Genomics 102 (2013) 84–95

Contents lists available at SciVerse ScienceDirect

Genomics

j ourna l homepage: www.e lsev ie r .com/ locate /ygeno

Review

Genomic aspects of NAFLD pathogenesis

Adviti Naik a, Rok Košir b,c, Damjana Rozman b,⁎a Faculty of Computer Sciences and Informatics, Tržaška Cesta 25, Ljubljana 1000, University of Ljubljana, Sloveniab Centre for Functional Genomics and Bio-Chips, Institute of Biochemistry, Faculty of Medicine, Zaloska 4, Ljubljana 1000, University of Ljubljana, Sloveniac DiaGenomi Ltd., Tehnoloski Park 19, 1000 Ljubljana, Slovenia

⁎ Corresponding author at: Centre for Functional GenE-mail address: damjana.rozman@mf.uni-lj.si (D. Ro

0888-7543/$ – see front matter © 2013 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.ygeno.2013.03.007

a b s t r a c t

a r t i c l e i n f o

Article history:Received 21 January 2013Accepted 22 March 2013Available online 29 March 2013

Keywords:Non-alcoholic fatty liver diseaseNASHHepatocellular carcinomaGenomicsGWASCandidate gene approaches

Non-alcoholic fatty liver disease (NAFLD) is themost predominant liver diseaseworldwide and hepaticmanifes-tation of the metabolic syndrome. Its histology spectrum ranges from steatosis, to steatohepatitis (NASH) thatcan further progress to cirrhosis and hepatocellular carcinoma (HCC). The increasing incidence of NAFLD hascontributed to rising numbers of HCC occurrences. NAFLD progression is governed by genetic susceptibility, en-vironmental factors, lifestyle and features of the metabolic syndrome, many of which overlap with HCC. Geneexpression profiling and genomewide association studies have identified novel disease pathways and polymor-phisms in genes that may be potential biomarkers of NAFLD progression. However, the multifactorial nature ofNAFLD and the limited number of sufficiently powered studies are among the current limitations for validatedbiomarkers of clinical utility. Further studies incorporating the links between circadian regulation and hepaticmetabolismmight represent an additional direction in the search for predictive biomarkers of liver disease pro-gression and treatment outcomes.

© 2013 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 851.1. NAFLD pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

2. Omic approaches aid in understanding liver pathologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 862.1. Genetic associations with NAFLD and HCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

2.1.1. Patatin-like phospholipase domain containing 3 (PNPLA3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 872.1.2. Manganese superoxide dismutase (MnSOD/SOD2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 872.1.3. Tumour necrosis factor α (TNFα) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 892.1.4. Glutathione s-transferases (GST) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 892.1.5. Peroxisome-proliferator-activated receptor α (PPARα) signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 892.1.6. Microsomal triglyceride transfer protein (MTTP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 892.1.7. Phosphatidylethanolamine methyltransferase (PEMT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 892.1.8. Apolipoprotein C-3 (ApoC3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 892.1.9. Apolipoprotein E (ApoE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 892.1.10. Adiponectin signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 902.1.11. Insulin receptor signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 902.1.12. Growth hormone (GH) and insulin-like growth factor (IGF) axis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 902.1.13. Interleukin 6 (IL-6) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 902.1.14. Drug metabolising enzymes (DMEs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 902.1.15. Other variant genes linked to NAFLD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

3. Conclusion and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 903.1. A future perspective: Disrupted circadian clock is another factor of liver diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

omics and Bio-Chips, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia.zman).

rights reserved.

85A. Naik et al. / Genomics 102 (2013) 84–95

1. Introduction

Non-alcoholic fatty liver disease (NAFLD) is awidely prevalent hepaticmetabolic disorder affecting up to 35% of western populations (Fig. 1),with an even higher risk in individuals with obesity and type 2 diabetes(T2D) [1–3]. NAFLD patients display a broad range of progressive stagesincluding the relatively benign steatosis, inflammation- and oxidativestress-induced non-alcoholic steatohepatitis (NASH), fibrosis and ulti-mately, hepatocellular carcinoma (HCC). The poor understanding ofNAFLD pathogenesis stems from its complex nature, governed byclose interactions between genetic factors and environmental cues.Recent studies have indicated the role of NAFLD as an emergingmenacein triggering HCC [4], which is the fifth most frequently diagnosed can-cer worldwide and the third leading cause of cancer deaths [5]. 70–85%of all diagnosed liver cancers are represented by HCC [6] whose risingincidence has been witnessed in many developed countries [7]. Whilehepatitis virus B and C (HBV/HCV) infections remain major risk factorsworldwide for HCC, a growing body of evidence clearly associates obe-sity, T2D, alcohol and tobacco abusewith HCC risk, at least in the UnitedStates [7,8]. A recent German study identified steatohepatitis as the un-derlying aetiology of HCC in 24% of patients compared to 23.3% withchronic HCV infection [9]. In addition to the development of HCC fromadvanced forms of NAFLD, steatosis has also been implicated in thedevelopment of HCC from cryptogenic cirrhosis [6] as well asnon-cirrhotic livers [10]. Thus, the proportion of NAFLD patients atrisk of developing HCC may be much higher than estimated to date,highlighting the importance of the elucidation of molecular pathwaysinvolved in the pathogenesis of NAFLD and malignant transformationto HCC.

1.1. NAFLD pathogenesis

The accumulation of triglycerides in the liver (>5% of steatoticcells in a liver section) is a histological hallmark of hepatic steatosis[11,12]. Patients diagnosed with NAFLD in the absence of other hepaticdisease aetiologies or excessive alcohol consumption also display lobu-lar inflammation, mild portal inflammation [13] and elevated levels ofalanine aminotransferase (ALT) in addition to hepatic steatosis [14].

Steatosis

S20-30% of generalwestern population

Steatohepatitis

NASH

Hepatocellular carcinoma

HCC

Cirrhosis

NASH-C

HBVHCV

Obesity≥70% of

obese people

up to 25%

Hh activation

Diabetes≥60% of diabetics

Inflammatory cytokines

Oxidative stress

Mitochondrial dysfunction

Expansion of progenitor hepatic cells

up to 25%10%

Fig. 1. Relationship between NAFLD and HCC. NAFLD development is closely linked todiabetes and obesity and is highly prevalent in patients with these metabolic dysfunc-tions. NAFLD may primarily manifest as steatosis, steatohepatitis or cirrhosis due toobesity-related metabolic complications or may progress through these stages overseveral decades. Recent evidence indicates that HCC can develop from any stage ofNAFLD, however, with different likelihoods. While HBV and HCV infections remainthe major risk factors for HCC development, NAFLD-related HCC will become an evenmore important risk factor in the near future due to the increasing prevalence ofNAFLD. Hh — hedgehog signalling pathway.

Isotopic studies in liver biopsies from obese, hypertriglyceridemic andhyperinsulinemic NAFLD patients have revealed elevated levels ofadipose-derived free fatty acid flux and de novo lipogenesis [15], alongwith compromised fatty acid oxidation and secretion [16].

The modulated expression and/or secretion of transcription factorsand cytokines respectively, influences downstream metabolic pathwaysand thus, plays a crucial role in NAFLD pathogenesis (Fig. 2). For example,sterol regulatory element-binding protein 1c (SREBP-1c/SREBF1) controlsthe expression of lipogenic genes. Elevated SREBP-1c correlates with he-patic steatosis in NAFLD patients [17]. The absence of carbohydrate-response element binding protein (ChREBP/MLXIPL) that regulatesglucosemetabolismand lipogenesis improves hepatic steatosis, indicat-ing that ChREBP is also linked to NAFLD [18]. Similarly, various PPARsubtypes are linked to the pathogenesis of NAFLD [19]. Apart fromtranscription factors involved in endogenous lipidmetabolism, nuclearreceptors regulating xenobiotic metabolism such as pregnane X recep-tor (PXR/NR1I2) and constitutive androstane receptor (CAR/NR1I3)and phase I and II drugmetabolizing enzymes also have altered profilesin NAFLD patients. The cross-talk between hepatic lipid and drugmetabolism and its impact on the development of NAFLD has beenreviewed in detail recently [20].

While the progression of benign steatosis is relatively low, about 2–5%of the general population progresses to NASH [3] (Fig. 1). Obesity(BMI > 30 kg/m2) increases the risk of NAFLD progression with a preva-lence of >25% of NASH patients among obese populations [2]. NASH ischaracterised by a steatotic histology, severe lobular inflammation andhepatocellular injury, such as the presence of ballooning, apoptosis or ne-crosis [13]. The progression from NAFLD to NASH results from at least 2hits (Fig. 2): the accumulation of triglycerides in the liver and the activa-tion of inflammatory, oxidative stress and fibrogenesis pathways [21,22],along with free fatty acid lipotoxicity [23,24]. Apart from lipotoxicity dueto the accumulation of fatty acids, ceramide and cholesterol are alsocrucial mediators of hepatic lipotoxicity [25]. The transcription factorSREBP2(SREBF2) that regulates cholesterol synthesis is elevated inhyperinsulinemia, which results in cholesterol accumulation, higherlevels of low-density lipoprotein receptor (LDLR) and reduced efflux offree cholesterol into the bile, thus cumulatively contributing to the pro-gression of NAFLD to NASH [26,27]. NASH patients have elevatedexpression of genes involved in stellate cell activation and fibrogenesisand downregulated expression of survival genes [28]. Oxidative stressdue to reactive oxygen species (ROS) resulting from mitochondrial dys-function, peroxisomal and microsomal fatty acid oxidation and lipid per-oxidation is a hallmark of NASH. Liver injury resulting from chronicexposure to drugs hampers themitochondrial role in fatty acid oxidation,promotes steatosis and activates the cascade of ROS generation and sub-sequent endoplasmic reticulum (ER) stress [20,25,29].

Among the cytokines, the adipose tissue secreted adipokines(adiponectin and leptin) are linked to progression from NAFLD toNASH. Adiponectin correlates negativelywith insulin resistance, plasmatriglycerides, LDL-cholesterol, hepatic fat content and progression toNASH in NAFLD patients [30–32]. A deficiency of leptin causes hyper-phagia, obesity and fatty liver, as shown in the ob/ob leptin-deficientmouse model. Leptin activates AMP-activated protein kinase (AMPK/PRKAA2) in the skeletal muscle and the liver resulting in the enhance-ment of fatty acid oxidation, hepatic glucose production and suppres-sion of lipogenesis, thus improving NAFLD [33,34]. Levels of solubleleptin receptor, which binds the circulating leptin, correlate with diabe-tes, fibrosis and NAFLD progression in morbidly obese patients [35].Other adipokines, such as resistin (RETN), visfatin (NAMPT) and retinolbinding protein 4 (RBP4) also appear as promising potential NAFLDmechanistic markers [36–39]. The cytokine tumour necrosis factor alpha(TNF-α) is elevated in NASH patients compared to patients with benignsteatosis [40]. It activates the transcription of other pro-inflammatorycytokines such as inhibitor of nuclear factor kappa-β (IKBKB) andnuclear factor kappa-B (NF-κB) [41] and enzymes involved in de novo li-pogenesis [42].

HE

PAT

IC S

TE

ATO

SIS

ST

EA

TOH

EPA

TIT

ISL

IVE

R F

IBR

OS

IS A

ND

HC

C

ADIPOSETISSUE

TNF-aIL-6

LipotoxicityROS

Lipid peroxidation

Anti-apoptotic

DNA damage Oncogenesis

Lipid output

VLDL,Ketones

Insulin resistanceand Obesity

Hyperinsulinemia

PlasmaFFA

Diet

SREBP-1c

ChREBP

PPAR-γ

Insulin

LXR

GlucoseGluco-

neogenesis FFA TAG

LIPID DROPLETSß-oxidation

12

6

39

Circadian clock

GR Glucocorticoids

Lipotoxicity

Cholesterol

SREBP-2

Bile acids

FFA

ROS

ApoptosisNecrosis

Lipid peroxidation

Ceramides

ER stress

Leptin

Hepatic stellate cell activation

Kupffer cell activation Collagen

Regeneration ofhepatocytes

TNF-aIL-6

TNF-aIL-6

Inflammation

Accumulation of triglycerides

AMPK

LeptinAdiponectin

Fig. 2. Molecular mediators involved in NAFLD pathogenesis.

