin-6-174G/C polymorphism is associated with non-alcoholic steatohepatitis.
      ItalianPoor selection of controls114 cases/79 controlsLiver biopsy in 59/114 cases
      LEPRrs6700986
      • Swellam M.
      • Hamdy N.
      Association of nonalcoholic fatty liver disease with a single nucleotide polymorphism on the gene encoding leptin receptor.
      Egyptian90 cases/30 controlsLiver biopsy
      rs1137100, rs1137101
      • Zain S.M.
      • Mohamed Z.
      • Mahadeva S.
      • et al.
      Impact of leptin receptor gene variants on risk of non-alcoholic fatty liver disease and its interaction with adiponutrin gene.
      Malaysian, Chinese, Indian144 cases/198 controlsLiver biopsy
      LYPLAL1rs12137855
      • Yuan C.
      • Lu L.
      • An B.
      • et al.
      Association between LYPLAL1 rs12137855 polymorphism with ultrasound-defined non-alcoholic fatty liver disease in a Chinese Han population.
      Chinese184 cases/114 controlsUS
      MBOAT7rs641738
      • Mancina R.M.
      • Dongiovanni P.
      • Petta S.
      • et al.
      The MBOAT7-TMC4 variant rs641738 increases risk of nonalcoholic fatty liver disease in individuals of European descent.
      American and European3854 total in Dallas Heart Study, 1149 total in Liver Biopsy CohortLiver biopsy and 1H-MRS
      NCANrs2228603
      • Gorden A.
      • Yang R.
      • Yerges-Armstrong L.M.
      • et al.
      Genetic variation at NCAN locus is associated with inflammation and fibrosis in non-alcoholic fatty liver disease in morbid obesity.
      AmericanBariatric cohort; 80% female subjects; Ethnicities not reported748 cases/344 controlsLiver biopsy
      PARVBrs5764455, rs6006473
      • Wu G.
      • Wang K.
      • Xue Y.
      • et al.
      Association of rs5764455 and rs6006473 polymorphisms in PARVB with liver damage of nonalcoholic fatty liver disease in Han Chinese population.
      Chinese60% male subjects384 cases/384 controlsUS
      PPP1R3Brs4240624
      • Hernaez R.
      • McLean J.
      • Lazo M.
      • et al.
      Association between variants in or near PNPLA3, GCKR, and PPP1R3B with ultrasound-defined steatosis based on data from the third National Health and Nutrition Examination Survey.
      AmericanPopulation-based4804 totalUS
      SAMM50rs738491, rs2143571, rs3761472
      • Chen L.
      • Lin Z.
      • Jiang M.
      • et al.
      Genetic variants in the SAMM50 gene create susceptibility to nonalcoholic fatty liver disease in a Chinese Han population.
      Chinese340 cases/452 controlsUS
      SLC2A1rs4658, rs841856
      • Vazquez-Chantada M.
      • Gonzalez-Lahera A.
      • Martinez-Arranz I.
      • et al.
      Solute carrier family 2 member 1 is involved in the development of nonalcoholic fatty liver disease.
      SpanishPoor selection of controls451 cases/304 controlsLiver biopsy
      SOD2rs4880
      • Huang Y.S.
      • Chang C.H.
      • Lin T.L.
      • et al.
      Genetic variations of superoxide dismutase 2 and cytochrome P450 2E1 in non-alcoholic steatohepatitis.
      Chinese131 cases/90 controlsLiver biopsy
      TCF7L2rs7903146
      • Musso G.
      • Gambino R.
      • Pacini G.
      • et al.
      Transcription factor 7-like 2 polymorphism modulates glucose and lipid homeostasis, adipokine profile, and hepatocyte apoptosis in NASH.
      Italian∼75% male subjects78 cases/156 controlsLiver biopsy in 34/78 cases
      TLR4rs4986790
      • Kiziltas S.
      • Ata P.
      • Colak Y.
      • et al.
      TLR4 gene polymorphism in patients with nonalcoholic fatty liver disease in comparison to healthy controls.
      Turkish119 cases/80 controlsLiver biopsy in 111/119 cases
      TRAILrs1131568, rs1131580
      • Yan X.
      • Xu L.
      • Qi J.
      • et al.
      sTRAIL levels and TRAIL gene polymorphisms in Chinese patients with fatty liver disease.
      Chinese84 cases/80 controlsUnreported
      UCP3rs11235972
      • Aller R.
      • De Luis D.A.
      • Izaola O.
      • et al.
      Role of -55CT polymorphism of UCP3 gene on non alcoholic fatty liver disease and insulin resistance in patients with obesity.
      Spanish39 casesLiver biopsy
      11β-HSD1rs2235543, rs12565406, rs4844880
      • Lutz S.Z.
      • Peter A.
      • Machicao F.
      • et al.
      Genetic variation in the 11beta-hydroxysteroid-dehydrogenase 1 gene determines NAFLD and visceral obesity.
      German100% Caucasian

      All subjects had family history of T2DM, BMI > 27, or impaired glucose tolerance testing
      327 total subjects1H-MRS
      CD14C159T polymorphism
      • Kapil S.
      • Duseja A.
      • Sharma B.K.
      • et al.
      Genetic polymorphism in CD14 gene, a co-receptor of TLR4 associated with non-alcoholic fatty liver disease.
      Indian64% male subjects200 NAFLD/50 controlsUS
      Omentin-1rs2274907
      • Kohan L.
      • Safarpur M.
      • Abdollahi H.
      Omentin-1 rs2274907 and resistin rs1862513 polymorphisms influence genetic susceptibility to nonalcoholic fatty liver disease.
      IranianSignificantly lower BMI in control group94 NAFLD/188 controlsUS
      PTPRDrs35929428
      • Nakajima S.
      • Tanaka H.
      • Sawada K.
      • et al.
      Polymorphism of receptor-type tyrosine-protein phosphatase delta gene in the development of non-alcoholic fatty liver disease.
      JapaneseSignificantly lower age in control group36 NAFLD/27 controlsLiver biopsy
      Resistinrs1862513
      • Kohan L.
      • Safarpur M.
      • Abdollahi H.
      Omentin-1 rs2274907 and resistin rs1862513 polymorphisms influence genetic susceptibility to nonalcoholic fatty liver disease.
      IranianSignificantly lower BMI in control group94 NAFLD/188 controlsUS
      a These studies have demonstrated no association with NAFLD.
      ABCC2, ATP-binding Cassette, Sub-family C, Member 2; ACSL4, Acyl-coA Synthase Long Chain Family Member 4; ADIPOR2, Adiponectin Receptor 2; ADRB2, Adrenergic Beta 2 Receptor; ADRB3, Adrenergic Beta 3 Receptor; AGTR1, Angiotensin II Type 1 Receptor; APOE, Apolipoprotein E; APPL1, Adaptor Protein, Phosphotyrosine Interacting with PH Domain and Leucine Zipper 1; CT, Computed Tomography; FADS1, Fatty Acid Desaturase 1; GCLC/GCLM, Glutamate-cysteine Ligase; 1H-MRS, Proton Magnetic Resonance Spectroscopy; IL-1, Interleukin-1; IL-6, Interleukin-6; LEPR, Leptin Receptor; LYPLAL1, Lysophospholipase-like 1; MBOAT7, Membrane Bound O-acyltransferase Domain-containing 7 Gene; NCAN, Neurocan; PARVB, Parvin Beta; PPP1R3B, Protein Phosphatase 1 Regulatory Subunit 3B; SAMM50, Sorting and Assembly Machinery Component 50; SLC2A1, Solute Carrier Family 2 Member 1; SOD2, Superoxide Dismutase 2, Mitochondrial; TCF7L2, Transcription Factor 7-like 2; TLR4, Toll-like Receptor 4; TRAIL, TNF-related Apoptosis-inducing Ligand; UCP3, Uncoupling Protein 3, Mitochondrial; 11β-HSD1, 11β-Hydroxysteroid Dehydrogenase Type 1; CD14, Coreceptor Cluster of Differentiation 14; PTPRD, Receptor-type Tyrosine-protein Phosphatase Delta; US, Ultrasonography.

