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Liver and Metabolic Syndrome

Dig Dis 2010;28:255–260 DOI: 10.1159/000282098

Genetic and Environmental Susceptibility to Non-Alcoholic Fatty Liver Disease

Christopher Paul Day 

Institute of Cellular Medicine, Newcastle University, Newcastle , UK

adults has NAFLD. The looming pandemic of obesity and type 2 diabetes coupled with the realisation that NAFLD is common in the paediatric population suggests that this prevalence is likely to increase further. Closely associated with the metabolic syndrome, the pathophysiology of NAFLD is centrally related to insulin resistance. Similar to diabetes and obesity, this liver component of the meta-bolic syndrome is thought to have a multi-factorial aetiol-ogy and, in common with its precursors, recent advances in DNA analysis technology have led to a growing inter-est in elucidating the genetic factors underlying the in-heritance of NAFLD.

The NAFLD spectrum ranges from simple steatosis (fatty liver), to non-alcoholic steatohepatitis (NASH) – characterised by liver cell injury, inflammation and fi-brosis – to cirrhosis, liver failure and hepatocellular car-cinoma. Since these histological features are identical to those seen in alcoholic liver disease, the diagnosis of NAFLD requires the exclusion of excessive alcohol in-take, typically less than 20 g per day for women and 30 g per day for men. As with alcoholic liver disease, it is clear that a large proportion of patients with risk factors for NAFLD develop hepatic steatosis. However, only a mi-nority develop more advanced disease (NASH, cirrhosis and hepatocellular carcinoma). Post-mortem and liver bi-opsy studies in obese cohorts undergoing bariatric sur-gery have demonstrated that only around 10–20% of even morbidly obese patients develop more than simple steato-

Key Words Non-alcoholic fatty liver disease � Non-alcoholic steatohepatitis � Free fatty acid

Abstract While the majority of those with non-alcoholic fatty liver dis-ease (NAFLD) will have simple hepatic steatosis, a minority will develop progressive steatohepatitis. Family studies and inter-ethnic variations in susceptibility suggest that genetic factors may be important in determining disease risk. Al-though no genetic associations with advanced NAFLD have been replicated in large studies, preliminary data suggest that polymorphisms in genes controlling lipid metabolism, pro-inflammatory cytokines, fibrotic mediators and oxida-tive stress may be associated with steatohepatitis and/or fi-brosis. Recent whole genome-wide scans have identified genes contributing to inherited susceptibility to steatosis and it seems likely that similar approaches will identify genes associated with disease progression in the near future.

Copyright © 2010 S. Karger AG, Basel

Introduction

Non-alcoholic fatty liver disease (NAFLD) is recog-nised as one of the commonest liver disorders seen by hepatologists [1] . It is estimated that one in three Western

Christopher Paul Day, MD, PhD, FRCP Faculty Office, The Medical School Framlington Place Newcastle upon Tyne NE2 4HH (UK) Tel. +44 191 222 7003, Fax +44 191 222 0723, E-Mail c.p.day   @   ncl.ac.uk

© 2010 S. Karger AG, Basel0257–2753/10/0281–0255$26.00/0

Accessible online at:www.karger.com/ddi

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sis [2, 3] . It is not clear what factors determine which pa-tients with obesity and insulin resistance develop ad-vanced NAFLD. Clearly environmental and genetic fac-tors are both likely to play a role.

Pathogenesis

Although new high throughput technology has made it possible for large quantities of genetic material to be analysed ‘hypothesis-free’ (through genome-wide scans), most of the available data on the genetic basis of NAFLD have been generated using classical hypothesis-based candidate gene allelic association studies. These hypoth-eses have been generated using the steps in pathogenesis. With an increasing number of studies on the pathogen-esis of NAFLD, the mechanisms of liver cell injury and fibrosis are becoming clearer with most attention cur-rently focused on free fatty acid (FFA)-induced cytokine, oxidative and endoplasmic reticulum stress-mediated in-jury, and non-inflammatory mediators of fibrosis [4–6] . Pro-inflammatory cytokines, including TNF- � , are pro-duced directly by hepatocytes in response to an increased supply of FFA and/or by adipose tissue macrophages that increase during obesity. Cytokines may also be produced by Kupffer cells in response to hepatocyte-derived cyto-kines or to gut-derived endotoxin. Oxidative stress in NAFLD arises principally via the increased oxidation of FFA by mitochondria, peroxisomes and microsomes. Low levels of adiponectin, an important anti-inflamma-tory anti-fibrotic cytokine produced by adipocytes, may also contribute to inflammation in NAFLD [7] . Fibrosis is thought to arise as part of the normal healing response to inflammation and injury, although evidence has sug-gested that factors related to obesity and insulin resis-tance, per se, may be directly fibrogenic. These include insulin and glucose (via stimulation of the release of con-nective tissue growth factor from hepatic stellate cells) and other adipokines synthesised and released by adipose tissue, including angiotensinogen, norepinephrine and leptin. Given these mechanisms, genetic and environ-mental risk factors for NAFLD seem likely to include fac-tors that influence hepatic FFA supply, the magnitude of oxidative stress, the release and effect of cytokines, and/or the severity of fibrosis.