86 A. Naik et al. / Genomics 102 (2013) 84–95

The inability of hepatocytes to regenerate and replace dead cells isthe proposed third hit in NAFLD progression, leading to an increasedpopulation of hepatocyte progenitor cells and prolonged infiltrationof inflammatory mediators [43]. These events are catalytic to hepaticfibrosis, cirrhosis and eventually hepatocellular carcinoma (Fig. 2).The presence of the metabolic syndrome and histological features ofNASH in cryptogenic cirrhosis patients implies the progression fromNASH to cirrhosis [44]. In addition, the development of HCC from NASHcoincided with the presence of cirrhosis in a Japanese population [45].However, studies inmicewith obesity-related steatosis showhyperplasiaand reduced cell death even in the absence of cirrhosis [46]. Molecularfactors involved in obesity and diabetes establish a tumour-promotingenvironment, which distinguishes the pathogenesis of NAFLD-relatedHCC to that of viral or other aetiologies [47]. In obesity, the adipose tissue

expansion leads to increased release of pro-inflammatory cytokinessuch as TNFα and interleukin-6 (IL6) that activate pro-oncogenic andanti-apoptotic pathways [39,47]. Ectopic deposition of fat in hepatocytesmay alter gene expression profiles through deregulation of cell signallingpathways and contribute to development of HCC [48]. Elevated levels ofperoxides and free radicals as a consequence of fatty acid oxidation cancause oxidative injury, endoplasmic reticulum stress and apoptosis [49].These events together promote liver inflammation and increase the riskof developing HCC.

2. Omic approaches aid in understanding liver pathologies

A large number of high resolution omic studies in NAFLD patientsand animal models have contributed to novel insights into the

87A. Naik et al. / Genomics 102 (2013) 84–95

molecular aspects of NAFLD [28,50,51]. Reverse-phase proteinmicroarrays indicated a deregulation of the insulin-receptor mediat-ed molecular signalling in white adipose tissue of progressiveNAFLD patients [52]. Specific phosphorylation and biomarker (e.g.caspase 9) signatures in omental fat could differentiate betweensteatotic and NASH obese patients. Serum metabolomics has identi-fied a panel of biomarkers that can differentiate the NAFLD/NASH dis-ease state from normal [53]. These findings are crucial in the search forpredictive non-invasive biomarkers as at present the liver biopsy is themost accurate method to diagnose early progression stages fromsteatosis to NASH. Recent advances in sequencing and expression pro-filing technologies, fine mapping and functional analysis of genomes,genome-wide association studies and applications of animal models,along with initiatives such as the Human Genome Project, HapMapand ENCODE, provided novel insights to dissect the pathogenesis ofNAFLD, as described further.

Male C57/BL6 mice that develop hepatic steatosis and obesity ona high-fat diet displayed altered expression of 309 genes comparedto the control diet, including genes of the lipogenesis and fatty acidoxidation pathways [50]. Rodents with orotic acid-induced steatosisdisplayed altered expression of genes involved in fatty acid, lipopro-tein and glucose metabolism and stress and inflammatory pathways[54].

Transcriptomic analyses in patients with different stages of NAFLDidentified differential expression of hepatic and adipose genes at pro-gressive stages of NAFLD [28,55]. While early stages of NAFLD wereaccompanied by upregulation of cell survival and liver regenerationgenes and downregulation of defence mechanisms against oxidativestress [28], NASH stages displayed upregulation of stellate cell activa-tion, inflammation, fibrogenesis and detoxification pathways centredaround TNFα, IL6, JUN and interferon γ (IFNG) regulation. Additional-ly, the survival factor ADP-ribosylation factor-like 6 interacting protein(ARL6IP/RAMER) and phase II enzyme sulfotransferase 1A2 (SULT1A2)were downregulated [28,55]. Gene expression analysis comparingCaucasians, who have a higher risk of NAFLD, and African-Americansindicated ethnic differences in gene expression patterns, especiallyamong NASH patients [56]. The most prominently upregulated genesin African-Americans are glutathione s-transferases GSTM-2, -4, and -5,acyl-CoA synthetase long-chain family member 4 (ACSL4), insulin-likegrowth factor 2 (IGF2) and cytochrome p450 3A4 (CYP3A4) [56].

Additionally, epigenetic changes andmicro RNAs have been linkedto liver pathologies. A study comparing miRNA expression in NASHpatients against controls identified a significant decrease in miR-122,which may account for insulin resistance, oxidative stress, apoptosisand inflammation in NASH [57]. miR-122 accounts for 70% of hepaticmiRNA and its inhibition in mice on a high-fat diet improved hepaticsteatosis, lowered plasma cholesterol and the expression of lipogenicgenes [58]. Other miRNAs such as miR-335, miR-181d and miR-10bhave been implicated in lipid homeostasis and adipocyte differentiation[59].

2.1. Genetic associations with NAFLD and HCC

The heritability of NAFLD and its complex nature have triggered theadvent of several candidate gene studies and a few genome-wide asso-ciation studies (GWAS) in NAFLD populations. Similarly, numerouscandidate-gene studies have reported associations between SNPs andthe presence of HCC, while limited GWAS studies have shed light intoadditional unsuspected loci that might be involved in hepatic carcino-genesis [60]. The variants identified in association with HCC and NAFLDshare some common molecular pathways such as oxidative stress, ironmetabolism, and inflammatory and immune response, suggesting thatgene variants associated with NAFLD could potentially modulate path-ways involved in HCC and vice versa [60]. A comparison of SNPs curatedfrom NAFLD-related literature searches and those reviewed by Nahon etal. [60] in the context of HCC, highlighted only 4 common SNPs: GSTM1

and SOD2 A16V from the oxidative stress response pathway, TNFG-238A involved in inflammatory/immune response and PNPLA3I148M, which potentially has a lipolytic role. However, it is importantto note that the HCC variants described by Nahon et al. were discoveredin studies that compared normal controls to HBV- or HCV-infected pa-tients or patientswith alcohol abuse. Studies designed to identify geneticrisks of NAFLD-triggeredHCC are limited and require amore focussed at-tention in the future [4,61].

The most prominent genetic variations associated with NAFLD arelisted in Table 1. Below we provide a detailed overview of polymor-phisms that have been associated with NAFLD. We begin by describ-ing the four overlapping polymorphisms associated with NAFLD andHCC in genes PNPLA3, SOD2, TNFα, and GSTM1, followed by other con-tributing SNPs in genes involved in lipid metabolism, inflammatorypathways, xenobiotic detoxification, etc.

2.1.1. Patatin-like phospholipase domain containing 3 (PNPLA3)PNPLA3 is one of the few examples that has been validated in sev-

eral populations and conclusively shown to associate with NAFLD.Although the functional roles of PNPLA3, which encodes adiponutrin,or its genetic variants have not been fully determined, it is known tohave lipase activity in vitro. The expression of PNPLA3 is regulated byChREBP and SREBP, implicating its function in glucose and lipid metab-olism [62]. Surprisingly, Pnpla3 knock-out (KO) mice or mice with in-creased Pnpla3 expression did not display lipolysis abnormalities orhepatic steatosis [63]. The activation of compensatory mechanismsthat may involve other members of the Pnpla family was not observedeither. Hence, developing tissue-specific Pnpla3-overexpressionmodelsmay be a more accurate approach to solve these inconsistencies.

PNPLA3was first identified as a susceptibility gene in a large studyincluding Hispanics, African-Americans and European-Americans fromthe Dallas Heart Study [64]. The rs738409[G] variant significantly asso-ciates with increased hepatic fat and inflammation whereas thers6006460[T] correlates with lower hepatic fat. This observation wasreplicated in a Finnish study [65] and a recent large-scale GWAS studygenotyping 2.4 million SNPs in 7100 individuals as a meta-analysisfrom several large population studies [66]. Moreover, studies in Italian,Chinese, Indian, Malay, Japanese and United Kingdom populations indi-cated that a homozygosity in the rs738409[G] variant predisposedpatients to NASH [67–69]. A study to determine if the NAFLD-associatedrs738409 variant in PNPLA3 also affects the prognosis of HCC in Japanesepatients concluded a higher incidence of the G/G genotype of rs738409PNPLA3 in NAFLD-related HCC [70]. The genotype was associated withhigher BMI and severe fibrosis but was found to affect HCC prognosisonly in the alcoholic liver disease patients' subgroup [70]. The G allele ofrs738409 PNPLA3was also over-represented in alcoholic/metabolic cir-rhosis Italian patientswith HCC [71], in accordancewith Takeuchi et al.,highlighting the potential predictive value of this polymorphism forHCC occurrence in cirrhotic patients.

2.1.2. Manganese superoxide dismutase (MnSOD/SOD2)As oxidative stress represents the second-hit in the development

of NASH, molecular mechanisms that lead to the generation of ROSmay contribute to NASH pathogenesis. SOD2 protects against ROSby detoxifying superoxides into oxygen and hydrogen peroxide. The1183 T/C polymorphism has been identified in the targeting sequencethat directs SOD2 to the mitochondria. The presence of TT genotype atthis position associates with NASH and with the degree of fibrosis inNAFLD patients, due to decreased transport of the protein into the mi-tochondria [72]. This association was further confirmed by familialanalysis using the transmission disequilibrium test in 55 informativefamilies and a case–control study in a larger European NAFLD popu-lation [73], both with biopsy-proven NASH [60]. A prospective study inalcoholic cirrhosis patients identified the presence of theAla16Val-SOD2allele as a susceptibility factor for HCC development and death, withfurther detrimental outcomes in combination with the G-463A

Table 1Genetic associations implicated in the pathogenesis of NAFLD. *Genes that have also been highlighted in association with HCC.

Gene Protein function Association with NAFLD

Patatin-like phospholipase domaincontaining 3 (PNPLA3)/adiponutrin *

• Lipolytic and lipogenic function in vitro• Gene expression enhanced by SREBP-1c

• I148M SNP significantly associated with hepatic fat content, ALT and ASTactivities, fibrosis and NASH. No correlation with insulin sensitivity observed[55,56,58–60,136].

• S453I associated with decreased risk of hepatic triglyceride accumulation.• Higher incidence of the G/G genotype of rs738409 PNPLA3 in NAFLD-relatedHCC patients, associated with higher BMI and severe fibrosis [63,64].

Superoxide dismutase 2 (SOD2) * • Detoxification of mitochondrial ROS • T/T genotype in mitochondrial targeting sequence is significantly prevalentin NASH patients [65].

• Frequency of T allele in the mitochondrial targeting sequence is proportionalto the degree of fibrosis in NAFLD patients [72].

• Ala16Val-SOD2 allele is a potential susceptibility factor for HCCdevelopment and death in alcoholic cirrhosis patients, with furtherdetrimental outcomes in combination with the G-463A MPO variant [73].

Tumour necrosis factor-alpha (TNF) * • Pro-inflammatory cytokine • Higher prevalence of the TNFα-238 polymorphism but not the 308 polymorphism,in NAFLD patients and was associated with increased insulin resistance[69,104–106,109].

• Meta-analysis showed that TNFα-308G/A, TNFα-238G/A and TNFα-863C/Apolymorphisms may associate with HCC in Asians populations [110].

Glutathione S-transferase (GST) * • Phase II detoxification enzyme• Defence against oxidative stress

• GSTM1-null genotype present at a higher frequency in Japanese NAFLDpopulation [120].

• GSTM1-null genotype associates with increased HCC risk in Asians, withfurther risk in the presence of dual GSTM1/GSTT1 null genotype [121].

Peroxisome proliferator-activatedreceptor α (PPARA)

• Activates fatty acid oxidation andhepatic lipid hydrolysis

• Ala227 PPARα variant is associated with lower fat indices in NAFLD patients[98].

PPARα co-activator 1-α (PPARGC1A) • Transcriptional co-activator of PPARγ• Regulates genes involved in energymetabolism

• Deficiency results in enhanced lipogenesis and hepatic steatosis in mice [100].• DNA methylation in PPARGC1A gene implicated in the degree of insulin resistancein NAFLD [99] and rs2290602 SNP in the PPARGC1A gene associated with theprogression of NAFLD to NASH in patients [102].

Microsomal triglyceride transferprotein (MTTP)

• Synthesis and secretion of VLDL in liver • -493 G/T SNP associated with NAFLD. GG homozygous patients have lowerMTTP activity, severe steatosis and more atherogenic postprandial lipidprofile in NASH patients, independent of insulin resistance [65,67].

Phosphatidylethanolamine methyltransferase(PEMT)

• Synthesis of phosphatidylcholine • V175M SNP confers susceptibility to NASH patients [68].

Apolipoprotein C-III (APOC3) • Inhibition of LPL • C482T and T455C variant carriers have 30% increased fasting plasma ApoC3and 60% increased fasting plasma triglycerides [75].

• 38% of C482T and T455C variant carriers displayed NAFLD compared to nonein the WT allele subjects [75].

Apolipoprotein E (APOE) • Plasma lipid transport protein. • APOE-ε3 allele has a significantly high incidence in NASH patients [81].• APOE-ε2 allele may be protective against NAFLD [82].

Adiponectin (ADIPOQ) • Insulin-sensitizing, anti-inflammatoryadipokine.

• G45T and G276T in ADIPO occur at a higher incidence in an Italian NAFLDcohort, however, contrasting observations made in Asian populations [88].

• ADIPOR2 rs767870 polymorphism is significantly associated with a 50%decrease in hepatic fat accumulation and decrease in serum γ-glutamyltransferase and triglyceride concentrations in a Finnish cohort [90].

Insulin receptor substrate 1 (IRS1) • Downstream regulator of insulin action • Gly172Arg polymorphism affects insulin receptor activity and is significantlyassociated with NAFLD disease severity in Caucasian NAFLD patients [85].

