INTRODUCTION
Nonalcoholic fatty liver disease (NAFLD) is one of the most common forms of chronic liver diseases and a cause of elevated serum aminotransferases worldwide. The prevalence of NAFLD in the general population of Western countries ranges from 20% to 30%[1-3]. Due to the alterations of diet structure and life style, the prevalence of NAFLD in developing countries has been increasing rapidly[4]. Recent studies, including one from our group indicate that the prevalence of NAFLD in Chinese population is about 15%[5-7]. The term NAFLD encompasses a morphological spectrum of diseases, ranging from simple fatty liver (SFL) to nonalcoholic steatohepatitis (NASH) and hepatic cirrhosis, which may progress to hepatocellular carcinoma (HCC). SFL generally has a benign prognosis. Only a minority of them develop NASH, which is characterized by inflammation, fibrosis and liver cell injury[8,9].
NAFLD has been shown to be associated with metabolic syndrome (MetS), which comprises obesity, type 2 diabetes, dyslipidemia and high blood pressure with insulin resistance being the central mechanism. NAFLD is presently considered the hepatic manifestation of MetS[5,6,8,10].
It is generally believed that environmental and genetic factors interact to produce NAFLD phenotype and determine its progression. However, the detailed pathogenesis that determines which individual develops NAFLD remains unclear. Recently, the emerging field of epigenetics shed lights on the pathogenesis of chronic liver disease including NAFLD[11,12]. Elucidation of genetic and epigenetic factors that predispose an individual to NAFLD may lead to development of noninvasive biomarkers for early diagnosis of NAFLD and may allow early preventive and therapeutic strategies for the people at the high risk. This review summarizes recent contributions to the field of the genetic and epigenetic variations that influence the development of NAFLD.
GENETIC VARIATIONS
Candidate gene studies
The genetic variations may result in conformational changes in the protein structures and functions of the genes. NAFLD is an exceedingly complex genetic disorder. Before 2008, the candidate genes based on the prior knowledge of MetS and NAFLD pathophysiology were selected for investigation[11,13]. In comparison with NAFLD, the relationships between the genotypes and phenotypes of MetS have been examined more extensively. A large number of single nucleotide polymorphisms (SNPs) at the genes encoding proteins involved in insulin resistance has been revealed to be associated with the development of MetS[14,15]. As there is substantial overlap in the pathogenesis of NAFLD and MetS, theoretically, many variations in candidate genes related to MetS may contribute to the pathogenesis of NAFLD: first, genes related to insulin resistance, such as adiponectin, resistin, insulin receptor, and peroxisome proliferatoractivated receptors-γ (PPAR-γ); second, genes influencing hepatic free fatty acid metabolism, such as hepatic lipase, leptin (or leptin receptor), adiponectin, microsomal triglyceride transfer protein, phosphatidylethanolamine N-methyltransferase (PEMT), PPAR-γ, cytochrome P 450, 2E1 and 4A; third, cytokine-related genes, such as tumor necrosis factor-α (TNF-α) and interleukin-10; fourth, genes affecting liver fibrogenic pathways, such as leptin, adiponectin, transforming growth factor beta1, connective tissue growth factor and angiotensinogen; and finally, genes encoding endotoxin receptors and oxidative stress responses, such as CD14, superoxide dismutase-2 and toll-like receptor-4. Among these genes, PPAR-γ, adiponectin, leptin and TNF-α were frequently reported in the field of MetS as well as NAFLD[11,13,16]. It is noted that one gene may have a number of SNPs at several nucleotide loci. For example, the SNPs at the PPAR-γ gene involved in MetS may occur at the loci of C-681G, C-689T, Pro12Ala, G67222A, A69208G, G81556T, T95872C, T115432G, C127599T and C161T, but only a few of them have been investigated extensively[17,18].