Environmental Factors Determining Disease Risk Alcohol intake, diet, exercise, small intestinal bacte-

rial overgrowth and obstructive sleep apnoea syndrome may play a role as environmental factors in NAFLD. Sev-

eral studies have suggested that moderate alcohol intake may protect against progressive NASH similar to the ben-efit in cardiovascular disease, presumably through its known insulin-sensitising effect [8] . A study has showna higher intake of saturated fat and a lower intake of the antioxidant vitamins C and E in obese patients with NASH compared to obese controls with no evidence of liver disease [9] . The effect of antioxidants is compatible with the putative role of oxidative stress in the pathogen-esis of NASH. Evidence supporting a role for bacterial overgrowth in the pathogenesis of ‘primary’, rather than post-intestinal bypass surgery, NASH comes largely from a study that reported a higher prevalence of small intes-tinal bacterial overgrowth in patients with NASH com-pared to healthy controls [10] . A recent study has demon-strated that NAFLD is associated with increased gut per-meability and small intestinal bacterial overgrowth [11] .

At least two studies have suggested that the severity of obesity-associated sleep apnoea syndrome influences the histological severity of NAFLD, suggesting a role forhypoxia as a ‘second hit’ [12] . Interestingly, a recent study has shown that children who are breast-fed are less likely to get NASH and develop liver fibrosis; furthermore, the risk reduces with every additional month of breast-feed-ing [13] . Although the exact method of protection is not known, breast-feeding has been shown to be beneficial in several illnesses and there is a possibility that metabolic conditioning or factors secreted in breast milk are useful in priming the individual for metabolic challenges par-ticularly in diet.

Genetic Factors in NAFLD As with all so-called ‘complex’ diseases, NAFLD seems

likely to be the result of an interaction between behav-iour, environment and several different genetic factors. Elucidation of genetic factors that predispose an individ-ual to NAFLD would therefore allow preventive strate-gies to be targeted early in those at higher risk. In addi-tion, determining genetic factors associated with disease risk should lead to the development of non-invasive bio-markers of early disease and the identification of treat-ment targets. A role for genetic factors in NAFLD is sug-gested mainly by family and inter-ethnic variation stud-ies. A recent familial aggregation study compared family members of overweight children with NAFLD to family members of children with a similar degree of obesity but without NAFLD [14] . Fatty liver was significantly more common in the siblings and parents of the pro-bands with NAFLD. Furthermore, liver fat faction was corre-lated with the body mass index more closely in the fami-

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Susceptibility to Non-Alcoholic Fatty Liver Disease

Dig Dis 2010;28:255–260 257

lies of the children with NAFLD compared to those with-out. Another study in monozygotic and dizygotic twins showed that serum ALT and fasting serum insulin intra-pair correlations were significantly higher in the mono-zygotic compared to the dizygotic twins [15] . Struben et al. [16] reported the co-existence of NASH and/or cryp-togenic cirrhosis in 7 out of 8 kindreds studied, while Willner et al. [17] found that 18% of 90 patients with NASH had an affected first-degree relative. Clearly, this clustering could simply be a reflection of the well-estab-lished heritability of the risk factors for NAFLD – obesity and insulin resistance. However, studies examining eth-nic differences in the prevalence of NAFLD and NAFLD-related ‘cryptogenic’ cirrhosis strongly suggest that sus-ceptibility to NAFLD, rather than to its risk factors, may have a genetic component [18, 19] .

Genes Influencing Hepatic FFA and Triglyceride Levels

Recognition of the role played by hepatic FFA in the pathogenesis of progressive liver disease implies that ge-netic factors regulating intra-hepatic FFA levels will in-fluence the risk of advanced NAFLD. Genetic factors de-termining the degree of obesity and insulin resistance would fall into this category in light of their effect on he-patic FFA supply. Recent evidence that the esterification of FFA to triacylglycerol (TAG) is a critical protective mechanism versus FFA-induced lipotoxicity [20] sug-gests that polymorphisms in genes encoding proteins in-volved in the synthesis, storage and export of TAG may play a role in NAFLD susceptibility.