Growth hormone (GH)/insulin-likegrowth factor-1 (IGF-1)

• Stimulates growth, regulates fatty acidoxidation, lipolysis, LDL clearance andgluconeogenesis

• NAFLD is common in patients with adult growth hormone deficiency (AGHD).• G allele of PPARγ, rs1800775, rs708272 and rs3764261 in CETP gene, T alleleof APOE SNP rs7412 and C allele of SNP rs35136575 in the APOE/C1/C4/C2gene cluster associated with altered serum lipid profiles in severe AGHDpatients [132].

• IGF1 and IGFBP3 polymorphisms are potentially associated with NAFLD-relatedchanges in IGF-1 and IGFBP-3 proteins [133,134].

Interleukin-6 (IL6) • Pro-inflammatory cytokine • IL6-174C variant present at a higher frequency in NASH than in NAFLD patientsand accompanied by increased insulin resistance in a Caucasian cohort [115].

Pregnane X receptor (PXR/NR1I2) • Nuclear receptor • rs2461823[A] and rs7643645[G]- containing haplotypes in the PXR geneassociated with disease severity and liver injury in a European NAFLDpopulation [117].

UDP-glucuronosyltransferases 1A1 (UGT1A1) • Phase II detoxification enzyme.• Anti-oxidative stress

• UGT1A1*6 allele associated with a lower risk of NAFLD in an obese Taiwanesecohort [118].

ABC- transporter ABCC2 • Xenobiotic transporter • SNPs rs17222723 and rs8187710 identified in association with NAFLD, liverhistology and disease severity [119].

Neurocan (NCAN) • Regulates cell adhesion and migration • rs2228603 SNP is associated with NAFLD and serum lipids [57].Glucokinase regulator (GCKR) • Inhibits glycogen synthesis • rs780094 SNP is associated with NAFLD and serum lipids [57].Lysophospholipase-like 1 (LYPLAL1) • Involved in triglyceride synthesis • rs12137855 SNP is associated with NAFLD and serum lipids [57].Group-specific component (GC) • Vitamin D–binding protein • rs222054 SNP associated with NAFLD in an adolescent population [124].

• Expression reduced by 83% in NAFLD liver biopsies. Serum and protein levelslower in progressive stages of NAFLD compared to controls [124].

Lymphocyte cytosolic protein 1 (LCP1) • Actin-binding protein • rs7324845 SNP associated with NAFLD in an adolescent population [124].• Gene expression increases by 300% in NAFLD liver biopsies [124].

Lipid phosphatase-related proteintype 4 (LPPR4)

• Neuron-specific• Dephosphorylates mediators inneuron development

• rs12743824 SNP associated with NAFLD in an adolescent population [124].

Solute carrier family 38 member8 (SLC38A8)

• Brain-specific putative sodium-coupledneutral amino acid-transporter

• rs11864146 SNP associated with NAFLD in an adolescent population [124].

88 A. Naik et al. / Genomics 102 (2013) 84–95

89A. Naik et al. / Genomics 102 (2013) 84–95

myeloperoxidase (MPO) variant [74]. This may be attributed to exces-sive hepatic iron accumulation associated with Ala16Val-SOD2, accom-panied by increased ROS-related oxidative stress and DNA damagecaused by excessive MPO activity [74].

2.1.3. Tumour necrosis factor α (TNFα)TNFα is a cytokine with broad functionality in inflammation, auto

immunity, tumour apoptosis and metabolic regulation [75]. Increasedlevels of TNFα correlate with insulin resistance and the activation ofinflammatory pathways, thus increasing the risk of NAFLD progres-sion to NASH [39,76]. Several polymorphisms have been identifiedin the TNFα gene promoter [77]. Candidate gene studies in an Italianpopulation identified a higher prevalence of the TNFα-238 promoterpolymorphism but not the -308 polymorphism in NAFLD patientscompared to controls [78]. Despite impaired insulin sensitivity, the-238 polymorphism was also associated with lower LDL-cholesterol,and comparatively lower BMI, indicating an inverse effect on the reg-ulation of glucose and lipid metabolism. Similar observations weremade in a Mexican and two separate Chinese cohorts that displayedan association between TNFα-238 and NAFLD/NASH [79–81]. A studyin Japanese population identified two different polymorphisms in theTNFα promoter, -1031C and -863A, that were present at a higher fre-quency in NASH patients compared to patients with simple steatosis[82]. The lack of association of the -238 and -308 polymorphisms inthe Japanese could be attributed to ethnic differences and the low fre-quency of variations at these positions in this population. Althoughother studies in Chinese populations failed to identify any of these asso-ciations,mainly due to the lack of statistical power [83], ameta-analysisof 8 studies evaluating polymorphisms in TNFα confirmed the associa-tion of the -238 polymorphismwithNAFLD, however the -308 polymor-phism was not identified as a susceptibility factor [84].

Another recentmeta-analysis to assess the association between TNFαpolymorphisms and HCC susceptibility showed that TNFα-308G/A,TNFα-238G/A and TNFα-863C/A polymorphisms may associate withHCC in Asians populations [85]. This observation is biologically plausi-ble in the context of tumour-promoting roles of TNFα in various can-cer types [86].

2.1.4. Glutathione s-transferases (GST)Glutathione s-transferases (GST) are phase II DMEs involved in

xenobiotic detoxification and protection against oxidative stress. Acase–control analysis indicated that the GSTM1-null genotype waspresent at a higher frequency in a Japanese NAFLD population [87].A meta-analysis suggests that the null genotype of GSTM1 associateswith increased HCC risk in Asians. Individuals with the dual GSTM1/GSTT1-null genotype have an even further susceptibility in developingHCC [88]. The GSTM1/GSTT1-null genotype has also been identified inassociation with several complex disorders featuring oxidative stress,inflammatory and detoxification irregularities, such as T2D [89], maleinfertility [90], etc. Thus, it seems plausible that GST-null polymor-phisms associate with characteristics such as oxidative stress and in-flammation, rather than with the disease in general.

2.1.5. Peroxisome-proliferator-activated receptor α (PPARα) signallingPPARαplays amajor regulatory role in lipidmetabolismby activating

fatty acid oxidation and lipid hydrolysis, thus preventing hepatic lipidaccumulation [19,91]. Adiponectin has an indirect effect on PPARαactivation [92]. Mice fed a methionine-, choline-deficient diet (MCD)develop steatosis, which is improved on treatment with PPARα-agonists [93]. Similarly, PPARα−/− mice develop hepatic steatosis ona high fat diet [94]. Thus, PPARα-agonist fibrates are used to treatpatients with elevated plasma triglycerides [95]. Several studies showa correlation between the Val227Ala variant of PPARα and low serumlipids [96,97]. A case–control comparison study of NAFLD patientshighlighted a potential protective role for the Val227Ala variant againstobesity. The subjects with the Ala227 variant appear to have lower fat

related indices, although no significant differences were identified inserum glucose, triglyceride and cholesterol levels [98]. The functionalrole of this SNP requires further elucidation.

PPARγ-coactivator 1-alpha (PGC1-α/PPARGC1A) is a systemicmodulator of energy homeostasis, a deficiency of which results in en-hanced lipogenesis and hepatic steatosis in mice [99]. The regulationof PGC1-α by sirtulin (SIRT1) and AMPK may possibly provide amechanism for its ability to sense metabolic deregulations [100]. Post-translational modifications such as DNA methylation in the PGC1-αgene associate with the degree of insulin resistance in NAFLD patientsand to the liver mitochondrial DNA content [101]. The T allele of theSNP rs2290602 in PPARGC1A was present at a significantly higher fre-quency in NASH patients compared to patients with simple steatosisandwas associated with decreased expression of the gene in a Japanesebiopsy-proven NAFLD population [102].

2.1.6. Microsomal triglyceride transfer protein (MTTP)MTTP is involved in the formation of VLDL particles from triglycer-

ides and apolipoprotein B (ApoB). A study in rats has indicated a roleof DNA methylation of theMttp promoter which decreased the expres-sion of Mttp gene on high fat diet [103]. The -493 G/T MTTP polymor-phism occurred at a higher incidence in a Japanese NASH populationcompared to healthy controls [72]. The G allele results in decreasedtranscription of the MTTP gene and increased accumulation of triglycer-ides in the liver. Furthermore, the degree of disease severity and plasmalipid profile correlated with the G/G genotype at -493 position of theMTTP gene in a cohort of NASH patients, independent of the level ofadipokines and insulin resistance [104]. However, due to the small num-ber of patients in this study these observations need to be replicated inlarger cohorts and different populations.

2.1.7. Phosphatidylethanolamine methyltransferase (PEMT)PEMT contributes to the synthesis of phosphatidylcholine from

phosphatidylethanolamine. Phosphatidylcholine is an essential compo-nent of VLDL formation for hepatic triglyceride secretion. Loss of functionof Pemt results in increased accumulation of lipids in mice [105]. TheV175M loss-of-function mutation in the human PEMT gene associateswith NAFLD patients [106]. This observation has been replicated in aChinese population [80]. Moreover, the presence of the polymorphismmay indicate susceptibility to NASH, as observed in a Japanese popula-tion of NASH patients compared to healthy controls [107].

2.1.8. Apolipoprotein C-3 (ApoC3)ApoC3, which is expressed in the liver, functions as an inhibitor of

lipoprotein lipase (LPL) that hydrolyses triglycerides in VLDL particlesand chylomicrons in the plasma. Its absence or over-expression re-sults in hyper- or hypo-triglyceridemia, respectively. In mice, insulininhibits ApoC3 expression via the nuclear exclusion of FOXO1,a transcription factor triggering the expression of ApoC3, which ex-plains the aberrant apoC3 production in the presence of insulin resis-tance [108]. 38% of APOC3 C482T and T455C variant carriers display anincrease in fasting plasma apoC3 and triglycerides in the presence ofNAFLD, compared to none in the wild-type allele carriers, as observedin a study on Asian Indian men that was further confirmed in men ofnon-Asian Indian descent [109]. However, no associations betweenhepatic triglyceride content and insulin resistance and the twoAPOC3 variants were identified in a much larger study on multiple eth-nic populations from theDallasHeart study and theAtherosclerosis Riskin Communities study (ARIC) [110] and separate studies in an obeseItalian [111] and Finnish cohort [112].

2.1.9. Apolipoprotein E (ApoE)ApoE is a major component of lipoprotein particles and its role in

the pathogenesis of NAFLD is widely accepted. Rodent studies indicatelowered susceptibility for the development of obesity and NAFLD in theabsence of ApoE [113,114]. APOE gene has variants ε2, ε3 and ε4. The

90 A. Naik et al. / Genomics 102 (2013) 84–95

occurrence of the APOE ε3 allele is significantly higher in biopsy-provenCaucasian NASH patients compared to controls [115], whereas the pres-ence of the APOE ε2 allele may be protective against NAFLD [116]. How-ever, these associations need to be confirmed in larger populations ofdifferent ethnicities.

2.1.10. Adiponectin signallingAdiponectin (ADIPOQ) has numerous systemic protective roles,

such as insulin-sensitization, anti-inflammatory, anti-fibrogenic andanti-atherogenic effects, ceramide catabolism and suppression of he-patic gluconeogenesis [39,117]. SNPs G45T and G276T of theadiponectin gene occur at a higher incidence in an Italian NAFLD co-hort compared to controls, together with a reduced postprandialadiponectin response and increased postprandial plasma triglycerides,free fatty acids and VLDL [118]. The study in Asian population did notconfirm the above results [119]. Adiponectin in the liver binds toadipoR2 receptor, which correlates negatively with insulin resistance[120].ADIPOR2 rs767870polymorphism significantly associateswith he-patic lipid accumulation, as observed in a Finnish cohort and confirmedin two independent studies [121]. The presence of the C allele in theADIPOR2 gene correlates to a 50% decrease in hepatic fat accumulationanda significant decrease in serumγ-glutamyl transferase and triglycer-ide concentrations after accounting for body mass index (BMI) [121].

2.1.11. Insulin receptor signallingInsulin resistance is a commonly observed characteristic in NAFLD

and NASH patients. Inflammatory factors target insulin receptor sub-strate (IRS) for ubiquitin-mediated degradation via activation of suppres-sors of cytokine signalling 3 (SOCS-3), thus causing a downregulationin insulin sensitivity [122]. NAFLD and T2D patients are usually treatedwith a PPARγ-targeted thiazolidinedione, an insulin-sensitizer that re-duces lipid release, induces lipid uptake and storage and suppresseshepatic gluconeogenesis [123]. Polymorphisms in the plasma cellantigen-1 (PC-1)/ectoenzyme nucleotide pyrophosphate phosphodies-terase 1 (ENPP1) (Lys121Gln) and in IRS1 (Gly172Arg), both affectinginsulin receptor activity, are significantly associated with NAFLD dis-ease severity in biopsy-proven Caucasian NAFLD patients [124].