      Epigenetic Modifications Implicated in NAFLD Pathogenesis

      Epigenetic changes consist in modifications at the transcriptional level affecting gene expression and phenotype. A number of epigenetic aberrations have been associated with NAFLD pathogenesis, causing alterations in lipid metabolism, Insulin Resistance (IR), dysfunction of ER and mitochondria, oxidative stress and inflammation. As opposed to targeting changes in the genetic sequence and analyzing SNPs associated with NAFLD, a growing interest in recent scientific studies have focused on mechanisms responsible for modified expression or translation of genes related to the disease process. Interestingly, it is unclear if the altered gene expression causes the disease or the disease itself drives the modifications of gene expression. Nevertheless, two major gene expression modifying mechanisms, MicroRNA (miRNA) and epigenetics are found to be associated with NAFLD.

      Analysis of miRNA Mediated Modifications

      miRNAs modulate gene expression via post-transcriptional mechanisms, regulating the main cellular processes, such as lipid metabolism, inflammation, apoptosis, cell growth and differentiation. Aberrant miRNA expression has been reported in a number of diseases including metabolic disorders,
      • Rottiers V.
      • Naar A.M.
      MicroRNAs in metabolism and metabolic disorders.
      • Jones A.
      • Danielson K.M.
      • Benton M.C.
      • et al.
      miRNA signatures of insulin resistance in obesity.
      whereas an increasing number of dysregulated miRNAs, implicated in fatty acid synthesis, uptake and storage of TGs or oxidation, have been recently identified in NAFLD.
      • Ferreira D.M.
      • Simao A.L.
      • Rodrigues C.M.
      • et al.
      Revisiting the metabolic syndrome and paving the way for microRNAs in non-alcoholic fatty liver disease.
      miRNAs have been the most comprehensively studied epigenetic mechanism in relation to NAFLD. There have been numerous miRNAs implicated in this pathology and demonstrated through mouse models, however only the associations found in humans have been listed in Table 4. In general, miRNAs listed here exhibit their action by modifying the expression or downstream effects of genes correlated with lipid metabolism, inflammation, and fibrogenesis. Presently, the most highly touted entity of this group has been miR-122, which constitutes over 70% of hepatic miRNA expression.
      • Elmen J.
      • Lindow M.
      • Schutz S.
      • et al.
      LNA-mediated microRNA silencing in non-human primates.
      One of its primary targets serves to decrease the expression of numerous genes critical for lipid metabolism, such as FASN, ACC2, SCD1, and ACLY.
      • Esau C.
      • Davis S.
      • Murray S.F.
      • et al.
      miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting.
      There have been three major studies examining the effects of this miRNA on NAFLD in human subjects. The first two demonstrated an association between miR-122 expression and NAFLD, however subsequent review exposed discordance between the levels miR-122 measured in hepatocytes and the serum.
      • Cermelli S.
      • Ruggieri A.
      • Marrero J.A.
      • et al.
      Circulating microRNAs in patients with chronic hepatitis C and non-alcoholic fatty liver disease.
      • Cheung O.
      • Puri P.
      • Eicken C.
      • et al.
      Nonalcoholic steatohepatitis is associated with altered hepatic MicroRNA expression.
      This phenomenon remained relatively unchallenged until the landmark investigation by Pirola et al.,
      • Pirola C.J.
      • Fernandez Gianotti T.
      • Castano G.O.
      • et al.
      Circulating microRNA signature in non-alcoholic fatty liver disease: from serum non-coding RNAs to liver histology and disease pathogenesis.
      Serum levels of miR-122 were once again demonstrated to be elevated in individuals with isolated steatosis or NASH as compared to controls. However, the hepatic expression was significantly downregulated in NAFLD, particularly in more severe cases such as NASH. Immunohistochemical staining revealed that miR-122 expression was restricted to the periphery of lipid-laden hepatocytes, suggesting they were in the process of exporting out of the hepatocytes into the circulation. Not only was this finding independently significant, but the serum levels of miR-122 were also found to correlate with hepatocellular ballooning and fibrosis in addition to serving as a superior biomarker for NAFLD than aminotransferases and CK-18, which has gained traction as a novel biomarker for liver disease. These groundbreaking findings suggest the export of miR-122 from hepatocytes into the circulation detects the underlying lack of inhibition on lipogenic gene expression and NAFLD pathology. miRNAs, such as miR-122, still demand further investigation, yet provide novel targets for NAFLD therapy in the future.
      Table 4Epigenetic and miRNA Targets Correlated with NAFLD.
      GeneStudy populationNotable cohort characteristicsSample sizeDiagnostic modality
      miR-16, 34a, 122
      • Elmen J.
      • Lindow M.
      • Schutz S.
      • et al.
      LNA-mediated microRNA silencing in non-human primates.
      American91% male Unreported ethnicities34 cases/19 controlsLiver biopsy
      miR-122
      • Cheung O.
      • Puri P.
      • Eicken C.
      • et al.
      Nonalcoholic steatohepatitis is associated with altered hepatic MicroRNA expression.
      American82% female 82% Caucasian25 NASH/25 controlsLiver biopsy
      miR-122, 192, 375
      • Pirola C.J.
      • Fernandez Gianotti T.
      • Castano G.O.
      • et al.
      Circulating microRNA signature in non-alcoholic fatty liver disease: from serum non-coding RNAs to liver histology and disease pathogenesis.
      ArgentineEthnicities not reported16 NASH/16 isolated steatosis/16 controlsLiver biopsy
      ACLY, GALNTL4, GRID1, IGF1, IGFBP2, IP6K3, PC, PLCG1, PRKCE
      • Ahrens M.
      • Ammerpohl O.
      • von Schonfels W.
      • et al.
      DNA methylation analysis in nonalcoholic fatty liver disease suggests distinct disease-specific and remodeling signatures after bariatric surgery.
      German and SwissMorbidly obese patients undergoing bariatric surgery45 cases/18 controlsLiver biopsy or histology from intraoperative sample
      CASP1, FGFR2, MAT1A, MTHFD2
      • Murphy S.K.
      • Yang H.
      • Moylan C.A.
      • et al.
      Relationship between methylome and transcriptome in patients with nonalcoholic fatty liver disease.
      American∼100% Caucasian56 cases/25 controlsLiver biopsy
      Collagen 1A1, PDGFα, PPARα, PPARδ, TGFβ1
      • Zeybel M.
      • Hardy T.
      • Robinson S.M.
      • et al.
      Differential DNA methylation of genes involved in fibrosis progression in non-alcoholic fatty liver disease and alcoholic liver disease.
      British100% male17 cases (no controls, just comparing mild to severe NAFLD)Liver biopsy
      MT-ND6
      • Pirola C.J.
      • Gianotti T.F.
      • Burgueno A.L.
      • et al.
      Epigenetic modification of liver mitochondrial DNA is associated with histological severity of nonalcoholic fatty liver disease.
      ArgentineEthnicities not reported45 cases/18 controlsLiver biopsy
      TET1, TET2
      • Pirola C.J.
      • Scian R.
      • Gianotti T.F.
      • et al.
      Epigenetic modifications in the biology of nonalcoholic fatty liver disease: the role of DNA hydroxymethylation and TET proteins.
      ArgentineEthnicities not reported67 cases/23 controlsLiver biopsy (only US in controls)
      TFAM
      • Sookoian S.
      • Rosselli M.S.
      • Gemma C.
      • et al.
      Epigenetic regulation of insulin resistance in nonalcoholic fatty liver disease: impact of liver methylation of the peroxisome proliferator-activated receptor gamma coactivator 1alpha promoter.
      ArgentineEthnicities not reported63 cases/11 controlsLiver biopsy
      lnc-JAM2-6
      • Sookoian S.
      • Rohr C.
      • Salatino A.
      • et al.
      Genetic variation in long noncoding RNAs and the risk of nonalcoholic fatty liver disease.
      ArgentineNone32 NAFL/32 NASH/32 controlsLiver biopsy
      PPARγ
      • Hardy T.
      • Zeybel M.
      • Day C.P.
      • et al.
      Plasma DNA methylation: a potential biomarker for stratification of liver fibrosis in non-alcoholic fatty liver disease.
      British2 subjects previously transplanted for “cirrhotic NAFLD” were included26 NAFLD/26 controlsLiver biopsy
      ACLY, ATP Citrate Lyase; CASP1, Caspase 1; Collagen 1A1, Collagen Type 1 Alpha 1; FGFR2, Fibroblast Growth Receptor 2; GALNTL4, UDP-N-acetyl-alpha-d-Galactosamine:Polypeptide N-Acetylgalactosaminyltransferase-like 4; GRID1, Glutamate Receptor, Ionotropic, Delta 1; HDAC3, Histone Deacetylase 3; HDAC8, Histone Deacetylase 8; IGF1, Insulin Like Growth Factor 1; IGFBP2, Insulin Like Growth Factor Binding Protein 2; IP6K3, Inositol Hexakisphosphate Kinase 3; MAT1A, Methionine Adenosyl Methyltransferase 1A, MT-ND6, Mitochondrially Encoded NADH Dehydrogenase 6; MTHFD2, Methylenetetrahydrofolate Dehydrogenase 2; PC, Pyruvate Carboxylase; PDGFα, Platelet Derived Growth Factor Alpha; PPARGC1α, PPARG Coactivator 1 Alpha; PLCG1, Phospholipase C Gamma 1; PPARα, Peroxisome Proliferator Activated Receptor Alpha; PPARδ, Peroxisome Proliferator Activated Receptor Delta, PRKCE, Protein Kinase C, Epsilon; SIRT1, Sirtuin 1; SIRT6, Sirtuin 6; TET1, Ten-Eleven Translocation Methylcytosine Dioxygenase 1; TET2, Ten-Eleven Translocation Methylcytosine Dioxygenase 2; TFAM, Transcription Factor A, Mitochondrial; TGFβ1, Transforming Growth Factor Beta 1; lncRNAs, Long Noncoding RNAs; PPARγ, Peroxisome Proliferator Activate Receptor Gamma; US, Ultrasonography.