There is evidence supporting the theory that these genetic factors account for considerable variability in susceptibility to NAFLD. The SNPs may increase or decrease the function of the target genes and their encoding proteins. We have previously demonstrated that many candidate genes’ SNPs mentioned above are associated with susceptibility to NAFLD. Some showed positive relationships (increased risk), i.e., TNF-α-238, adiponectin-45, leptin-2548, PPAR-γ-161 and PEMT-175. Other SNPs demonstrated a negative association (decreased risk), i.e., adiponectin-276 and hepatic lipase-514. Two were not relevant, i.e., TNF-α-380 and PPAR-γ coactivator-1a-482[19]. Gene variations might affect the pathogenesis of NAFLD via blood cytokines (such as leptin and adiponectin) and insulin resistance pathways[19,20]. Although many pathobiological candidacies of SNPs were reported, most studies in literature have not been well validated by larger replication cohorts. The findings in candidate gene studies might be influenced by specific ethnic groups or environmental conditions.
Genome-wide association studies
Since the introduction of genome-wide association studies (GWASs) to investigate genomic variations, there have been significant advances in our understanding of human genome and its clinical sequelae over a range of diseases. More than 3.1 million SNPs have been identified so far. The International HapMap Project (http://hapmap.ncbi.nlm.nih.gov) has characterized patterns of SNPs across individuals from diverse ethnic backgrounds[16,21,22]. Although a number of GWASs has been published in the field of MetS (type 2 diabetes and insulin resistance)[23], and other liver diseases (HCC, hepatitis B, hepatitis C, drug-induced liver injury and primary biliary cirrhosis)[16,24,25], only a few studies were carried out on NAFLD.
In 2008, the first GWAS on NAFLD was reported by Romeo et al[26]. In this population-based study, noninvasive proton magnetic resonance spectroscopy (1H-MRS) was applied to assess hepatic steatosis. Totally, 2111 individuals comprising a mixed population of Hispanic, African American, and European American were enrolled. Nonsynonymous sequence variations of 9229 SNPs were identified in NAFLD group compared with normal controls. An allele of patatin like phospholipase domain containing family member A3 (PNPLA3, SNP rs738409, encoding I148M), also termed adiponutrin, on chromosome 22 was shown to be strongly associated with increased hepatic fat levels and hepatic inflammation. This allele was most common in Hispanics, the group most susceptible to NAFLD, with hepatic fat content being more than twofold higher in G homozygous subjects than in non-carriers. G allele frequency was lower in people of European descents and lowest in African Americans, the group found to have the lowest level of hepatic triglyceride accumulation. These findings were validated by another GWAS. Totally 1117 individuals with histologically confirmed NAFLD were genotyped for six SNPs relevant to hepatic fat levels and liver enzymes. PNPLA3 was significantly associated with steatosis, portal inflammation, lobular inflammation, Mallory-Denk bodies, NAFLD activity score and fibrosis[27]. Subsequently, the extension of the hepatic phenotype associated with the PNPLA3 genotype was independently replicated in both adult and pediatric subjects with simple steatosis, NASH and NASH-related fibrosis using different laboratory techniques[28-33]. There was evidence that carriers of PNPLA3 exhibited more severe steatohepatitis and higher levels of fibrosis. PNPLA3 was consistent with the concept of NASH rather than the broader features of the MetS, such as body mass index, dyslipidemia, and type 2 diabetes mellitus. The influence of PNPLA3 on hepatic steatosis was not through insulin resistance pathway as assessed by hyperinsulinaemic, euglycaemic clamp and oral glucose tolerance testing[27-29,32,34,35]. Recently a meta-analysis enrolling 16 studies (2937 subjects) was performed to evaluate the association of PNPLA3 with NAFLD. The results showed that PNPLA3 exerted a strong influence not only on liver fat accumulation (the GG homozygous subjects had a 73% higher lipid fat content compared with CC ones), but also on higher susceptibility to liver disorders (GG homozygous subjects had 3.24-fold higher risk of higher necro-inflammatory scores and 3.2-fold higher risk of developing fibrosis compared with CC homozygous ones). The PNPLA3 GG genotype vs the CC genotype was associated with a 28% increase in alanine transaminase (ALT) level. NASH was more frequently observed in GG than in CC homozygous subjects (odds ratio 3.488, 95%CI: 1.859-6.545). Nevertheless, carrying GG alleles did not seem to increase the risk of severe histological features[36]. In a clinical study recruiting 302 subjects with 1H-MRS-confirmed NAFLD whose genotyping was determined with TaqMan polymerase chain reaction (PCR), a SNP (rs767870) at adiponectin receptors 2 (ADIPOR2), but not at ADIPOR1 and PPAR gene, was found to link to a higher liver fat content. In this study, PNPLA3 was not tested[37].