2.1.12. Growth hormone (GH) and insulin-like growth factor (IGF) axisNAFLD and other characteristics of the metabolic syndrome are

common in patients with adult growth hormone deficiency (AGHD)syndrome and hypopituitarism. GH positively regulates fatty acid oxida-tion, lipolysis, LDL clearance and gluconeogenesis [125]. A majorityof these effects are propagated via the interaction of GH withIGF-1, which is a catabolic hormone secreted by hepatocytes on GH-stimulation. The bioavailability of IGF-1 is regulated by IGF-bindingprotein 3 (IGFBP-3) [126]. Studies in rodents have also identifiedGH-independent IGF-1 effects in improving NASH by downregulatingoxidative stress [127]. Both obese and AGHD patients with NAFLD andinsulin resistance display low levels of GH and IGF-1 and an increasein GH-binding protein (GHBP) and IGFBP-3 that correlate with thestage of hepatic steatosis and fibrosis [128,129]. The frequency and se-verity of fatty liver negatively correlate with GH levels in hypopituitarymales with features of the metabolic syndrome [130]. Furthermore,GH-replacement therapy ameliorates oxidative stress in NASH patientswith AGHD [131]. Several SNPs associatedwith serum lipid profileswereidentified in patients with severe AGHD and hypopituitarism in a candi-date gene association study [132]. The G allele of PPARγwas associatedwith low LDL-cholesterol. Minor alleles of rs1800775, rs708272 andrs3764261 in the CETP gene are correlated with increased serumHDL-cholesterol, the T allele of APOE SNP rs7412 was associated withlow total cholesterol and LDL-cholesterol and the C allele of SNPrs35136575 in the APOE/C1/C4/C2 gene cluster was associated with in-creased serum triglyceride levels in these patients. Although associationof these SNPs with hepatic fat content was not studied, they might rep-resent potential candidates in the context of NAFLD development.

Moreover, IGF1 and IGFBP3 polymorphisms could potentially be associat-edwith NAFLD-related changes in IGF-1 and IGFBP-3 proteins [133,134].

2.1.13. Interleukin 6 (IL-6)Inflammationis is well defined as the second hit in NASH develop-

ment. Elevated IL-6 associateswith inflammation and insulin resistancevia proteosomal degradation of IRS-1 [122,135,136]. Circulating levelsof IL-6 are increased in individuals with T2D and obesity [136,137]. Ina Caucasian cohort the IL6-174C variant occurred at a higher frequencyin NASH than in NAFLD patients and was accompanied by increased in-sulin resistance, in accordance with several studies that suggested theassociation of theC allelewith T2D, insulin resistance and othermarkersof the metabolic syndrome [138].

2.1.14. Drug metabolising enzymes (DMEs)Differential expression of DMEs in NAFLD patients is mainly due

to the presence of common factors that govern lipid and xenobioticmetabolism [139]. In healthy conditions the nuclear receptors andphase II DMEs provide defence against hepatic oxidative stress. PXRis a nuclear receptor that regulates the expression of several DMEsand is also activated by endobiotics. Its activation in mice results inhepatic steatosis [140]. rs2461823[A] and rs7643645[G]-containinghaplotypes in the PXR gene are associated with disease severity andliver injury in a European NAFLD population [141].

The roles of GSTM, a phase II DME, and SNPs in the encoding geneassociated with NAFLD and HCC are described in Section 2.1.4. The phaseII DME UDP-glucuronosyltransferases 1A1 (UGT1A1) that protect cellsagainst reactive oxygen species were also candidates for genetic associ-ation studies in an obese Taiwanese paediatric cohort, where theUGT1A1*6 allele is associated with a lower risk of NAFLD [142]. SNPsrs17222723 and rs8187710 in the ABC- transporter ABCC2 are associatedwith NAFLD, liver histology and disease severity [143].

2.1.15. Other variant genes linked to NAFLDNovel genetic variants were identified in association with com-

puter tomography (CT)-measured hepatic steatosis [66]. These includeneurocan (NCAN, rs2228603), the gene encoding a protein regulatingcell adhesion and migration, glucokinase regulator (GCKR, rs780094),which inhibits glycogen synthesis, and lysophospholipase-like 1(LYPLAL1, rs12137855) that is involved in triglyceride synthesis. Thesevariants display association patterns with histologic NAFLD, serumlipids and glycaemic and anthropometric traits. A GWAS in adolescentsdiagnosed with NAFLD identified SNP associations to two liver-specificgenes (the group-specific component (GC, rs222054) and lymphocytecytosolic protein 1 (LCP1, rs7324845)), two neuron-specific genes(lipid phosphatase-related protein type 4 (LPPR4, rs12743824)) and asolute carrier family 38 member 8 (SLC38A8, rs11864146). This studyfurther confirmed the significant differential expression of GC andLCP1 in a NAFLD cohort [144]. Further functional characterisation ofproteins encoded by these genes is required to dissect their molecularrole in NAFLD pathogenesis.

3. Conclusion and future directions

The multi-faceted, complex, non-Mendelian nature of NAFLD andHCC shows the heterogeneous nature of the disease in terms of diseaseprogression, severity, susceptibility in different ethnic populations andtreatment responses. Although GWAS and candidate gene approachesidentified several susceptibility factors associated with NAFLD, only afew (in the case of NAFLD only PNPLA3) have been confirmed in multi-ple populations. Most studies suffer from lack of statistical power, repli-cation in different populations, well-matched samples and the presenceof heterogeneities in disease stages. Non-additive effects, such as dom-inance, epistasis, environmental and lifestyle factors, hinder identifica-tion of genes associated with NAFLD pathogenesis. As the invasiveliver biopsy technique remains the gold standard to diagnose NASH, a

91A. Naik et al. / Genomics 102 (2013) 84–95

majority of the current studies are based on ultrasound identification ofthe disease stage which lacks accuracy due to its inability to separatesteatosis from early stages of NASH. Large-scale inter-protocol agree-ments and standardization of grading criteria for different stages ofliver diseases are needed to reduce the current technical variability[145]. Further development of combined polymorphism risk-scoringalgorithms, in combination with whole genome sequencing, could en-able identification of high risk groups for preventative care and earlydetection of the disease.

3.1. A future perspective: Disrupted circadian clock is another factor ofliver diseases

Awide array of physiological processes exhibit a circadian (~24 h)rhythm represented by transcriptional and translational feedbackloops that govern internal clocks. In the liver, the circadian rhythmresults from an interplay of cis-regulatory elements [146]. Consequent-ly, it is believed that the intrinsic gene regulatory networks determinethe hepatic clock, whereas systemic cues, such as light–dark cycles,serve only to fine-tune the rhythm. Several circadian transcription fac-tors that can switch the gene expression on and off have been identifiedbut knowledge regarding global changes of rhythmic transcription atthe genome level is only starting to be revealed [147]. Circadian pro-cesses at the body level include the sleep/wake cycle, body tempera-ture, blood pressure, hormone secretion, etc. In the liver, the crucialendogenous metabolic pathways (metabolism of glucose, fatty acids,cholesterol, amino acids, etc.) and detoxification of xenobiotics repre-sent thousands of genes that are orchestrated by themolecular circadianclock [147–149]. The concentration of several bloodmetabolites, includ-ing cholesterol [150] and corticosterone [151], the mouse analogue ofcortisol, changes over 24-hour periods. It is thus not surprising than anintact circadian clock is essential for themaintenance of body homeosta-sis [149] and that disruption of the clock leads to desynchronization ofmetabolism and consequently to pathologies, including obesity and can-cer [152]. Furthermore, clock genes have been identified as potentialtherapeutic targets. A small molecule KL001 binds to CRY1 and preventsits degradation, resulting in lengthening of the circadian period. Stabili-zation of CRY has important metabolic consequences since it inhibitshepatic gluconeogenesis [153].

The major causes of clock disruption are chronic lifestyle distur-bances, such as professional jet lag or long-term shift work. Here,humans and mice seem to share similar mechanisms since circadiandesynchrony also promotes metabolic disruption in a mouse modelof shift work [154], where Per1 and Per2 regulate the physiologicalresponse of metabolism associated genes to sleep disruption [155].As relations between the hepatic clock and metabolic disturbancesmay be difficult to assess in humans, mousemodels remain importanttools [156–160]. Animals with mutations in clock genes have providedkey insights into the inter-dependence between the circadian clockand metabolism [161]. One of the most striking proofs of the essentialconnection between clock and metabolism in humans arises from a14-year prospective study in Japan in which about 7000 steel industryworkers were routinely examined for general health and blood lipid pa-rameters. The study established the adverse effects of alternating shiftwork on lipid metabolism, leading to a statistically significant rise intotal blood cholesterol [162]. The consequences of the clock disruptionfor gut systems diseases, including the effect on the liver, have alsobeen reviewed [163] but a direct link between clock disruption andliver diseases has been shown only in mouse models. Hepatic fibrosisin mice leads to alterations in the circadian rhythm of hepatic clockgenes where the daily rhythm of Cry2 was markedly decreased andassociated with loss of circadian rhythm of Ppara and cytochrome P450reductase Por [164]. Moreover, the circadian disruption acceleratedhepatic carcinogenesis in mice, suggesting that persistent circadiancoordination can slow down and/or revert cancer development [165].

In addition to circadian disruption, mutations in the core clockgenes might also result in disease susceptibility and progression, orinfluence treatment outcomes. Hawkins et al. [166] discovered con-siderable differences in minor allele frequency across ethnic groupsfor nonsynonymous coding variants in clock genes PER1, PER2, PER3,CLOCK and TIMELESS. Due to limitations of this study (only 95 subjectsof various ethnic groups) the link to disease could not be dissected. Inanother study, a functional polymorphism in PER3, a circadian repressor,is associated with the prognosis of HCC patients treated with trans-catheter arterial chemoembolization. Patients carrying at least one variantallele of rs2640908 had a significantly lower risk of deathwhen comparedwith homozygouswild-type allele patients [167]. Another study reportedthe link between common genetic variations of the CLOCK gene and sus-ceptibility to NAFLD [168]. The study included 136 patients with NAFLDand 64 healthy individuals and showed that CLOCK variant haplotypefrequencies significantly differ between NAFLD patients and controls.

Novel indications point to histone modification enzymes as addi-tional gene targets associated with circadian rhythm-related disorders.Circadianmodification of histone regions in daily-activated genes high-lights epigenetic modification as a key node regulated by the clock. Thehistone-remodelling enzymeMLL3was found tomodulate hundreds ofepigenetically targeted liver circadian “output” genes, especially thesein the one-carbon metabolism pathway [147].MLL3 is thus a good can-didate linking the circadian clock and liver diseases. It is plausible to be-lieve that other circadian rhythm-related genes and metabolites mightin the future become recognized as promising non-invasive markersthat would be useful in determining the stages and prognosis of liverdiseases.

Acknowledgments

This work was supported by the FP7 FightingDrugFailure MarieCurie Initial Training Network grant 238132, by Slovenian ResearchAgency grants J7-4053 and P1-0104 and by funds from Diagenomid.o.o.

References

[1] N.C. Leite, G.F. Salles, A.L. Araujo, C.A. Villela-Nogueira, C.R. Cardoso, Prevalenceand associated factors of non-alcoholic fatty liver disease in patients with type-2diabetes mellitus, Liver Int. 29 (2009) 113–119.

[2] M. Machado, P. Marques-Vidal, H. Cortez-Pinto, Hepatic histology in obesepatients undergoing bariatric surgery, J. Hepatol. 45 (2006) 600–606.

[3] N. Chalasani, Z. Younossi, J.E. Lavine, A.M. Diehl, E.M. Brunt, K. Cusi, M. Charlton,A.J. Sanyal, The diagnosis and management of non-alcoholic fatty liver disease:practice Guideline by the American Association for the Study of Liver Diseases,American College of Gastroenterology, and the American GastroenterologicalAssociation, Hepatology 55 (2012) 2005–2023.

[4] G. Baffy, E.M. Brunt, S.H. Caldwell, Hepatocellular carcinoma in non-alcoholicfatty liver disease: an emerging menace, J. Hepatol. 56 (2012) 1384–1391.

[5] A. Jemal, F. Bray, M.M. Center, J. Ferlay, E.Ward, D. Forman, Global cancer statistics,CA Cancer J. Clin. 61 (2011) 69–90.

[6] H.B. El-Serag, Hepatocellular carcinoma, N. Engl. J. Med. 365 (2011) 1118–1127.[7] W. Blonski, D.S. Kotlyar, K.A. Forde, Non-viral causes of hepatocellular carcino-

ma, World J. Gastroenterol. 16 (2010) 3603–3615.[8] S.C. Larsson, A. Wolk, Overweight, obesity and risk of liver cancer: a meta-

analysis of cohort studies, Br. J. Cancer 97 (2007) 1005–1008.[9] J. Ertle, A. Dechene, J.P. Sowa, V. Penndorf, K. Herzer, G. Kaiser, J.F. Schlaak, G.

Gerken, W.K. Syn, A. Canbay, Non-alcoholic fatty liver disease progresses tohepatocellular carcinoma in the absence of apparent cirrhosis, Int. J. Cancer128 (2011) 2436–2443.