      Analysis of Epigenetic Modifications

      While the seemingly small amount of data regarding miRNAs has yet provided new insight into this pathologic mechanism, epigenetic modifications have been characterized to an even lesser degree. Such epigenetic modifications are driven by DNA methylation. Defects within this epigenetic mechanism have not been studied as thoroughly as other genetic variants, but many have been correlated with NAFLD. Much like for the miRNAs, there has been a great deal of work in mouse models, but only those significant studies performed in human subjects
      • Elmen J.
      • Lindow M.
      • Schutz S.
      • et al.
      LNA-mediated microRNA silencing in non-human primates.
      • Cheung O.
      • Puri P.
      • Eicken C.
      • et al.
      Nonalcoholic steatohepatitis is associated with altered hepatic MicroRNA expression.
      • Pirola C.J.
      • Fernandez Gianotti T.
      • Castano G.O.
      • et al.
      Circulating microRNA signature in non-alcoholic fatty liver disease: from serum non-coding RNAs to liver histology and disease pathogenesis.
      • Ahrens M.
      • Ammerpohl O.
      • von Schonfels W.
      • et al.
      DNA methylation analysis in nonalcoholic fatty liver disease suggests distinct disease-specific and remodeling signatures after bariatric surgery.
      • Murphy S.K.
      • Yang H.
      • Moylan C.A.
      • et al.
      Relationship between methylome and transcriptome in patients with nonalcoholic fatty liver disease.
      • Zeybel M.
      • Hardy T.
      • Robinson S.M.
      • et al.
      Differential DNA methylation of genes involved in fibrosis progression in non-alcoholic fatty liver disease and alcoholic liver disease.
      • Pirola C.J.
      • Gianotti T.F.
      • Burgueno A.L.
      • et al.
      Epigenetic modification of liver mitochondrial DNA is associated with histological severity of nonalcoholic fatty liver disease.
      • Pirola C.J.
      • Scian R.
      • Gianotti T.F.
      • et al.
      Epigenetic modifications in the biology of nonalcoholic fatty liver disease: the role of DNA hydroxymethylation and TET proteins.
      • Sookoian S.
      • Rosselli M.S.
      • Gemma C.
      • et al.
      Epigenetic regulation of insulin resistance in nonalcoholic fatty liver disease: impact of liver methylation of the peroxisome proliferator-activated receptor gamma coactivator 1alpha promoter.
      have been outlined here in Table 4. The first study analyzed morbidly obese patients undergoing bariatric surgery through a retrospective case–control study.
      • Ahrens M.
      • Ammerpohl O.
      • von Schonfels W.
      • et al.
      DNA methylation analysis in nonalcoholic fatty liver disease suggests distinct disease-specific and remodeling signatures after bariatric surgery.
      After histologic analysis of livers taken from both NAFLD and control patients, a correlation was found between the methylation of several genes with the existence of NAFLD. However, this data is very selective in that the subjects were morbidly obese and undergoing major surgical intervention. Furthermore, the control samples in this study were taken from subjects undergoing major oncologic surgery. This garners data in a very highly specific clinical scenario and may not accurately provide insight to the general population of patients with NAFLD. The next two major studies established a correlation between methylation of several target genes and the diagnosis of NAFLD based upon liver biopsy is presented in Table 4.
      • Murphy S.K.
      • Yang H.
      • Moylan C.A.
      • et al.
      Relationship between methylome and transcriptome in patients with nonalcoholic fatty liver disease.
      • Zeybel M.
      • Hardy T.
      • Robinson S.M.
      • et al.
      Differential DNA methylation of genes involved in fibrosis progression in non-alcoholic fatty liver disease and alcoholic liver disease.
      However, a great portion of this data vested its analysis in the epigenetic differences among various degrees of NAFLD severity, or increased fibrosis. Furthermore, it has recently been shown that DNA methylation as detected in the plasma can serve as an accurate biomarker for hepatic fibrosis in NAFLD.
      • Hardy T.
      • Zeybel M.
      • Day C.P.
      • et al.
      Plasma DNA methylation: a potential biomarker for stratification of liver fibrosis in non-alcoholic fatty liver disease.
      Certainly there is promise for NAFLD association with epigenetic mechanisms such as these, but many more studies with stronger methodology and a higher level of evidence must be completed in order to confirm this association.
      Lastly, there has been novel exploration into the remaining genome and its influence on NAFLD development and severity. Analysis of Long Noncoding RNAs (lncRNA) have provided significant intrigue in regards to their transcriptional and epigenetic influence over this pathophysiology. Most recently, one specific lncRNA, lnc-JAM2-6, was associated with NAFLD and disease severity, potentially through interactions with oncogenes MAFK and JUND and the transcription factor CEBPB that is central to the inflammatory mechanism.
      • Sookoian S.
      • Rohr C.
      • Salatino A.
      • et al.
      Genetic variation in long noncoding RNAs and the risk of nonalcoholic fatty liver disease.
      This study represents a landmark trial in terms of genomic analysis among NAFLD subjects and may have pioneered methodology for future studies.
      Emerging single-cell epigenomic methods are being developed with the exciting potential to transform our knowledge of gene regulation. High-throughput sequencing has revolutionized the field of epigenetics with methods for genome-wide mapping of DNA methylation, histone modifications, chromatin accessibility, and chromosome conformation. Initially, the input requirements for these methods meant that samples containing hundreds of thousands or millions of cells were required; but in the last couple of years this has changed with numerous epigenetic features now assayable at the single-cell level.
      • Clark S.J.
      • Lee H.J.
      • Smallwood S.A.
      • et al.
      Single-cell epigenomics: powerful new methods for understanding gene regulation and cell identity.