Although most studies supported the association between PNPLA3 and NAFLD, a few reports failed to validate this finding. In a GWAS enrolling 236 women with biopsy-confirmed NAFLD, no association for any feature of NAFLD with PNPLA3 was found. Another SNP (rs2645424) on chromosome 8 in the farnesyl diphosphate farnesyl transferase 1 gene, generating an enzyme with a role in cholesterol biosynthesis, was identified to relate to the severity of NAFLD histology including NAFLD activity score, liver fibrosis, lobular inflammation as well as increased ALT[38].
The results from GWASs shed light on the understanding of the genetics in NAFLD, as the loci identified are frequently novel and have not previously been implicated. However, such findings require further detailed studies both to determine the activity and to validate the causality, as neither biological functions nor pathogenic mechanisms of these genetic variations are known.
EPIGENETIC MODIFICATIONS
During the past decade, the role of epigenetic mechanisms in the pathogenesis of disease has been increasingly recognized. Epigenetic modification, mainly including microRNAs (miRNAs, miRs), DNA methylation, histone modification and ubiquitination, refers to phenotypic changes caused by the mechanism that is unrelated to changes in the underlying DNA sequence. As an adaptive mechanism to alteration of genetic and environmental signal patterns and epigenetic regulation, which allows fine-tuning gene expression, is essential for the proper maintenance of cellular homeostasis. Disruption of the balance will lead to the development of a wide range of disorders. So far, epigenetic research has mainly focused on cancer, cardiovascular disease, mental illness and autoimmune disease. The roles of epigenetics in the pathogenesis of NAFLD are largely unknown[39]. Among epigenetic modifications, miRs are studied most extensively in NAFLD. miRs are small naturally occurring single stranded RNA molecules regulating mRNA degradation or translation inhibition, subsequently altering protein expression of target genes. One miR can target multiple genes (multiplicity) and multiple miRs may target a single gene (cooperativity). Since the first discovery in 1993, many miRs in various organisms have been determined. To date, more than 1420 miRs have been identified in humans (miRBase v17). (http://www.mirbase.org/)[40,41]. The expression of miRs is both organ-specific and dependent on the stage of development. miRs influence at least one-third of all human transcripts and are known regulators of important cellular processes, e.g., cell metabolism, cell proliferation, apoptosis, immune function, tissue development and differentiation[42,43]. It has recently been shown that some 100 miRs are differentially expressed in human NASH. These miRs have diverse functions involved in the pathogenesis of NAFLD, including metabolisms of lipid and glucose, regulations of the unfolded protein response, endoplasmic reticulum stress, oxidative stress, cellular differentiation, inflammation, apoptosis and so on[44,45]. In a clinical study, the miR profiles of 15 patients with biopsy-proven NASH and 15 controls with normal liver histology were investigated. Out of a total of 474 tested miRs, 46 were differentially expressed in NASH with 23 being up-regulated (in particular, miR-34a and miR-146b), and 23 being down-regulated (in particular, miR-122). These differentially expressed miRs were further validated by quantitative real-time PCR[45].