[10] M.P. Bralet, J.M. Regimbeau, P. Pineau, S. Dubois, G. Loas, F. Degos, D. Valla, J.Belghiti, C. Degott, B. Terris, Hepatocellular carcinoma occurring in nonfibroticliver: epidemiologic and histopathologic analysis of 80 French cases, Hepatology32 (2000) 200–204.

[11] K. Fon Tacer, D. Rozman, Nonalcoholic fatty liver disease: focus on lipoproteinand lipid deregulation, J. Lipids 2011 (2011).

[12] G. Lorbek, D. Rozman, Cholesterol and Inflammation at the Crossroads ofNon-alcoholic Fatty Liver Disease (NAFLD) and Atherogenesis, in: S. Parthasarathy(Ed.), Atherogenesis, InTech, 2012, pp. 281–304.

[13] E.M. Brunt, D.G. Tiniakos, Histopathology of nonalcoholic fatty liver disease,World J. Gastroenterol. 16 (2010) 5286–5296.

[14] E.M. Brunt, Nonalcoholic steatohepatitis, Semin. Liver Dis. 24 (2004) 3–20.

92 A. Naik et al. / Genomics 102 (2013) 84–95

[15] K.L. Donnelly, C.I. Smith, S.J. Schwarzenberg, J. Jessurun, M.D. Boldt, E.J. Parks,Sources of fatty acids stored in liver and secreted via lipoproteins in patientswith nonalcoholic fatty liver disease, J. Clin. Invest. 115 (2005) 1343–1351.

[16] M. Kohjima, M. Enjoji, N. Higuchi, M. Kato, K. Kotoh, T. Yoshimoto, T. Fujino, M.Yada, R. Yada, N. Harada, R. Takayanagi, M. Nakamuta, Re-evaluation of fattyacid metabolism-related gene expression in nonalcoholic fatty liver disease,Int. J. Mol. Med. 20 (2007) 351–358.

[17] Z.X. Yang, W. Shen, H. Sun, Effects of nuclear receptor FXR on the regulation ofliver lipid metabolism in patients with non-alcoholic fatty liver disease, Hepatol.Int. 4 (2010) 741–748.

[18] R. Dentin, F. Benhamed, I. Hainault, V. Fauveau, F. Foufelle, J.R. Dyck, J. Girard, C.Postic, Liver-specific inhibition of ChREBP improves hepatic steatosis and insulinresistance in ob/ob mice, Diabetes 55 (2006) 2159–2170.

[19] E.R. Kallwitz, A. McLachlan, S.J. Cotler, Role of peroxisome proliferators-activatedreceptors in the pathogenesis and treatment of nonalcoholic fatty liver disease,World J. Gastroenterol. 14 (2008) 22–28.

[20] A. Naik, A. Belič, U.M. Zanger, D. Rozman, Molecular interactions between NAFLDand xenobiotic metabolism, Front. Genet. 4 (2013) 2.

[21] C.P. Day, O.F. James, Steatohepatitis: a tale of two “hits”? Gastroenterology 114(1998) 842–845.

[22] C.P. Day, From fat to inflammation, Gastroenterology 130 (2006) 207–210.[23] A.E. Feldstein, N.W. Werneburg, A. Canbay, M.E. Guicciardi, S.F. Bronk, R.

Rydzewski, L.J. Burgart, G.J. Gores, Free fatty acids promote hepatic lipotoxicityby stimulating TNF-alpha expression via a lysosomal pathway, Hepatology 40(2004) 185–194.

[24] K. Yamaguchi, L. Yang, S. McCall, J. Huang, X.X. Yu, S.K. Pandey, S. Bhanot, B.P.Monia, Y.X. Li, A.M. Diehl, Inhibiting triglyceride synthesis improves hepaticsteatosis but exacerbates liver damage andfibrosis in obesemicewith nonalcoholicsteatohepatitis, Hepatology 45 (2007) 1366–1374.

[25] N. Anderson, J. Borlak, Molecular mechanisms and therapeutic targets insteatosis and steatohepatitis, Pharmacol. Rev. 60 (2008) 311–357.

[26] F. Caballero, A. Fernandez, A.M. De Lacy, J.C. Fernandez-Checa, J. Caballeria, C.Garcia-Ruiz, Enhanced free cholesterol, SREBP-2 and StAR expression in humanNASH, J. Hepatol. 50 (2009) 789–796.

[27] D.M. Van Rooyen, C.Z. Larter, W.G. Haigh, M.M. Yeh, G. Ioannou, R. Kuver, S.P.Lee, N.C. Teoh, G.C. Farrell, Hepatic free cholesterol accumulates in obese,diabetic mice and causes non-alcoholic steatohepatitis, Gastroenterology 141 (4)(2011) 1393–1403.

[28] Z.M. Younossi, A. Baranova, K. Ziegler, L. Del Giacco, K. Schlauch, T.L. Born, H.Elariny, F. Gorreta, A. VanMeter, A. Younoszai, J.P. Ong, Z. Goodman, V.Chandhoke, A genomic and proteomic study of the spectrum of nonalcoholicfatty liver disease, Hepatology 42 (2005) 665–674.

[29] N.M. Borradaile, X. Han, J.D. Harp, S.E. Gale, D.S. Ory, J.E. Schaffer, Disruption ofendoplasmic reticulum structure and integrity in lipotoxic cell death, J. LipidRes. 47 (2006) 2726–2737.

[30] M. Cnop, P.J. Havel, K.M. Utzschneider, D.B. Carr, M.K. Sinha, E.J. Boyko, B.M.Retzlaff, R.H. Knopp, J.D. Brunzell, S.E. Kahn, Relationship of adiponectin tobody fat distribution, insulin sensitivity and plasma lipoproteins: evidence forindependent roles of age and sex, Diabetologia 46 (2003) 459–469.

[31] E. Bugianesi, U. Pagotto, R. Manini, E. Vanni, A. Gastaldelli, R. de Iasio, E.Gentilcore, S. Natale, M. Cassader, M. Rizzetto, R. Pasquali, G. Marchesini, Plasmaadiponectin in nonalcoholic fatty liver is related to hepatic insulin resistanceand hepatic fat content, not to liver disease severity, J. Clin. Endocrinol. Metab.90 (2005) 3498–3504.

[32] J.M. Hui, A. Hodge, G.C. Farrell, J.G. Kench, A. Kriketos, J. George, Beyond insulinresistance in NASH: TNF-alpha or adiponectin? Hepatology 40 (2004) 46–54.

[33] F. Andreelli, M. Foretz, C. Knauf, P.D. Cani, C. Perrin, M.A. Iglesias, B. Pillot, A.Bado, F. Tronche, G. Mithieux, S. Vaulont, R. Burcelin, B. Viollet, Liver adenosinemonophosphate-activated kinase-alpha2 catalytic subunit is a key target for thecontrol of hepatic glucose production by adiponectin and leptin but not insulin,Endocrinology 147 (2006) 2432–2441.

[34] Y. Minokoshi, Y.B. Kim, O.D. Peroni, L.G. Fryer, C. Muller, D. Carling, B.B. Kahn,Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase,Nature 415 (2002) 339–343.

[35] V. Medici, M.R. Ali, S. Seo, C.A. Aoki, L. Rossaro, K. Kim, W.D. Fuller, T.J. Vidovszky,W. Smith, J.X. Jiang, K. Maganti, P.J. Havel, A. Kamboj, R. Ramsamooj, N.J. Torok,Increased soluble leptin receptor levels in morbidly obese patients with insulinresistance and nonalcoholic fatty liver disease, Obesity (Silver Spring) 18 (2010)2268–2273.

[36] N.S. Singhal, R.T. Patel, Y. Qi, Y.S. Lee, R.S. Ahima, Loss of resistin ameliorateshyperlipidemia and hepatic steatosis in leptin-deficient mice, Am. J. Physiol.Endocrinol. Metab. 295 (2008) E331–E338.

[37] C. Pagano, G. Soardo, C. Pilon, C. Milocco, L. Basan, G. Milan, D. Donnini, D.Faggian, M. Mussap, M. Plebani, C. Avellini, G. Federspil, L.A. Sechi, R. Vettor,Increased serum resistin in nonalcoholic fatty liver disease is related to liverdisease severity and not to insulin resistance, J. Clin. Endocrinol. Metab. 91 (2006)1081–1086.

[38] G. Perseghin, R. Bonfanti, S. Magni, G. Lattuada, F. De Cobelli, T. Canu, A. Esposito,P. Scifo, G. Ntali, F. Costantino, L. Bosio, F. Ragogna, A. Del Maschio, G. Chiumello,L. Luzi, Insulin resistance andwhole body energy homeostasis in obese adolescentswith fatty liver disease, Am. J. Physiol. Endocrinol. Metab. 291 (2006) E697–E703.

[39] F. Marra, C. Bertolani, Adipokines in liver diseases, Hepatology 50 (2009) 957–969.[40] J. Crespo, A. Cayon, P. Fernandez-Gil, M. Hernandez-Guerra, M. Mayorga, A.

Dominguez-Diez, J.C. Fernandez-Escalante, F. Pons-Romero, Gene expression oftumor necrosis factor alpha and TNF-receptors, p55 and p75, in nonalcoholicsteatohepatitis patients, Hepatology 34 (2001) 1158–1163.

[41] H. Tilg, G.S. Hotamisligil, Nonalcoholic fatty liver disease: cytokine–adipokineinterplay and regulation of insulin resistance, Gastroenterology 131 (2006)934–945.

[42] A. Wree, A. Kahraman, G. Gerken, A. Canbay, Obesity affects the liver — the linkbetween adipocytes and hepatocytes, Digestion 83 (2011) 124–133.

[43] J. Jou, S.S. Choi, A.M. Diehl, Mechanisms of disease progression in nonalcoholicfatty liver disease, Semin. Liver Dis. 28 (2008) 370,379.

[44] S.H. Caldwell, D.M. Crespo, The spectrum expanded: cryptogenic cirrhosis andthe natural history of non-alcoholic fatty liver disease, J. Hepatol. 40 (2004)578–584.

[45] M. Shimada, E. Hashimoto, M. Taniai, K. Hasegawa, H. Okuda, N. Hayashi, K.Takasaki, J. Ludwig, Hepatocellular carcinoma in patients with non-alcoholicsteatohepatitis, J. Hepatol. 37 (2002) 154–160.

[46] S. Yang, H.Z. Lin, J. Hwang, V.P. Chacko, A.M. Diehl, Hepatic hyperplasia innoncirrhotic fatty livers: is obesity-related hepatic steatosis a premalignant con-dition? Cancer Res. 61 (2001) 5016–5023.

[47] F. Stickel, C. Hellerbrand, Non-alcoholic fatty liver disease as a risk factor forhepatocellular carcinoma:mechanisms and implications, Gut 59 (2010) 1303–1307.

[48] M. Vinciguerra, F. Carrozzino, M. Peyrou, S. Carlone, R. Montesano, R. Benelli, M.Foti, Unsaturated fatty acids promote hepatoma proliferation and progressionthrough downregulation of the tumor suppressor PTEN, J. Hepatol. 50 (2009)1132–1141.

[49] Y. Wei, D. Wang, F. Topczewski, M.J. Pagliassotti, Saturated fatty acids induceendoplasmic reticulum stress and apoptosis independently of ceramide inliver cells, Am. J. Physiol. Endocrinol. Metab. 291 (2006) E275–E281.

[50] I.A. Kirpich, L.N. Gobejishvili, M. Bon Homme, S. Waigel, M. Cave, G. Arteel, S.S.Barve, C.J. McClain, I.V. Deaciuc, Integrated hepatic transcriptome and proteomeanalysis of mice with high-fat diet-induced nonalcoholic fatty liver disease,J. Nutr. Biochem. 22 (2011) 38–45.

[51] X. Zhang, J. Yang, Y. Guo, H. Ye, C. Yu, C. Xu, L. Xu, S. Wu, W. Sun, H. Wei, X. Gao,Y. Zhu, X. Qian, Y. Jiang, Y. Li, F. He, Functional proteomic analysis of nonalcoholicfatty liver disease in rat models: enoyl-coenzyme a hydratase down-regulationexacerbates hepatic steatosis, Hepatology 51 (2010) 1190–1199.

[52] V.S. Calvert, R. Collantes, H. Elariny, A. Afendy, A. Baranova, M. Mendoza, Z.Goodman, L.A. Liotta, E.F. Petricoin, Z.M. Younossi, A systems biology approachto the pathogenesis of obesity-related nonalcoholic fatty liver disease usingreverse phase protein microarrays for multiplexed cell signaling analysis,Hepatology 46 (2007) 166–172.

[53] S.C. Kalhan, L. Guo, J. Edmison, S. Dasarathy, A.J. McCullough, R.W. Hanson, M.Milburn, Plasma metabolomic profile in nonalcoholic fatty liver disease, Metab.Clin. Exp. 60 (2011) 404–413.