The first genome-wide association study in NAFLD has recently demonstrated that an allele in patatin-like phospholipase 3 ( PNPLA3; rs738409: C 1 G) was strongly associated with increased hepatic fat levels (p = 5.9 ! 10 –10 ) and with hepatic inflammation as indicated by se-rum ALT levels (p = 3.7 ! 10 –4 ). The allele was most common in Hispanics, which is the group most suscep-tible to NAFLD. Hepatic fat content was more than two-fold higher in PNPLA3 rs738409 homozygotes than in non-carriers [21] . This finding has been replicated in sev-eral other studies [22] and also shown to be associated with histological liver inflammation [23] and alcoholic liver disease [24] . The precise function of adiponutrin, the protein encoded by PNPLA3, is unknown; however, it appears to have lipogenic transacetylase activity. These data support a central role for lipid processing in the pathogenesis of steatosis and progressive liver disease.

Microsomal triglyceride transfer protein (MTP) is critical for the synthesis and secretion of very-low-densi-ty lipoprotein in the liver and intestine and a frameshift mutation in the gene is associated with abetalipoprotein-aemia. A G 1 T single nucleotide polymorphism (SNP) at position 493 in the 5 � promoter region has been associ-ated with lower levels of transcription resulting in lower MTP levels and failure to excrete TAG from the liver. Ev-idence has been presented that patients with NAFLD ho-mozygous for the G allele have increased steatosis and histological NASH grade compared to heterozygous pa-tients or patients homozygous for the high activity allele [25, 26] . Unfortunately, the study that included histologi-cal classification of NAFLD included only 63 patients [25] and, therefore, its conclusions should be interpreted with caution. Two small Japanese studies have reported an as-sociation between NAFLD and a loss-of-function SNP in the gene encoding phosphatidylethanolamine methyl-transferase (PEMT), which is involved in phosphatidyl-choline synthesis required for very-low-density lipopro-tein synthesis. The largest of these studies included 107 patients with biopsy-proven NASH, and demonstrated that the phosphatidylethanolamine methyltransferase V175M allele was more common in NASH patients com-pared to controls, and that those with the V175M allele among the NASH patients had the lowest BMI, suggest-ing they were more genetically predisposed to developing NASH [27] . Clearly, larger studies of these and other SNPs in genes encoding proteins involved in regulating intra-hepatic FFA flux and TAG synthesis, storage and export are required.

Genes Influencing Oxidative Stress

Genes that may influence the magnitude and effect of oxidative stress in NAFLD include the HFE gene and the gene encoding superoxide dismutase (SOD)-2. With re-spect to HFE , an initial study from Australia showed that 31% of 51 patients with NASH possessed at least one copy of the C282Y HFE mutation compared to only 13% of con-trols [28] , and a study of 126 patients with histologically diagnosed NASH reported that the C282Y mutation was associated with bridging fibrosis or cirrhosis [29] . How-ever, an Italian study of 263 consecutive patients with NAFLD has reported a prevalence of the C282Y and H63D mutations identical to the locally matched popu-lation (blood donors) [30] . Furthermore, amongst the NAFLD patients, liver iron content was no different in patients with and without the mutations, and the severity

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of fibrosis was unrelated either to liver iron content or to HFE genotype.

With respect to SOD-2, a small Japanese study has re-ported that the valine-encoding SNP genotype is associ-ated with advanced histology, and this has been replicat-ed in a preliminary report from the UK [31] . A further study from the latter group using both case-control and intra-familial association methodology reported a con-sistent association between a functional SNP in the mito-chondrial targeting sequence of SOD-2 and fibrosis in NAFLD. The case control study showed the presence of significant fibrosis (stage 1 1) increased with the number of Val (T) alleles (Ala/Ala 23%, Ala/Val 37%, Val/Val 45%; p = 0.034). There was no association with hepato-cyte ballooning. In the family study, 55 families were ‘in-formative’ in that one or both parents were heterozygous for the SNP. In these families the Val allele was transmit-ted on 47 of 76 possible occasions (62%), whereas the C allele was transmitted on only 29 of 76 occasions (38%;p = 0.038) [32] .