The miR-122, a highly abundant miR in the liver, has caught most attention in liver diseases. Accounting for nearly 70% of all miRs in the liver, miR-122 is significantly under-expressed (63%) in NASH subjects compared to controls[45,46]. In addition to its role in lipid and cholesterol metabolism, miR-122 has been shown to promote adipocyte differentiation[42]. Subsequently, the roles of miR-122 in the pathogenesis of NAFLD were confirmed by a number of studies. Inhibition of miR-122 in a diet-induced obesity mouse model with an antisense oligonucleotide treatment resulted in decreased mRNA expression of acetyl-coenzyme-A carboxylase-2, fatty acid synthetase, sterol regulatory element binding proteins 1-c, 2, stearoyl-CoA desaturase and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG CoA) reductase, all of which were key lipogenic factors in human NASH. The histology also showed substantial improvement in liver steatosis[47]. The results were validated by another study in mice, in which the plasma cholesterol level, hepatic fatty-acid and cholesterol synthesis rate as well as HMG-CoA reductase level were significantly decreased after silencing miR-122[42]. All these findings strongly suggested the significance of miR-122 in the regulation of lipid metabolism and the contribution to the development of NAFLD. A further study suggested that miR-122 was closely linked to the output system of the circadian clock by regulating circadianly expressed genes[48]. Besides miR-122, some miRs have been demonstrated to be involved in NAFLD development. miR-34a and miR-146b were shown to be significantly over-expressed (99% and 80%, respectively) in human NASH[45]. The expression of miR-335 in the liver and white adipose tissue was up-regulated in mice. The increased miR-335 expression was associated with increased body, liver and white adipose tissue weight, as well as elevated hepatic triglyceride and cholesterol levels. Furthermore, hepatic miR-335 level was closely correlated with the expression of adipocyte differentiation markers, i.e., PPAR-α and FAS in adipocyte[49]. The presence of miR-181d significantly decreased lipid droplets in the liver (60%), and subsequently reduced cellular triglyceride and cholesterol[50]. miR-10b regulated steatosis level through PPAR-α pathway in a steatotic hepatocyte (L02 cell line) model. The post-transcriptional regulation of PPAR-α by miR-10b was maintained by a single binding site[51].
Aberrant methylation patterns of genomic DNA have been studied in many diseases. Hypermethylation of CpG islands is generally associated with gene silencing, and hypomethylation of global genomic DNA affects genomic stability. Hypermethylation of multiple genes in CpG islands has been demonstrated in human HCC, in which CpG island methylator phenotype was involved in the promoter hypermethylation of multiple genes[52]. However, the relation of DNA methylation to NAFLD development has not been well documented. A recent study enrolling 63 NAFLD patients confirmed by liver biopsies and 11 controls showed a tight interaction between the presence of NAFLD and hepatic DNA methylation of CpG in PPAR-γ coactivator 1α (PPARGC1A) and mitochondrial transcription factor A (TFAM) promoters. The proportion of DNA methylation in PPARGC1A and TFAM was significantly higher in the NAFLD livers than in the controls. However, the histological severity and activity scores of NAFLD were not correlated to methylation level and methylated DNA/unmethylated DNA ratio either in PPARGC1A or TFAM promoter[53]. The development of hepatic steatosis in a mouse model was accompanied by prominent epigenetic abnormalities, which comprised pronounced loss of genomic and repetitive sequences cytosine methylation, increased level of repeat-associated transcripts, aberrant histone modifications and alterations in expression of the maintenance DNA methyltransferase 1 (DNMT1) and de novo DNMT3A proteins in the livers[54].
Ubiquitination and sumoylation (sumo: abbreviation of small ubiquitin-like protein) are recently demonstrated to be novel forms of post-translational modifications (PTMs). PTMs of transcription factors through the course of protein processing play important roles in controlling many biological events[55]. The research of ubiquitination related to NAFLD is just at the beginning. In a study investigating the hepatic gene networks in morbidly obese patients with NAFLD, hepatic fibrosis signaling was found to be the most significant pathway in the up-regulated NAFLD gene cluster, whereas the endoplasmic reticulum stress and protein ubiquitination pathways to be the most significant pathways in the down-regulated NAFLD gene cluster[56]. Besides ubiquitination, transcription factors can undergo several types of PTMs, including acetylation, phosphorylation, and glycosylation. Little is known about their role in NAFLD so far[55].
In conclusion, environmental and genetic factors interact to produce NAFLD phenotype and to determine its progression. This review summarizes the current knowledge of genetic and epigenetic determinations on NAFLD. Genetic variations (e.g., SNPs) account for only a small fraction of environmental and heritable disease risks, whereas epigenetic modifications (e.g., miRs, DNA methylation histone modifications and ubiquitination) affect a bigger proportion of disease phenotypes. The investigation into the potential roles of epigenetics in NAFLD is just at the beginning and needs to be refined. The accumulation of genetic and epigenetic knowledge related to NAFLD has provided novel insight into disease pathogenesis, and may help to develop new diagnostic biomarkers and therapeutic targets for NAFLD management.