[54] J.L. Griffin, S.A. Bonney, C. Mann, A.M. Hebbachi, G.F. Gibbons, J.K. Nicholson, C.C.Shoulders, J. Scott, An integrated reverse functional genomic and metabolic ap-proach to understanding orotic acid-induced fatty liver, Physiol. Genomics 17(2004) 140–149.

[55] A. Baranova, K. Schlauch, H. Elariny, M. Jarrar, C. Bennett, C. Nugent, S.J. Gowder,Z. Younoszai, R. Collantes, V. Chandhoke, Z.M. Younossi, Gene expression patternsin hepatic tissue and visceral adipose tissue of patients with non-alcoholic fattyliver disease, Obes. Surg. 17 (2007) 1111–1118.

[56] M. Stepanova, N. Hossain, A. Afendy, K. Perry, Z.D. Goodman, A. Baranova, Z.Younossi, Hepatic gene expression of Caucasian and African-American patientswith obesity-related non-alcoholic fatty liver disease, Obes. Surg. 20 (2010)640–650.

[57] O. Cheung, P. Puri, C. Eicken, M.J. Contos, F. Mirshahi, J.W. Maher, J.M. Kellum, H.Min, V.A. Luketic, A.J. Sanyal, Nonalcoholic steatohepatitis is associated withaltered hepatic microRNA expression, Hepatology 48 (2008) 1810–1820.

[58] C. Esau, S. Davis, S.F. Murray, X.X. Yu, S.K. Pandey, M. Pear, L. Watts, S.L. Booten,M. Graham, R. McKay, A. Subramaniam, S. Propp, B.A. Lollo, S. Freier, C.F.Bennett, S. Bhanot, B.P. Monia, miR-122 regulation of lipid metabolism revealedby in vivo antisense targeting, Cell Metab. 3 (2006) 87–98.

[59] Y.Y. Li, Genetic and epigenetic variants influencing the development ofnonalcoholic fatty liver disease, World J. Gastroenterol. 18 (2012) 6546–6551.

[60] P. Nahon, J. Zucman-Rossi, Single nucleotide polymorphisms and risk of hepato-cellular carcinoma in cirrhosis, J. Hepatol. 57 (2012) 663–674.

[61] D.M. Torres, S.A. Harrison, Nonalcoholic steatohepatitis and noncirrhotic hepa-tocellular carcinoma: fertile soil, Semin. Liver Dis. 32 (2012) 30–38.

[62] C. Dubuquoy, C. Robichon, F. Lasnier, C. Langlois, I. Dugail, F. Foufelle, J. Girard,A.F. Burnol, C. Postic, M. Moldes, Distinct regulation of adiponutrin/PNPLA3gene expression by the transcription factors ChREBP and SREBP1c in mouseand human hepatocytes, J. Hepatol. 55 (2011) 145–153.

[63] M.K. Basantani, M.T. Sitnick, L. Cai, D.S. Brenner, N.P. Gardner, J.Z. Li, G.Schoiswohl, K. Yang, M. Kumari, R.W. Gross, R. Zechner, E.E. Kershaw,Pnpla3/Adiponutrin deficiency in mice does not contribute to fatty liver diseaseor metabolic syndrome, J. Lipid Res. 52 (2011) 318–329.

[64] S. Romeo, J. Kozlitina, C. Xing, A. Pertsemlidis, D. Cox, L.A. Pennacchio, E.Boerwinkle, J.C. Cohen, H.H. Hobbs, Genetic variation in PNPLA3 confers suscep-tibility to nonalcoholic fatty liver disease, Nat. Genet. 40 (2008) 1461–1465.

[65] A. Kotronen, L.E. Johansson, L.M. Johansson, C. Roos, J. Westerbacka, A. Hamsten,R. Bergholm, P. Arkkila, J. Arola, T. Kiviluoto, R.M. Fisher, E. Ehrenborg, M.Orho-Melander, M. Ridderstrale, L. Groop, H. Yki-Jarvinen, A common variantin PNPLA3, which encodes adiponutrin, is associated with liver fat content inhumans, Diabetologia 52 (2009) 1056–1060.

[66] E.K. Speliotes, L.M. Yerges-Armstrong, J. Wu, R. Hernaez, L.J. Kim, C.D. Palmer, V.Gudnason, G. Eiriksdottir, M.E. Garcia, L.J. Launer, M.A. Nalls, J.M. Clark, B.D.Mitchell, A.R. Shuldiner, J.L. Butler, M. Tomas, U. Hoffmann, S.J. Hwang, J.M.Massaro, C.J. O'Donnell, D.V. Sahani, V. Salomaa, E.E. Schadt, S.M. Schwartz,

93A. Naik et al. / Genomics 102 (2013) 84–95

D.S. Siscovick, B.F. Voight, J.J. Carr, M.F. Feitosa, T.B. Harris, C.S. Fox, A.V. Smith,W.H. Kao, J.N. Hirschhorn, I.B. Borecki, Genome-wide association analysis iden-tifies variants associated with nonalcoholic fatty liver disease that have distincteffects on metabolic traits, PLoS Genet. 7 (2011) e1001324.

[67] L. Valenti, A. Al-Serri, A.K. Daly, E. Galmozzi, R. Rametta, P. Dongiovanni, V.Nobili, E. Mozzi, G. Roviaro, E. Vanni, E. Bugianesi, M. Maggioni, A.L. Fracanzani, S.Fargion, C.P. Day, Homozygosity for the patatin-like phospholipase-3/adiponutrinI148M polymorphism influences liver fibrosis in patients with nonalcoholic fattyliver disease, Hepatology 51 (2010) 1209–1217.

[68] T. Kawaguchi, Y. Sumida, A. Umemura, K. Matsuo, M. Takahashi, T. Takamura, K.Yasui, T. Saibara, E. Hashimoto, M. Kawanaka, S. Watanabe, S. Kawata, Y. Imai, M.Kokubo, T. Shima, H. Park, H. Tanaka, K. Tajima, R. Yamada, F. Matsuda, D. JapanStudy Group of Nonalcoholic Fatty Liver, Genetic polymorphisms of the humanPNPLA3 gene are strongly associated with severity of non-alcoholic fatty liverdisease in Japanese, PLoS One 7 (2012) e38322.

[69] S.M. Zain, R. Mohamed, S. Mahadeva, P.L. Cheah, S. Rampal, R.C. Basu, Z.Mohamed, A multi-ethnic study of a PNPLA3 gene variant and its associationwith disease severity in non-alcoholic fatty liver disease, Hum. Genet. 131 (7)(2012) 1145–1152.

[70] Y. Takeuchi, F. Ikeda, Y. Moritou, H. Hagihara, T. Yasunaka, K. Kuwaki, Y. Miyake,H. Ohnishi, S. Nakamura, H. Shiraha, A. Takaki, Y. Iwasaki, K. Nouso, K.Yamamoto, The impact of patatin-like phospholipase domain-containing pro-tein 3 polymorphism on hepatocellular carcinoma prognosis, J. Gastroenterol.48 (3) (2012) 405–412.

[71] E. Falleti, C. Fabris, S. Cmet, A. Cussigh, D. Bitetto, E. Fontanini, E. Fornasiere, S.Bignulin, E. Fumolo, E. Bignulin, M. Pirisi, P. Toniutto, PNPLA3 rs738409C/G poly-morphism in cirrhosis: relationship with the aetiology of liver disease and hepa-tocellular carcinoma occurrence, Liver Int. 31 (2011) 1137–1143.

[72] C. Namikawa, Z. Shu-Ping, J.R. Vyselaar, Y. Nozaki, Y. Nemoto, M. Ono, N.Akisawa, T. Saibara, M. Hiroi, H. Enzan, S. Onishi, Polymorphisms of microsomaltriglyceride transfer protein gene and manganese superoxide dismutase gene innon-alcoholic steatohepatitis, J. Hepatol. 40 (2004) 781–786.

[73] A. Al-Serri, Q.M. Anstee, L. Valenti, V. Nobili, J.B. Leathart, P. Dongiovanni, J.Patch, A. Fracanzani, S. Fargion, C.P. Day, A.K. Daly, The sod2 c47t polymorphisminfluences nafld fibrosis severity: evidence from case–control and intra-familialallele association studies, J. Hepatol. 56 (2) (2011) 448–454.

[74] P. Nahon, A. Sutton, P. Rufat, M. Ziol, H. Akouche, C. Laguillier, N. Charnaux, N.Ganne-Carrie, V. Grando-Lemaire, G. N'Kontchou, J.C. Trinchet, L. Gattegno, D.Pessayre, M. Beaugrand, Myeloperoxidase and superoxide dismutase 2 poly-morphisms comodulate the risk of hepatocellular carcinoma and death in alco-holic cirrhosis, Hepatology 50 (2009) 1484–1493.

[75] K. Pfeffer, Biological functions of tumor necrosis factor cytokines and theirreceptors, Cytokine Growth Factor Rev. 14 (2003) 185–191.

[76] B. Antuna-Puente, B. Feve, S. Fellahi, J.P. Bastard, Adipokines: the missing linkbetween insulin resistance and obesity, Diabetes Metab. 34 (2008) 2–11.

[77] T. Qidwai, F. Khan, Tumour necrosis factor gene polymorphism and diseaseprevalence, Scand. J. Immunol. 74 (2011) 522–547.

[78] L. Valenti, A.L. Fracanzani, P. Dongiovanni, G. Santorelli, A. Branchi, E. Taioli, G. Fiorelli,S. Fargion, Tumor necrosis factor alpha promoter polymorphisms and insulin resis-tance in nonalcoholic fatty liver disease, Gastroenterology 122 (2002) 274–280.

[79] K. Trujillo-Murillo, F.J. Bosques-Padilla, I. Calderón-Lozano, S. Navar-Vizcarra, E.Garza-González, A. Niderhauser-García, J.P. Flores-Gutiérrez, P. Zorrilla-Blanco,R. Salinas-Garza, A.M. Rivas-Estilla, H.G. Martínez-Rodríguez, Association oftumor necrosis factor α and manganese superoxide dismutase polymorphismsin patients with non-alcoholic steatohepatitis from northeast Mexico, OpenHepatol. J. 3 (2011) 1–6.

[80] Y.J. Zhou, Y.Y. Li, Y.Q. Nie, H. Yang, Q. Zhan, J. Huang, S.L. Shi, X.B. Lai, H.L. Huang,Influence of polygenetic polymorphisms on the susceptibility to non-alcoholicfatty liver disease of Chinese people, J. Gastroenterol. Hepatol. 25 (2010) 772–777.

[81] Z.W. Hu, H.B. Luo, Y.M. Xu, J.W. Guo, X.L. Deng, Y.W. Tong, X. Tang, Tumor necrosisfactor-alpha gene promoter polymorphisms in Chinese patients with nonalcoholicfatty liver diseases, Acta Gastroenterol Belg 72 (2009) 215–221.

[82] K. Tokushige, M. Takakura, N. Tsuchiya-Matsushita, M. Taniai, E. Hashimoto, K.Shiratori, Influence of TNF gene polymorphisms in Japanese patients withNASH and simple steatosis, J. Hepatol. 46 (2007) 1104–1110.

[83] V.W. Wong, G.L. Wong, S.W. Tsang, A.Y. Hui, A.W. Chan, P.C. Choi, W.Y. So, A.M.Tse, F.K. Chan, J.J. Sung, H.L. Chan, Genetic polymorphisms of adiponectin andtumor necrosis factor-alpha and nonalcoholic fatty liver disease in Chinese people,J. Gastroenterol. Hepatol. 23 (2008) 914–921.

[84] J.K.Wang, Z.W. Feng, Y.C. Li, Q.Y. Li, X.Y. Tao, Association of tumor necrosis factor-αgene promoter polymorphism at sites -308 and -238 with non-alcoholic fatty liverdisease: a meta-analysis, J. Gastroenterol. Hepatol. 27 (2012) 670–676.

[85] Y. Wei, F. Liu, B. Li, X. Chen, Y. Ma, L. Yan, T. Wen, M. Xu, W. Wang, J. Yang, Poly-morphisms of tumor necrosis factor-alpha and hepatocellular carcinoma risk: aHuGE systematic review and meta-analysis, Dig. Dis. Sci. 56 (2011) 2227–2236.

[86] G.M. Anderson, M.T. Nakada, M. DeWitte, Tumor necrosis factor-alpha in thepathogenesis and treatment of cancer, Curr. Opin. Pharmacol. 4 (2004) 314–320.

[87] M. Hori, K. Oniki, T. Nakagawa, K. Takata, S. Mihara, T. Marubayashi, K. Nakagawa,Association between combinations of glutathione-S-transferase M1, T1 and P1 ge-notypes and non-alcoholic fatty liver disease, Liver Int. 29 (2009) 164–168.

[88] B. Wang, G. Huang, D. Wang, A. Li, Z. Xu, R. Dong, D. Zhang, W. Zhou, Nullgenotypes of GSTM1 and GSTT1 contribute to hepatocellular carcinoma risk:evidence from an updated meta-analysis, J. Hepatol. 53 (2010) 508–518.