      Pharmacogenomics

      Although genetic makeup of an individual does not fully explain the disease phenotype and natural history of NAFLD, the utility of genetic data on assessing the responsiveness to various therapeutic interventions in NAFLD is emerging. This personalized treatment, or pharmacogenomics, supplies a pragmatic treatment option in NAFLD given the heterogeneity of the disease phenotype, with contributions of host and environmental factors. Furthermore, the genotype of an individual has a potential to change the metabolism of dietary components and also the pharmacodynamics of the medications used for NAFLD patients. For example, de novo lipogenesis and hepatic fat content in patients with I148M variant, or GG polymorphism, of the PNPLA3 gene have been associated with intake of sugars and sweetened beverages
      • Davis J.N.
      • Le K.A.
      • Walker R.W.
      • et al.
      Increased hepatic fat in overweight Hispanic youth influenced by interaction between genetic variation in PNPLA3 and high dietary carbohydrate and sugar consumption.
      • Sevastianova K.
      • Santos A.
      • Kotronen A.
      • et al.
      Effect of short-term carbohydrate overfeeding and long-term weight loss on liver fat in overweight humans.
      and hepatotoxicity due to imbalance of the ratio of omega-6 to omega-3 fatty acids.
      • Santoro N.
      • Savoye M.
      • Kim G.
      • et al.
      Hepatic fat accumulation is modulated by the interaction between the rs738409 variant in the PNPLA3 gene and the dietary omega6/omega3 PUFA intake.
      On the other hand, this variant was also associated with reduced hepatic steatosis and fat content with increased consumption of vegetables in diet
      • Nobili V.
      • Liccardo D.
      • Bedogni G.
      • et al.
      Influence of dietary pattern, physical activity, and I148M PNPLA3 on steatosis severity in at-risk adolescents.
      and with weight loss.
      • Sevastianova K.
      • Kotronen A.
      • Gastaldelli A.
      • et al.
      Genetic variation in PNPLA3 (adiponutrin) confers sensitivity to weight loss-induced decrease in liver fat in humans.
      Although most studies have shown benefit of statins across the entire NAFLD spectrum, this benefit is blunted in patients with GG polymorphism of the PNPLA3 gene.
      • Dongiovanni P.
      • Petta S.
      • Mannisto V.
      • et al.
      Statin use and non-alcoholic steatohepatitis in at risk individuals.
      Potential beneficial effects of statins in NAFLD and other chronic liver diseases are based on low quality RCTs, and needs further studies before can be routinely recommended.
      • Kamal S.
      • Khan M.A.
      • Seth A.
      • et al.
      Beneficial effects of statins on the rates of hepatic fibrosis, hepatic decompensation, and mortality in chronic liver disease: a systematic review and meta-analysis.
      In another study, a marginal association was shown between V433M genotype of CYP4F2, a primary component of vitamin E metabolism, and improvement in NAFLD activity score without impact on vitamin E levels, suggesting another potential mechanism of this benefit in patients with this specific genotype.
      • Athinarayanan S.
      • Wei R.
      • Zhang M.
      • et al.
      Genetic polymorphism of cytochrome P450 4F2, vitamin E level and histological response in adults and children with nonalcoholic fatty liver disease who participated in PIVENS and TONIC clinical trials.
      As the fields of genetic testing and therapies for NAFLD evolve, pharmacogenomics and personalized therapy is going to become increasingly more relevant in tailoring the diet, exercise regimen, and use of pharmacological treatment options for managing patients with NAFLD.

      Conclusion

      The exact mechanism of NAFLD pathogenesis still remains intricately obscure with interplay among environmental factors, individual genetic variants, or alterations in the intestinal microbiome to provide an environment which is susceptible for the development of NAFLD. Several genetic variants have been implicated in this disease process, however their specificity remains unknown. Certain genetic variants, such as PNPLA3 rs738409 and TM6SF2 rs58542926, have been repeatedly demonstrated to be closely linked in this disease process, however it is critical to note that causality cannot be established from these studies. While currently there is a great deal of unknown, identification of known genetic variants will help tailor treatment strategies for these high risk patients in the future. Rigorous prospective investigation of these genetic variants are needed in biopsy proven NAFLD patients in order to firmly establish which of those genetic components serve as the primary culprit of NAFLD pathogenesis, in addition to the progression of NASH to advanced fibrosis, cirrhosis and HCC.

      Conflicts of Interest

      SKS has received Grant/research support from Biotest, Conatus, Genfit, Gilead Sciences, Intercept, and Shire; served on the Advisory board or as consultant for Abbvie, Gilead Sciences, and Intercept; and on the speakers bureau for Intercept, and Alexion. The other authors declare no related conflicts of interest.

      Acknowledgments

      We would like to thank Jennifer N. Gentry, CMI for her brilliant work with the medical illustrations she was able to provide. We also wish to thank Dr. Pamela B. Sylvestre, MD of University of Tennessee Health Science Center for generously providing slides in order to visualize NAFLD histology. In addition, we would like to acknowledge Wilfried J.J. Karmaus, MD, MPH for his genetic and epidemiologic expertise in the editing of this paper.