Genes Influencing the Response to Endotoxin

Evidence supporting a role for endotoxin-mediated cytokine release in the pathogenesis of NAFLD, together with the identification of promoter polymorphisms in genes encoding endotoxin receptors, has recently sug-gested an alternative set of ‘candidates’ to explain genetic susceptibility to advanced fatty liver diseases. CD14, a li-popolysaccharide receptor on monocytes, macrophages and neutrophils, has no intracellular domain, but en-hances signalling through toll-like receptor-4 (TLR4), another lipopolysaccharide receptor. A C 1 T polymor-phism is present at position –159 in the CD14 promoter, with the TT genotype associated with increased levels of soluble and membrane CD14 [33] . A preliminary study in NASH has reported an association with the CD14 poly-morphism, but not with the TLR4 polymorphism or SNPs in the gene encoding the NOD2 cytosolic receptor for the peptidoglycan components of bacterial cell walls [34] .

Genes Influencing the Release or Effect of Cytokines

Excess TNF- � production is a typical feature in alco-holic liver disease. The first association between a cyto-kine gene polymorphism and alcoholic liver disease was reported between alcoholic hepatitis and a polymor-

phism at position –238 in the TNF- � promoter region, and this polymorphism has subsequently been associated with NASH [35] . The functional significance of the poly-morphism is, however, unclear and the associations may well be either spurious or due to linkage disequilibrium with another true ‘disease-associated polymorphism’ on chromosome 6. In a Japanese study, 102 patients were an-alysed for several TNF- � SNPs and the serum level of soluble TNF receptor-2. This study showed that the level of sTNFr-2 was significantly higher in NASH patients when compared to those with simple steatosis or controls. The carrier frequencies of polymorphisms at –1031C and –863A in the promoter region were significantly higher in NASH than those with simple steatosis. However, there was no significant difference between those with NAFLD and the control population [36] . This small study clearly requires replication before any conclusions on the role of TNF- � as a NASH susceptibility gene can be drawn.

Genes Influencing the Severity of Fibrosis

Genes encoding proteins involved in fibrogenesis or fibrinolysis in the liver are clearly candidates for a role in NAFLD-related fibrosis [37] . Obvious candidates would include the polymorphic genes encoding transforming growth factor (TGF)- � 1, connective tissue growth factor, matrix metalloproteinase 3, PPAR- � , DDX5, CPT1A and various fibrogenic adipocytokines including angiotensin II. One relevant study in this regard is a report that obese patients possessing both the high TGF- � 1 and angioten-sinogen producing SNPs may be more susceptible to ad-vanced fibrosis. However interpretation of this study is limited by very small numbers [38] . A recent study has shown that several polymorphisms in the angiotensin II receptor 1 gene may be associated with NASH and he-patic fibrosis, in keeping with a few studies showing ben-efit of the angiotensin receptor blockers in the treatment of NAFLD. Five SNPs have been detected, one (rs 3772622 in the AngIIr1 gene) in particular has been significantly associated with the hepatic fibrosis index [39] . A recent study also showed that a loss-of-function polymorphism in the Kruppel-like factor (KLF) 6 gene is associated with advanced NAFLD [40] . In this study three European pop-ulations of patients with NAFLD were used to show that KLF6-IVS1-27G 1 A polymorphism resulted in less fibro-sis compared to patients homozygous for the wild-type allele. This may reflect the role of KLF6 in hepatic stellate cell activation in response to oxidative stress and TGF- � 1.

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Conclusion

Genetic and environmental risk factors determine why only a minority of obese, insulin resistant individu-als progress from simple steatosis to inflammation and fibrosis. Single studies have suggested that dietary satu-rated fat and antioxidant intake and small bowel bacte-rial overgrowth may play a role in disease progression. Family studies and inter-ethnic variations in susceptibil-ity suggest that genetic factors are important in deter-mining the risk of NAFLD. Recent genome-wide scans have demonstrated a role for a polymorphism in the PNPLA3 gene encoding adiputrin in steatosis severity and a possible role in NASH; however, no genetic asso-ciations with advanced NAFLD have been replicated in large studies in any populations. Preliminary data sug-gest that polymorphisms in genes encoding MTP, SOD-2, the CD14 endotoxin receptor, TNF- � , TGF- � and angio-tensinogen may play a role in disease progression. It is clear that future studies examining susceptibility to NAFLD need to be considerably larger than those per-

formed thus far if we are to come up with environmental and genetic associations that are robust enough to guide targeted treatment and prevention strategies. These stud-ies are critically dependent on the collection of large numbers of well-phenotyped cases and controls, which almost certainly requires national and multi-national collaborations. With respect to genetic studies, in the fu-ture the choice of candidate genes is likely to be further extended by: (a) genome and proteome expression studies in tissue from patients with various stages of disease, (b) QTL mapping in animal models and (c) mouse mutagen-esis studies.

Disclosure Statement

The author declares that no financial or other conflict of inter-est exists in relation to the content of the article.

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