[89] J. Zhang, H. Liu, H. Yan, G. Huang, B. Wang, Null genotypes of GSTM1 and GSTT1contribute to increased risk of diabetes mellitus: a meta-analysis, Gene 518 (2)(2013) 405–411.

[90] H.P. Kan, F.L. Wu, W.B. Guo, Y.Z. Wang, J.P. Li, Y.Q. Huang, S.G. Li, J.P. Liu, Nullgenotypes of GSTM1 and GSTT1 contribute to male factor infertility risk: ameta-analysis, Fertil. Steril. 99 (3) (2012) 690–696.

[91] M. Rakhshandehroo, B. Knoch, M. Muller, S. Kersten, Peroxisomeproliferator-activated receptor alpha target genes, PPAR Res. 2010 (2010),http://dx.doi.org/10.1155/2010/612089, (Epub).

[92] T. Yamauchi, J. Kamon, Y. Ito, A. Tsuchida, T. Yokomizo, S. Kita, T. Sugiyama, M.Miyagishi, K. Hara, M. Tsunoda, K. Murakami, T. Ohteki, S. Uchida, S. Takekawa, H.Waki, N.H. Tsuno, Y. Shibata, Y. Terauchi, P. Froguel, K. Tobe, S. Koyasu, K. Taira, T.Kitamura, T. Shimizu, R. Nagai, T. Kadowaki, Cloning of adiponectin receptors thatmediate antidiabetic metabolic effects, Nature 423 (2003) 762–769.

[93] T. Nagasawa, Y. Inada, S. Nakano, T. Tamura, T. Takahashi, K. Maruyama, Y.Yamazaki, J. Kuroda, N. Shibata, Effects of bezafibrate, PPAR pan-agonist, andGW501516, PPARdelta agonist, on development of steatohepatitis in mice feda methionine- and choline-deficient diet, Eur. J. Pharmacol. 536 (2006) 182–191.

[94] S.S. Lee, T. Pineau, J. Drago, E.J. Lee, J.W. Owens, D.L. Kroetz, P.M. Fernandez-Salguero, H. Westphal, F.J. Gonzalez, Targeted disruption of the alpha isoformof the peroxisome proliferator-activated receptor gene in mice results in abol-ishment of the pleiotropic effects of peroxisome proliferators, Mol. Cell. Biol.15 (1995) 3012–3022.

[95] C.R. Sirtori, G. Franceschini, Effects of fibrates on serum lipids and atherosclerosis,Pharmacol. Ther. 37 (1988) 167–191.

[96] K. Yamakawa-Kobayashi, H. Ishiguro, T. Arinami, R. Miyazaki, H. Hamaguchi, AVal227Ala polymorphism in the peroxisome proliferator activated receptoralpha (PPARalpha) gene is associated with variations in serum lipid levels,J. Med. Genet. 39 (2002) 189–191.

[97] H. Naito, O. Yamanoshita, M. Kamijima, T. Katoh, T. Matsunaga, C.H. Lee, H. Kim,T. Aoyama, F.J. Gonzalez, T. Nakajima, Association of V227A PPARalpha polymor-phism with altered serum biochemistry and alcohol drinking in Japanese men,Pharmacogenet. Genomics 16 (2006) 569–577.

[98] S. Chen, Y. Li, S. Li, C. Yu, A Val227Ala substitution in the peroxisome proliferatoractivated receptor alpha (PPAR alpha) gene associated with non-alcoholic fattyliver disease and decreased waist circumference and waist-to-hip ratio,J. Gastroenterol. Hepatol. 23 (2008) 1415–1418.

[99] T.C. Leone, J.J. Lehman, B.N. Finck, P.J. Schaeffer, A.R. Wende, S. Boudina, M.Courtois, D.F. Wozniak, N. Sambandam, C. Bernal-Mizrachi, Z. Chen, J.O.Holloszy, D.M. Medeiros, R.E. Schmidt, J.E. Saffitz, E.D. Abel, C.F. Semenkovich,D.P. Kelly, PGC-1alpha deficiency causes multi-system energy metabolicderangements: muscle dysfunction, abnormal weight control and hepaticsteatosis, PLoS Biol. 3 (2005) e101.

[100] C. Canto, J. Auwerx, PGC-1alpha, SIRT1 and AMPK, an energy sensing networkthat controls energy expenditure, Curr. Opin. Lipidol. 20 (2009) 98–105.

[101] S. Sookoian, M.S. Rosselli, C. Gemma, A.L. Burgueno, T. Fernandez Gianotti, G.O.Castano, C.J. Pirola, Epigenetic regulation of insulin resistance in nonalcoholicfatty liver disease: impact of liver methylation of the peroxisome proliferator-activated receptor gamma coactivator 1alpha promoter, Hepatology 52 (2010)1992–2000.

[102] M. Yoneda, K. Hotta, Y. Nozaki, H. Endo, T. Uchiyama, H. Mawatari, H. Iida, S.Kato, K. Hosono, K. Fujita, K. Yoneda, H. Takahashi, H. Kirikoshi, N. Kobayashi,M. Inamori, Y. Abe, K. Kubota, S. Saito, S. Maeyama, K. Wada, A. Nakajima, Asso-ciation between PPARGC1A polymorphisms and the occurrence of nonalcoholicfatty liver disease (NAFLD), BMC Gastroenterol. 8 (2008) 27.

[103] X. Chang, H. Yan, J. Fei, M. Jiang, H. Zhu, D. Lu, X. Gao, Berberine reduces meth-ylation of the MTTP promoter and alleviates fatty liver induced by a high-fat dietin rats, J. Lipid Res. 51 (2010) 2504–2515.

[104] R. Gambino, M. Cassader, G. Pagano, M. Durazzo, G. Musso, Polymorphism inmicrosomal triglyceride transfer protein: a link between liver disease andatherogenic postprandial lipid profile in NASH? Hepatology 45 (2007) 1097–1107.

[105] A.A. Noga, Y. Zhao, D.E. Vance, An unexpected requirement for phosphatidyleth-anolamine N-methyltransferase in the secretion of very low density lipopro-teins, J. Biol. Chem. 277 (2002) 42358–42365.

[106] J. Song, K.A. da Costa, L.M. Fischer, M. Kohlmeier, L. Kwock, S. Wang, S.H. Zeisel,Polymorphism of the PEMT gene and susceptibility to nonalcoholic fatty liverdisease (NAFLD), FASEB J. 19 (2005) 1266–1271.

[107] H. Dong, J. Wang, C. Li, A. Hirose, Y. Nozaki, M. Takahashi, M. Ono, N. Akisawa, S.Iwasaki, T. Saibara, S. Onishi, The phosphatidylethanolamine N-methyltransferasegene V175M single nucleotide polymorphism confers the susceptibility to NASHin Japanese population, J. Hepatol. 46 (2007) 915–920.

[108] J. Altomonte, L. Cong, S. Harbaran, A. Richter, J. Xu, M. Meseck, H.H. Dong, Foxo1mediates insulin action on apoC-III and triglyceride metabolism, J. Clin. Invest.114 (2004) 1493–1503.

[109] K.F. Petersen, S. Dufour, A. Hariri, C. Nelson-Williams, J.N. Foo, X.M. Zhang, J.Dziura, R.P. Lifton, G.I. Shulman, Apolipoprotein C3 gene variants in nonalcoholicfatty liver disease, N. Engl. J. Med. 362 (2010) 1082–1089.

[110] J. Kozlitina, E. Boerwinkle, J.C. Cohen, H.H. Hobbs, Dissociation between APOC3variants, hepatic triglyceride content and insulin resistance, Hepatology 53(2011) 467–474.

[111] F. Sentinelli, S. Romeo, C. Maglio, M. Incani, M.A. Burza, F. Scano, F. Coccia, E. Cossu, F.Leonetti, M.G. Baroni, Lack of effect of apolipoprotein C3 polymorphisms on indices ofliver steatosis, lipid profile and insulin resistance in obese Southern Europeans, LipidsHealth Dis. 10 (2011) 93.

[112] J. Hyysalo, I. Stojkovic, A. Kotronen, A. Hakkarainen, K. Sevastianova, J. Makkonen,N. Lundbom, A. Rissanen, R.M. Krauss, O. Melander, M. Orho-Melander, H.Yki-Jarvinen, Genetic variation in PNPLA3 but not APOC3 influences liver fatin non-alcoholic fatty liver disease, J. Gastroenterol. Hepatol. 27 (2012)951–956.

94 A. Naik et al. / Genomics 102 (2013) 84–95

[113] E.A. Karavia, D.J. Papachristou, I. Kotsikogianni, I. Giopanou, K.E. Kypreos, Defi-ciency in apolipoprotein E has a protective effect on diet-induced nonalcoholicfatty liver disease in mice, FEBS J. 278 (17) (2011) 3119–3129.

[114] J. Gao, H. Katagiri, Y. Ishigaki, T. Yamada, T. Ogihara, J. Imai, K. Uno, Y. Hasegawa,M. Kanzaki, T.T. Yamamoto, S. Ishibashi, Y. Oka, Involvement of apolipoprotein Ein excess fat accumulation and insulin resistance, Diabetes 56 (2007) 24–33.

[115] A. Sazci, G. Akpinar, C. Aygun, E. Ergul, O. Senturk, S. Hulagu, Association of apo-lipoprotein E polymorphisms in patients with non-alcoholic steatohepatitis, Dig.Dis. Sci. 53 (2008) 3218–3224.

[116] M.D. Demirag, H.I. Onen, M.Y. Karaoguz, I. Dogan, T. Karakan, A. Ekmekci, G. Guz,Apolipoprotein E gene polymorphism in nonalcoholic fatty liver disease, Dig.Dis. Sci. 52 (2007) 3399–3403.

[117] W.L. Holland, R.A. Miller, Z.V. Wang, K. Sun, B.M. Barth, H.H. Bui, K.E. Davis, B.T.Bikman, N. Halberg, J.M. Rutkowski, M.R. Wade, V.M. Tenorio, M.-S. Kuo, J.T.Brozinick, B.B. Zhang, M.J. Birnbaum, S.A. Summers, P.E. Scherer, Receptor-mediatedactivation of ceramidase activity initiates the pleiotropic actions of adiponectin,Nat. Med. 17 (2011) 55–63.

[118] G. Musso, R. Gambino, F. De Michieli, M. Durazzo, G. Pagano, M. Cassader,Adiponectin gene polymorphisms modulate acute adiponectin response todietary fat: possible pathogenetic role in NASH, Hepatology 47 (2008) 1167–1177.

[119] A.J. Hooper, L.A. Adams, J.R. Burnett, Genetic determinants of hepatic steatosis inman, J. Lipid Res. 52 (2011) 593–617.

[120] T. Kadowaki, T. Yamauchi, Adiponectin and adiponectin receptors, Endocr. Rev.26 (2005) 439–451.

[121] A. Kotronen, H. Yki-Jarvinen, A. Aminoff, R. Bergholm, K.H. Pietilainen, J.Westerbacka, P.J. Talmud, S.E. Humphries, A. Hamsten, B. Isomaa, L. Groop, M.Orho-Melander, E. Ehrenborg, R.M. Fisher, Genetic variation in the ADIPOR2gene is associated with liver fat content and its surrogate markers in three inde-pendent cohorts, Eur. J. Endocrinol. 160 (2009) 593–602.

[122] L. Rui, M. Yuan, D. Frantz, S. Shoelson, M.F. White, SOCS-1 and SOCS-3 blockinsulin signaling by ubiquitin-mediated degradation of IRS1 and IRS2, J. Biol.Chem. 277 (2002) 42394–42398.

[123] E. Vanni, E. Bugianesi, A. Kotronen, S. De Minicis, H. Yki-Jarvinen, G.Svegliati-Baroni, From the metabolic syndrome to NAFLD or vice versa? Dig.Liver Dis. 42 (2010) 320–330.

[124] P. Dongiovanni, L. Valenti, R. Rametta, A.K. Daly, V. Nobili, E. Mozzi, J.B. Leathart,A. Pietrobattista, A.D. Burt, M. Maggioni, A.L. Fracanzani, E. Lattuada, M.A. Zappa,G. Roviaro, G. Marchesini, C.P. Day, S. Fargion, Genetic variants regulating insulinreceptor signalling are associated with the severity of liver damage in patientswith non-alcoholic fatty liver disease, Gut 59 (2010) 267–273.

[125] Y. Takahashi, Essential roles of growth hormone (GH) and insulin-like growthfactor-I (IGF-I) in the liver, Endocr. J. 59 (2012) 955–962.

[126] Z. Laron, Insulin-like growth factor 1 (IGF-1): a growth hormone, Mol. Pathol. 54(2001) 311–316.

[127] H. Nishizawa, M. Takahashi, H. Fukuoka, G. Iguchi, R. Kitazawa, Y. Takahashi,GH-independent IGF-I action is essential to prevent the development ofnonalcoholic steatohepatitis in a GH-deficient rat model, Biochem. Biophys.Res. Commun. 423 (2012) 295–300.