      References

        • Caldwell S.
        • Argo C.
        The natural history of non-alcoholic fatty liver disease.
        Dig Dis. 2010; 28: 162-168
        • Matteoni C.A.
        • Younossi Z.M.
        • Gramlich T.
        • et al.
        Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity.
        Gastroenterology. 1999; 116: 1413-1419
        • Adams L.A.
        • Lymp J.F.
        • St Sauver J.
        • et al.
        The natural history of nonalcoholic fatty liver disease: a population-based cohort study.
        Gastroenterology. 2005; 129: 113-121
        • Angulo P.
        • Kleiner D.E.
        • Dam-Larsen S.
        • et al.
        Liver fibrosis, but no other histologic features, associates with long-term outcomes of patients with nonalcoholic fatty liver disease.
        Gastroenterology. 2015;
        • Alpers D.H.
        • Sabesin S.M.
        • White H.M.
        Fatty liver; biochemical and clinical aspects.
        in: Shiff L. Shiff E. Disease of the Liver. Lippincott, Philadelphia1993: 825-855
        • Sanyal A.J.
        • Campbell-Sargent C.
        • Mirshahi F.
        • et al.
        Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities.
        Gastroenterology. 2001; 120: 1183-1192
        • Marchesini G.
        • Pagotto U.
        • Bugianesi E.
        • et al.
        Low ghrelin concentrations in nonalcoholic fatty liver disease are related to insulin resistance.
        J Clin Endocrinol Metab. 2003; 88: 5674-5679
        • Chitturi S.
        • Abeygunasekera S.
        • Farrell G.C.
        • et al.
        NASH and insulin resistance: insulin hypersecretion and specific association with the insulin resistance syndrome.
        Hepatology. 2002; 35: 373-379
        • Lotta L.A.
        • Gulati P.
        • Day F.R.
        • et al.
        Integrative genomic analysis implicates limited peripheral adipose storage capacity in the pathogenesis of human insulin resistance.
        Nat Genet. 2017; 49: 17-26
        • Gastaldelli A.
        • Cusi K.
        • Pettiti M.
        • et al.
        Relationship between hepatic/visceral fat and hepatic insulin resistance in nondiabetic and type 2 diabetic subjects.
        Gastroenterology. 2007; 133: 496-506
        • Lomonaco R.
        • Ortiz-Lopez C.
        • Orsak B.
        • et al.
        Effect of adipose tissue insulin resistance on metabolic parameters and liver histology in obese patients with nonalcoholic fatty liver disease.
        Hepatology. 2012; 55: 1389-1397
        • Pagano G.
        • Pacini G.
        • Musso G.
        • et al.
        Nonalcoholic steatohepatitis, insulin resistance, and metabolic syndrome: further evidence for an etiologic association.
        Hepatology. 2002; 35: 367-372
        • Koek G.H.
        • Liedorp P.R.
        • Bast A.
        The role of oxidative stress in non-alcoholic steatohepatitis.
        Clin Chim Acta. 2011; 412: 1297-1305
        • Kujoth G.C.
        • Hiona A.
        • Pugh T.D.
        • et al.
        Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging.
        Science. 2005; 309: 481-484
        • Lee J.
        • Ozcan U.
        Unfolded protein response signaling and metabolic diseases.
        J Biol Chem. 2014; 289: 1203-1211
        • Ozcan U.
        • Cao Q.
        • Yilmaz E.
        • et al.
        Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes.
        Science. 2004; 306: 457-461
        • Fuchs M.
        • Sanyal A.J.
        Lipotoxicity in NASH.
        J Hepatol. 2012; 56: 291-293
        • Polyzos S.A.
        • Kountouras J.
        • Mantzoros C.S.
        Adipokines in nonalcoholic fatty liver disease.
        Metabolism. 2016; 65: 1062-1079
        • Flier J.S.
        • Maratos-Flier E.
        Biology of obesity.
        in: Kasper D. Fauci A. Hauser S. Harrison's Principles of Internal Medicine. 19the ed. McGraw-Hill Education, New York, NY2015
        • Miele L.
        • Valenza V.
        • La Torre G.
        • et al.
        Increased intestinal permeability and tight junction alterations in nonalcoholic fatty liver disease.
        Hepatology. 2009; 49: 1877-1887
        • Vijay-Kumar M.
        • Aitken J.D.
        • Carvalho F.A.
        • et al.
        Metabolic syndrome and altered gut microbiota in mice lacking Toll-like receptor 5.
        Science. 2010; 328: 228-231
        • Shapiro M.D.
        Rare genetic disorders altering lipoproteins.
        in: De Groot L.J. Chrousos G. Dungan K. Feingold K.R. Grossman A. Hershman J.M. Koch C. Korbonits M. McLachlan R. New M. Purnell J. Rebar R. Singer F. Vinik A. Endotext. MDText.com, Inc., South Dartmouth, MA2000
        • Erwin A.L.
        The role of sebelipase alfa in the treatment of lysosomal acid lipase deficiency.
        Therap Adv Gastroenterol. 2017; 10: 553-562
        • Loomba R.
        • Schork N.
        • Chen C.H.
        • et al.
        Heritability of hepatic fibrosis and steatosis based on a prospective twin study.
        Gastroenterology. 2015; 149: 1784-1793
        • Stender S.
        • Kozlitina J.
        • Nordestgaard B.G.
        • et al.
        Adiposity amplifies the genetic risk of fatty liver disease conferred by multiple loci.
        Nat Genet. 2017; 49: 842-847
        • Romeo S.
        • Kozlitina J.
        • Xing C.
        • et al.
        Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.
        Nat Genet. 2008; 40: 1461-1465
        • Chalasani N.
        • Guo X.
        • Loomba R.
        • et al.
        Genome-wide association study identifies variants associated with histologic features of nonalcoholic Fatty liver disease.
        Gastroenterology. 2010; 139 (1576.e1–6): 1567-1576
        • Speliotes E.K.
        • Yerges-Armstrong L.M.
        • Wu J.
        • et al.
        Genome-wide association analysis identifies variants associated with nonalcoholic fatty liver disease that have distinct effects on metabolic traits.
        PLoS Genet. 2011; 7: e1001324
        • Kawaguchi T.
        • Sumida Y.
        • Umemura A.
        • et al.
        Genetic polymorphisms of the human PNPLA3 gene are strongly associated with severity of non-alcoholic fatty liver disease in Japanese.
        PLoS ONE. 2012; 7: e38322
        • Adams L.A.
        • White S.W.
        • Marsh J.A.
        • et al.
        Association between liver-specific gene polymorphisms and their expression levels with nonalcoholic fatty liver disease.
        Hepatology. 2013; 57: 590-600
        • Kitamoto T.
        • Kitamoto A.
        • Yoneda M.
        • et al.
        Genome-wide scan revealed that polymorphisms in the PNPLA3, SAMM50, and PARVB genes are associated with development and progression of nonalcoholic fatty liver disease in Japan.