[128] A. Fusco, L. Miele, A. D'Uonnolo, A. Forgione, L. Riccardi, C. Cefalo, A. Barini, A.Bianchi, A. Giampietro, V. Cimino, R. Landolfi, A. Grieco, L. De Marinis,Nonalcoholic fatty liver disease is associated with increased GHBP and reducedGH/IGF-I levels, Clin. Endocrinol. (Oxf.) 77 (2012) 531–536.

[129] T. Ichikawa, K. Nakao, K. Hamasaki, R. Furukawa, S. Tsuruta, Y. Ueda, N. Taura, H.Shibata, M. Fujimoto, K. Toriyama, K. Eguchi, Role of growth hormone,insulin-like growth factor 1 and insulin-like growth factor-binding protein 3 indevelopment of non-alcoholic fatty liver disease, Hepatol. Int. 1 (2007)287–294.

[130] J.W. Hong, J.Y. Kim, Y.E. Kim, E.J. Lee, Metabolic parameters and nonalcoholicfatty liver disease in hypopituitary men, Horm. Metab. Res. 43 (2011) 48–54.

[131] Y. Takahashi, K. Iida, K. Takahashi, S. Yoshioka, H. Fukuoka, R. Takeno, M.Imanaka, H. Nishizawa, M. Takahashi, Y. Seo, Y. Hayashi, T. Kondo, Y. Okimura,H. Kaji, R. Kitazawa, S. Kitazawa, K. Chihara, Growth hormone reversesnonalcoholic steatohepatitis in a patient with adult growth hormone deficiency,Gastroenterology 132 (2007) 938–943.

[132] E.J. Barbosa, C.A. Glad, A.G. Nilsson, H. Filipsson Nystrom, G. Gotherstrom, P.A.Svensson, I. Vinotti, B.A. Bengtsson, S. Nilsson, C.L. Boguszewski, G. Johannsson,Genotypes associated with lipid metabolism contribute to differences in serumlipid profile of GH-deficient adults before and after GH replacement therapy, Eur.J. Endocrinol. 167 (2012) 353–362.

[133] A.V. Patel, I. Cheng, F. Canzian, L. Le Marchand, M.J. Thun, C.D. Berg, J. Buring, E.E.Calle, S. Chanock, F. Clavel-Chapelon, D.G. Cox, M. Dorronsoro, L. Dossus, C.A.Haiman, S.E. Hankinson, B.E. Henderson, R. Hoover, D.J. Hunter, R. Kaaks, L.N.Kolonel, P. Kraft, J. Linseisen, E. Lund, J. Manjer, C. McCarty, P.H. Peeters, M.C.Pike, M. Pollak, E. Riboli, D.O. Stram, A. Tjonneland, R.C. Travis, D.Trichopoulos, R. Tumino, M. Yeager, R.G. Ziegler, H.S. Feigelson, IGF-1, IGFBP-1,and IGFBP-3 polymorphisms predict circulating IGF levels but not breast cancerrisk: findings from the Breast and Prostate Cancer Cohort Consortium (BPC3),PLoS One 3 (2008) e2578.

[134] O. Fletcher, L. Gibson, N. Johnson, D.R. Altmann, J.M. Holly, A. Ashworth, J. Peto,S. Silva Idos, Polymorphisms and circulating levels in the insulin-like growthfactor system and risk of breast cancer: a systematic review, Cancer Epidemiol.Biomarkers Prev. 14 (2005) 2–19.

[135] J.J. Senn, P.J. Klover, I.A. Nowak, T.A. Zimmers, L.G. Koniaris, R.W. Furlanetto, R.A.Mooney, Suppressor of cytokine signaling-3 (SOCS-3), a potential mediator ofinterleukin-6-dependent insulin resistance in hepatocytes, J. Biol. Chem. 278(2003) 13740–13746.

[136] P. Dandona, A. Aljada, A. Chaudhuri, P. Mohanty, R. Garg, Metabolic syndrome: acomprehensive perspective based on interactions between obesity, diabetes,and inflammation, Circulation 111 (2005) 1448–1454.

[137] J.C. Pickup, G.D. Chusney, S.M. Thomas, D. Burt, Plasma interleukin-6, tumournecrosis factor alpha and blood cytokine production in type 2 diabetes, LifeSci. 67 (2000) 291–300.

[138] L. Carulli, I. Canedi, S. Rondinella, S. Lombardini, D. Ganazzi, S. Fargion, M. DePalma, A. Lonardo, M. Ricchi, M. Bertolotti, N. Carulli, P. Loria, Genetic polymor-phisms in non-alcoholic fatty liver disease: interleukin-6-174G/C polymorphismis associated with non-alcoholic steatohepatitis, Dig. Liver Dis. 41 (2009)823–828.

[139] A. Naik, A. Belič, U.M. Zanger, D. Rozman, Molecular interactions between NAFLDand xenobiotic metabolism, Front. Gene 4 (2).

[140] J. Zhou, Y. Zhai, Y. Mu, H. Gong, H. Uppal, D. Toma, S. Ren, R.M. Evans, W. Xie,A novel pregnane X receptor-mediated and sterol regulatory element-bindingprotein-independent lipogenic pathway, J. Biol. Chem. 281 (2006) 15013–15020.

[141] S. Sookoian, G.O. Castano, A.L. Burgueno, T.F. Gianotti, M.S. Rosselli, C.J. Pirola,The nuclear receptor PXR gene variants are associated with liver injury innonalcoholic fatty liver disease, Pharmacogenet. Genomics 20 (2010) 1–8.

[142] Y.C. Lin, P.F. Chang, F.C. Hu, M.H. Chang, Y.H. Ni, Variants in the UGT1A1 geneand the risk of pediatric nonalcoholic fatty liver disease, Pediatrics 124 (2009)e1221–e1227.

[143] S. Sookoian, G. Castano, T.F. Gianotti, C. Gemma, C.J. Pirola, Polymorphismsof MRP2 (ABCC2) are associated with susceptibility to nonalcoholic fatty liverdisease, J. Nutr. Biochem. 20 (2009) 765–770.

[144] L.A. Adams, S.W. White, J.A. Marsh, S.J. Lye, K.L. Connor, R. Maganga, O.T.Ayonrinde, J.K. Olynyk, T.A. Mori, L.J. Beilin, L.J. Palmer, C.E. Pennell, Associationbetween liver-specific gene polymorphisms and their expression levels withnon-alcoholic fatty liver disease, Hepatology 57 (2) (2013) 590–600.

[145] Z.M. Younossi, M. Stepanova, N. Rafiq, H. Makhlouf, Z. Younoszai, R. Agrawal, Z.Goodman, Pathologic criteria for nonalcoholic steatohepatitis: interprotocolagreement and ability to predict liver-related mortality, Hepatology 53 (2011)1874–1882.

[146] A. Korenčič, G. Bordyugov, R. Košir, D. Rozman, M. Goličnik, H. Herzel, The inter-play of cis-regulatory elements rules circadian rhythms in mouse liver, PLoS One7 (2012) e46835.

[147] U.K. Valekunja, R.S. Edgar, M. Oklejewicz, G.T. van der Horst, J.S. O'Neill, F.Tamanini, D.J. Turner, A.B. Reddy, Histone methyltransferase MLL3 contributesto genome-scale circadian transcription, Proc. Natl. Acad. Sci. U. S. A. 110 (4)(2013) 1554–1559.

[148] U.P. Zmrzljak, D. Rozman, Circadian regulation of the hepatic endobiotic andxenobitoic detoxification pathways: the time matters, Chem. Res. Toxicol. 25(2012) 811–824.

[149] C.B. Green, J.S. Takahashi, J. Bass, The meter of metabolism, Cell 134 (2008)728–742.

[150] J. Ačimovič, R. Košir, D. Kastelec, M. Perše, G. Majdič, D. Rozman, K. Košmelj, M.Goličnik, Circadian rhythm of cholesterol synthesis in mouse liver: a statisticalanalysis of the post-squalene metabolites in wild-type and Crem-knock-outmice, Biochem. Biophys. Res. Commun. 408 (2011) 635–641.

[151] R. Košir, U.P. Zmrzljak, T. Bele, J. Acimovic, M. Perse, G. Majdic, C. Prehn, J.Adamski, D. Rozman, Circadian expression of steroidogenic cytochromes P450in the mouse adrenal gland-involvement of cAMP-responsive elementmodulator in epigenetic regulation of Cyp17a1, FEBS J. 279 (2012)1584–1593.

[152] S. Sahar, P. Sassone-Corsi, Metabolism and cancer: the circadian clock connection,Nat. Rev. Cancer 9 (2009) 886–896.

[153] T. Hirota, J.W. Lee, P.C. St John, M. Sawa, K. Iwaisako, T. Noguchi, P.Y.Pongsawakul, T. Sonntag, D.K. Welsh, D.A. Brenner, F.J. Doyle, P.G. Schultz, S.A.Kay, Identification of small molecule activators of cryptochrome, Science 337(2012) 1094–1097.

[154] J.L. Barclay, J. Husse, B. Bode, N. Naujokat, J. Meyer-Kovac, S.M. Schmid, H.Lehnert, H. Oster, Circadian desynchrony promotes metabolic disruption in amouse model of shiftwork, PLoS One 7 (2012) e37150.

[155] J. Husse, S.C. Hintze, G. Eichele, H. Lehnert, H. Oster, Circadian clock genes Per1and Per2 regulate the response of metabolism-associated transcripts to sleepdisruption, PLoS One 7 (2012) e52983.

[156] G. Lorbek, M. Lewinska, D. Rozman, Cytochrome P450s in the synthesis ofcholesterol and bile acids — from mouse models to human diseases, FEBS J.279 (9) (2011) 1516–1533.

[157] S. Horvat, J. McWhir, D. Rozman, Defects in cholesterol synthesis genes in mouseand in humans: lessons for drug development and safer treatments, DrugMetab. Rev. 43 (2011) 69–90.

[158] M.J. Prasai, R.S. Mughal, S.B. Wheatcroft, M.T. Kearney, P.J. Grant, E.M. Scott,Diurnal variation in vascular and metabolic function in diet-induced obesity:divergence of insulin resistance and loss of clock rhythm, Diabetes (2013), [Elec-tronic publication ahead of print].

[159] K. Takahashi, T. Yamada, S. Tsukita, K. Kaneko, Y. Shirai, Y. Munakata, Y. Ishigaki,J. Imai, K. Uno, Y. Hasegawa, S. Sawada, Y. Oka, H. Katagiri, Chronic mild stressalters circadian expressions of molecular clock genes in the liver, Am. J. Physiol.Endocrinol. Metab. 304 (2013) E301–E309.

[160] Y.Q. Xu, D. Zhang, T. Jin, D.J. Cai, Q. Wu, Y. Lu, J. Liu, C.D. Klaassen, Diurnalvariation of hepatic antioxidant gene expression in mice, PLoS One 7 (2012)e44237.

[161] O. Froy, The circadian clock and metabolism, Clin. Sci. (Lond.) 120 (2011) 65–72.[162] M. Dochi, Y. Suwazono, K. Sakata, Y. Okubo, M. Oishi, K. Tanaka, E. Kobayashi, K.

Nogawa, Shift work is a risk factor for increased total cholesterol level: a 14-year

95A. Naik et al. / Genomics 102 (2013) 84–95

prospective cohort study in 6886 male workers, Occup. Environ. Med. 66 (2009)592–597.

[163] P.C. Konturek, T. Brzozowski, S.J. Konturek, Stress and the gut: pathophysiology,clinical consequences, diagnostic approach and treatment options, J. Physiol.Pharmacol. 62 (2011) 591–599.

[164] P. Chen, X. Kakan, J. Zhang, Altered circadian rhythm of the clock genes in fibroticlivers induced by carbon tetrachloride, FEBS Lett. 584 (2010) 1597–1601.

[165] E. Filipski, P. Subramanian, J. Carrière, C. Guettier, H. Barbason, F. Lévi, Circadiandisruption accelerates liver carcinogenesis in mice, Mutat. Res. 680 (2009)95–105.

[166] G.A. Hawkins, D.A. Meyers, E.R. Bleecker, A.I. Pack, Identification of coding poly-morphisms in human circadian rhythm genes PER1, PER2, PER3, CLOCK, ARNTL,CRY1, CRY2 and TIMELESS in a multi-ethnic screening panel, DNA Seq. 19(2008) 44–49.

[167] B. Zhao, J. Lu, J. Yin, H. Liu, X. Guo, Y. Yang, N. Ge, Y. Zhu, H. Zhang, J. Xing, A func-tional polymorphism in PER3 gene is associated with prognosis in hepatocellularcarcinoma, Liver Int. 32 (2012) 1451–1459.

[168] S. Sookoian, G. Castaño, C. Gemma, T.F. Gianotti, C.J. Pirola, Common genetic var-iations in CLOCK transcription factor are associated with nonalcoholic fatty liverdisease, World J. Gastroenterol. 13 (2007) 4242–4248.