        Hum Genet. 2013; 132: 783-792
        • Feitosa M.F.
        • Wojczynski M.K.
        • North K.E.
        • et al.
        The ERLIN1-CHUK-CWF19L1 gene cluster influences liver fat deposition and hepatic inflammation in the NHLBI Family Heart Study.
        Atherosclerosis. 2013; 228: 175-180
        • Kozlitina J.
        • Smagris E.
        • Stender S.
        • et al.
        Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease.
        Nat Genet. 2014; 46: 352-356
        • Shang X.R.
        • Song J.Y.
        • Liu F.H.
        • et al.
        GWAS-identified common variants with nonalcoholic fatty liver disease in Chinese children.
        J Pediatr Gastroenterol Nutr. 2015; 60: 669-674
        • DiStefano J.K.
        • Kingsley C.
        • Craig Wood G.
        • et al.
        Genome-wide analysis of hepatic lipid content in extreme obesity.
        Acta Diabetol. 2015; 52: 373-382
        • Dongiovanni P.
        • Donati B.
        • Fares R.
        • et al.
        PNPLA3 I148M polymorphism and progressive liver disease.
        World J Gastroenterol. 2013; 19: 6969-6978
        • Smagris E.
        • BasuRay S.
        • Li J.
        • et al.
        Pnpla3I148M knockin mice accumulate PNPLA3 on lipid droplets and develop hepatic steatosis.
        Hepatology. 2015; 61: 108-118
        • Kalia H.S.
        • Gaglio P.J.
        The prevalence and pathobiology of nonalcoholic fatty liver disease in patients of different races or ethnicities.
        Clin Liver Dis. 2016; 20: 215-224
        • Santoro N.
        • Zhang C.K.
        • Zhao H.
        • et al.
        Variant in the glucokinase regulatory protein (GCKR) gene is associated with fatty liver in obese children and adolescents.
        Hepatology. 2012; 55: 781-789
        • Li H.
        • Xu R.
        • Peng X.
        • et al.
        Association of glucokinase regulatory protein polymorphism with type 2 diabetes and fasting plasma glucose: a meta-analysis.
        Mol Biol Rep. 2013; 40: 3935-3942
        • Wang J.
        • Guo X.F.
        • Yu S.J.
        • et al.
        Adiponectin polymorphisms and non-alcoholic fatty liver disease risk: a meta-analysis.
        J Gastroenterol Hepatol. 2014; 29: 1396-1405
        • Li M.R.
        • Zhang S.H.
        • Chao K.
        • et al.
        Apolipoprotein C3 (-455T>C) polymorphism confers susceptibility to nonalcoholic fatty liver disease in the Southern Han Chinese population.
        World J Gastroenterol. 2014; 20: 14010-14017
        • Zhang H.
        • Chen L.
        • Xin Y.
        • et al.
        Apolipoprotein c3 gene polymorphisms are not a risk factor for developing non-alcoholic Fatty liver disease: a meta-analysis.
        Hepat Mon. 2014; 14: e23100
        • Zain S.M.
        • Mohamed Z.
        • Mohamed R.
        Common variant in the glucokinase regulatory gene rs780094 and risk of nonalcoholic fatty liver disease: a meta-analysis.
        J Gastroenterol Hepatol. 2015; 30: 21-27
        • Sun M.Y.
        • Zhang L.
        • Shi S.L.
        • et al.
        Associations between methylenetetrahydrofolate reductase (MTHFR) polymorphisms and non-alcoholic fatty liver disease (NAFLD) risk: a meta-analysis.
        PLOS ONE. 2016; 11: e0154337
        • Li L.
        • Wang S.J.
        • Shi K.
        • et al.
        Correlation between MTP -493G>T polymorphism and non-alcoholic fatty liver disease risk: a meta-analysis.
        Genet Mol Res. 2014; 13: 10150-10161
        • Tan H.L.
        • Mohamed R.
        • Mohamed Z.
        • et al.
        Phosphatidylethanolamine N-methyltransferase gene rs7946 polymorphism plays a role in risk of nonalcoholic fatty liver disease: evidence from meta-analysis.
        Pharmacogenet Genomics. 2016; 26: 88-95
        • Sookoian S.
        • Pirola C.J.
        Meta-analysis of the influence of I148M variant of patatin-like phospholipase domain containing 3 gene (PNPLA3) on the susceptibility and histological severity of nonalcoholic fatty liver disease.
        Hepatology. 2011; 53: 1883-1894
        • Zhang L.
        • You W.
        • Zhang H.
        • et al.
        PNPLA3 polymorphisms (rs738409) and non-alcoholic fatty liver disease risk and related phenotypes: a meta-analysis.
        J Gastroenterol Hepatol. 2015; 30: 821-829
        • Xu R.
        • Tao A.
        • Zhang S.
        • et al.
        Association between patatin-like phospholipase domain containing 3 gene (PNPLA3) polymorphisms and nonalcoholic fatty liver disease: a HuGE review and meta-analysis.
        Sci Rep. 2015; 5: 9284
        • Wang J.
        • Guo X.
        • Wu P.
        • et al.
        Association between the Pro12Ala polymorphism of PPAR-gamma gene and the non-alcoholic fatty liver disease: a meta-analysis.
        Gene. 2013; 528: 328-334
        • Lee Y.H.
        • Bae S.C.
        • Song G.G.
        Meta-analysis of associations between the peroxisome proliferator-activated receptor-gamma Pro12Ala polymorphism and susceptibility to nonalcoholic fatty liver disease, rheumatoid arthritis, and psoriatic arthritis.
        Genet Test Mol Biomarkers. 2014; 18: 341-348
        • Musso G.
        • Cassader M.
        • Bo S.
        • et al.
        Sterol regulatory element-binding factor 2 (SREBF-2) predicts 7-year NAFLD incidence and severity of liver disease and lipoprotein and glucose dysmetabolism.
        Diabetes. 2013; 62: 1109-1120
        • Pirola C.J.
        • Sookoian S.
        The dual and opposite role of the TM6SF2-rs58542926 variant in protecting against cardiovascular disease and conferring risk for nonalcoholic fatty liver: a meta-analysis.
        Hepatology. 2015; 62: 1742-1756
        • Goffredo M.
        • Caprio S.
        • Feldstein A.E.
        • et al.
        Role of TM6SF2 rs58542926 in the pathogenesis of nonalcoholic pediatric fatty liver disease: a multiethnic study.
        Hepatology. 2016; 63: 117-125
        • Chamoun Z.
        • Vacca F.
        • Parton R.G.
        • et al.
        PNPLA3/adiponutrin functions in lipid droplet formation.
        Biol Cell. 2013; 105: 219-233
        • He S.
        • McPhaul C.
        • Li J.Z.
        • et al.
        A sequence variation (I148M) in PNPLA3 associated with nonalcoholic fatty liver disease disrupts triglyceride hydrolysis.
        J Biol Chem. 2010; 285: 6706-6715
        • Huang Y.
        • Cohen J.C.
        • Hobbs H.H.
        Expression and characterization of a PNPLA3 protein isoform (I148M) associated with nonalcoholic fatty liver disease.
        J Biol Chem. 2011; 286: 37085-37093
        • Basantani M.K.
        • Sitnick M.T.
        • Cai L.
        • et al.
        Pnpla3/adiponutrin deficiency in mice does not contribute to fatty liver disease or metabolic syndrome.
        J Lipid Res. 2011; 52: 318-329
        • Liu Y.L.
        • Reeves H.L.
        • Burt A.D.
        • et al.
        TM6SF2 rs58542926 influences hepatic fibrosis progression in patients with non-alcoholic fatty liver disease.
        Nat Commun. 2014; 5: 4309
        • Mahdessian H.
        • Taxiarchis A.
        • Popov S.
        • et al.
        TM6SF2 is a regulator of liver fat metabolism influencing triglyceride secretion and hepatic lipid droplet content.
        Proc Natl Acad Sci U S A. 2014; 111: 8913-8918
        • Sookoian S.
        • Castano G.
        • Gianotti T.F.
        • et al.
        Polymorphisms of MRP2 (ABCC2) are associated with susceptibility to nonalcoholic fatty liver disease.
        J Nutr Biochem. 2009; 20: 765-770
        • Kotronen A.
        • Yki-Jarvinen H.
        • Aminoff A.
        • et al.
        Genetic variation in the ADIPOR2 gene is associated with liver fat content and its surrogate markers in three independent cohorts.
        Eur J Endocrinol. 2009; 160: 593-602
        • Iwamoto N.
        • Ogawa Y.
        • Kajihara S.
        • et al.
        Gln27Glu beta2-adrenergic receptor variant is associated with hypertriglyceridemia and the development of fatty liver.
        Clin Chim Acta. 2001; 314: 85-91
        • Nozaki Y.
        • Saibara T.
        • Nemoto Y.
        • et al.
        Polymorphisms of interleukin-1 beta and beta 3-adrenergic receptor in Japanese patients with nonalcoholic steatohepatitis.
        Alcohol Clin Exp Res. 2004; 28: 106s-110s
        • Yoneda M.
        • Hotta K.
        • Nozaki Y.
        • et al.
        Association between angiotensin II type 1 receptor polymorphisms and the occurrence of nonalcoholic fatty liver disease.
        Liver Int. 2009; 29: 1078-1085
        • Sazci A.
        • Akpinar G.
        • Aygun C.
        • et al.
        Association of apolipoprotein E polymorphisms in patients with non-alcoholic steatohepatitis.
        Dig Dis Sci. 2008; 53: 3218-3224
        • Wang B.
        • Wang B.
        • Wang Y.
        • et al.
        Association of APPL1 gene polymorphism with non-alcoholic fatty liver disease susceptibility in a Chinese Han population.
        Clin Lab. 2015; 61: 1659-1666
        • Wang L.
        • Athinarayanan S.
        • Jiang G.
        • et al.
        Fatty acid desaturase 1 gene polymorphisms control human hepatic lipid composition.
        Hepatology. 2015; 61: 119-128
        • Oliveira C.P.
        • Stefano J.T.
        • Cavaleiro A.M.
        • et al.
        Association of polymorphisms of glutamate-cystein ligase and microsomal triglyceride transfer protein genes in non-alcoholic fatty liver disease.
        J Gastroenterol Hepatol. 2010; 25: 357-361
        • Carulli L.
        • Canedi I.
        • Rondinella S.
        • et al.
        Genetic polymorphisms in non-alcoholic fatty liver disease: interleukin-6-174G/C polymorphism is associated with non-alcoholic steatohepatitis.
        Dig Liver Dis. 2009; 41: 823-828
        • Swellam M.
        • Hamdy N.
        Association of nonalcoholic fatty liver disease with a single nucleotide polymorphism on the gene encoding leptin receptor.
        IUBMB Life. 2012; 64: 180-186
        • Zain S.M.
        • Mohamed Z.
        • Mahadeva S.
        • et al.
        Impact of leptin receptor gene variants on risk of non-alcoholic fatty liver disease and its interaction with adiponutrin gene.
        J Gastroenterol Hepatol. 2013; 28: 873-879
        • Mancina R.M.
        • Dongiovanni P.
        • Petta S.
        • et al.
        The MBOAT7-TMC4 variant rs641738 increases risk of nonalcoholic fatty liver disease in individuals of European descent.
        Gastroenterology. 2016; 150 (1219–1230.e6)
        • Gorden A.
        • Yang R.
        • Yerges-Armstrong L.M.
        • et al.
        Genetic variation at NCAN locus is associated with inflammation and fibrosis in non-alcoholic fatty liver disease in morbid obesity.
        Hum Hered. 2013; 75: 34-43
        • Wu G.
        • Wang K.
        • Xue Y.
        • et al.
        Association of rs5764455 and rs6006473 polymorphisms in PARVB with liver damage of nonalcoholic fatty liver disease in Han Chinese population.
        Gene. 2016; 575: 270-275
        • Hernaez R.
        • McLean J.
        • Lazo M.
        • et al.
        Association between variants in or near PNPLA3, GCKR, and PPP1R3B with ultrasound-defined steatosis based on data from the third National Health and Nutrition Examination Survey.
        Clin Gastroenterol Hepatol. 2013; 11 (1183-1190.e2)
        • Vazquez-Chantada M.
        • Gonzalez-Lahera A.
        • Martinez-Arranz I.
        • et al.
        Solute carrier family 2 member 1 is involved in the development of nonalcoholic fatty liver disease.
        Hepatology. 2013; 57: 505-514
        • Huang Y.S.
        • Chang C.H.
        • Lin T.L.
        • et al.
        Genetic variations of superoxide dismutase 2 and cytochrome P450 2E1 in non-alcoholic steatohepatitis.
        Liver Int. 2014; 34: 931-936
        • Musso G.
        • Gambino R.
        • Pacini G.
        • et al.
        Transcription factor 7-like 2 polymorphism modulates glucose and lipid homeostasis, adipokine profile, and hepatocyte apoptosis in NASH.
        Hepatology. 2009; 49: 426-435
        • Kiziltas S.
        • Ata P.
        • Colak Y.
        • et al.
        TLR4 gene polymorphism in patients with nonalcoholic fatty liver disease in comparison to healthy controls.
        Metab Syndr Relat Disord. 2014; 12: 165-170
        • Yan X.
        • Xu L.
        • Qi J.
        • et al.
        sTRAIL levels and TRAIL gene polymorphisms in Chinese patients with fatty liver disease.
        Immunogenetics. 2009; 61: 551-556
        • Aller R.
        • De Luis D.A.
        • Izaola O.
        • et al.
        Role of -55CT polymorphism of UCP3 gene on non alcoholic fatty liver disease and insulin resistance in patients with obesity.
        Nutr Hosp. 2010; 25: 572-576
        • Yuan C.
        • Lu L.
        • An B.
        • et al.
        Association between LYPLAL1 rs12137855 polymorphism with ultrasound-defined non-alcoholic fatty liver disease in a Chinese Han population.
        Hepat Mon. 2015; 15: e33155
        • Chen L.
        • Lin Z.
        • Jiang M.
        • et al.
        Genetic variants in the SAMM50 gene create susceptibility to nonalcoholic fatty liver disease in a Chinese Han population.
        Hepat Mon. 2015; 15: e31076
        • Rottiers V.
        • Naar A.M.
        MicroRNAs in metabolism and metabolic disorders.
        Nat Rev Mol Cell Biol. 2012; 13: 239-250
        • Jones A.
        • Danielson K.M.
        • Benton M.C.
        • et al.
        miRNA signatures of insulin resistance in obesity.
        Obesity (Silver Spring). 2017;
        • Ferreira D.M.
        • Simao A.L.
        • Rodrigues C.M.
        • et al.
        Revisiting the metabolic syndrome and paving the way for microRNAs in non-alcoholic fatty liver disease.
        FEBS J. 2014; 281: 2503-2524
        • Elmen J.
        • Lindow M.
        • Schutz S.
        • et al.
        LNA-mediated microRNA silencing in non-human primates.
        Nature. 2008; 452: 896-899
        • Esau C.
        • Davis S.
        • Murray S.F.
        • et al.
        miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting.
        Cell Metab. 2006; 3: 87-98
        • Cermelli S.
        • Ruggieri A.
        • Marrero J.A.
        • et al.
        Circulating microRNAs in patients with chronic hepatitis C and non-alcoholic fatty liver disease.
        PLoS ONE. 2011; 6: e23937
        • Cheung O.
        • Puri P.
        • Eicken C.
        • et al.
        Nonalcoholic steatohepatitis is associated with altered hepatic MicroRNA expression.
        Hepatology. 2008; 48: 1810-1820
        • Pirola C.J.
        • Fernandez Gianotti T.
        • Castano G.O.
        • et al.
        Circulating microRNA signature in non-alcoholic fatty liver disease: from serum non-coding RNAs to liver histology and disease pathogenesis.
        Gut. 2015; 64: 800-812
        • Ahrens M.
        • Ammerpohl O.
        • von Schonfels W.
        • et al.
        DNA methylation analysis in nonalcoholic fatty liver disease suggests distinct disease-specific and remodeling signatures after bariatric surgery.
        Cell Metab. 2013; 18: 296-302
        • Murphy S.K.
        • Yang H.
        • Moylan C.A.
        • et al.
        Relationship between methylome and transcriptome in patients with nonalcoholic fatty liver disease.
        Gastroenterology. 2013; 145: 1076-1087
        • Zeybel M.
        • Hardy T.
        • Robinson S.M.
        • et al.
        Differential DNA methylation of genes involved in fibrosis progression in non-alcoholic fatty liver disease and alcoholic liver disease.
        Clin Epigenet. 2015; 7: 25
        • Pirola C.J.
        • Gianotti T.F.
        • Burgueno A.L.
        • et al.
        Epigenetic modification of liver mitochondrial DNA is associated with histological severity of nonalcoholic fatty liver disease.
        Gut. 2013; 62: 1356-1363
        • Pirola C.J.
        • Scian R.
        • Gianotti T.F.
        • et al.
        Epigenetic modifications in the biology of nonalcoholic fatty liver disease: the role of DNA hydroxymethylation and TET proteins.
        Medicine (Baltimore). 2015; 94: e1480
        • Sookoian S.
        • Rosselli M.S.
        • Gemma C.
        • et al.
        Epigenetic regulation of insulin resistance in nonalcoholic fatty liver disease: impact of liver methylation of the peroxisome proliferator-activated receptor gamma coactivator 1alpha promoter.
        Hepatology. 2010; 52: 1992-2000
        • Hardy T.
        • Zeybel M.
        • Day C.P.
        • et al.
        Plasma DNA methylation: a potential biomarker for stratification of liver fibrosis in non-alcoholic fatty liver disease.
        Gut. 2017; 66: 1321-1328
        • Sookoian S.
        • Rohr C.
        • Salatino A.
        • et al.
        Genetic variation in long noncoding RNAs and the risk of nonalcoholic fatty liver disease.
        Oncotarget. 2017; 8: 22917-22926
        • Clark S.J.
        • Lee H.J.
        • Smallwood S.A.
        • et al.
        Single-cell epigenomics: powerful new methods for understanding gene regulation and cell identity.
        Genome Biol. 2016; 17: 72
        • Davis J.N.
        • Le K.A.
        • Walker R.W.
        • et al.
        Increased hepatic fat in overweight Hispanic youth influenced by interaction between genetic variation in PNPLA3 and high dietary carbohydrate and sugar consumption.
        Am J Clin Nutr. 2010; 92: 1522-1527
        • Sevastianova K.
        • Santos A.
        • Kotronen A.
        • et al.
        Effect of short-term carbohydrate overfeeding and long-term weight loss on liver fat in overweight humans.
        Am J Clin Nutr. 2012; 96: 727-734
        • Santoro N.
        • Savoye M.
        • Kim G.
        • et al.
        Hepatic fat accumulation is modulated by the interaction between the rs738409 variant in the PNPLA3 gene and the dietary omega6/omega3 PUFA intake.
        PLoS ONE. 2012; 7: e37827
        • Nobili V.
        • Liccardo D.
        • Bedogni G.
        • et al.
        Influence of dietary pattern, physical activity, and I148M PNPLA3 on steatosis severity in at-risk adolescents.
        Genes Nutr. 2014; 9: 392
        • Sevastianova K.
        • Kotronen A.
        • Gastaldelli A.
        • et al.
        Genetic variation in PNPLA3 (adiponutrin) confers sensitivity to weight loss-induced decrease in liver fat in humans.
        Am J Clin Nutr. 2011; 94: 104-111
        • Dongiovanni P.
        • Petta S.
        • Mannisto V.
        • et al.
        Statin use and non-alcoholic steatohepatitis in at risk individuals.
        J Hepatol. 2015; 63: 705-712
        • Kamal S.
        • Khan M.A.
        • Seth A.
        • et al.
        Beneficial effects of statins on the rates of hepatic fibrosis, hepatic decompensation, and mortality in chronic liver disease: a systematic review and meta-analysis.
        Am J Gastroenterol. 2017;
        • Athinarayanan S.
        • Wei R.
        • Zhang M.
        • et al.
        Genetic polymorphism of cytochrome P450 4F2, vitamin E level and histological response in adults and children with nonalcoholic fatty liver disease who participated in PIVENS and TONIC clinical trials.
        PLOS ONE. 2014; 9: e95366
        • Lutz S.Z.
        • Peter A.
        • Machicao F.
        • et al.
        Genetic variation in the 11beta-hydroxysteroid-dehydrogenase 1 gene determines NAFLD and visceral obesity.
        J Clin Endocrinol Metab. 2016; 101: 4743-4751
        • Kapil S.
        • Duseja A.
        • Sharma B.K.
        • et al.
        Genetic polymorphism in CD14 gene, a co-receptor of TLR4 associated with non-alcoholic fatty liver disease.
        World J Gastroenterol. 2016; 22: 9346-9355
        • Kohan L.
        • Safarpur M.
        • Abdollahi H.
        Omentin-1 rs2274907 and resistin rs1862513 polymorphisms influence genetic susceptibility to nonalcoholic fatty liver disease.
        Mol Biol Res Commun. 2016; 5: 11-17
        • Nakajima S.
        • Tanaka H.
        • Sawada K.
        • et al.
        Polymorphism of receptor-type tyrosine-protein phosphatase delta gene in the development of non-alcoholic fatty liver disease.
        J Gastroenterol Hepatol. 2017;