Research ArticleThe SOD2 C47T polymorphism influences NAFLD fibrosis severity: Evidence from case-control and intra-familial allele association studies
Introduction
Non-alcoholic fatty liver disease (NAFLD), widely considered to be the hepatic manifestation of the metabolic syndrome, encompasses a spectrum of liver disease from simple steatosis (fatty liver) through non-alcoholic steatohepatitis (NASH) to fibrosis and ultimately cirrhosis in the absence of alcohol abuse [1]. The prevalence of NAFLD is rapidly increasing and it is now the most common cause of chronic liver disease in Western countries [2]. Despite its high prevalence, only a minority of patients with steatosis progress to develop inflammation and less than a quarter of subjects with NAFLD ever progress beyond steatosis to significant fibrosis and cirrhosis [3], [4].
NAFLD is best considered a complex disease trait where subtle inter-patient variations including host genetic factors and environment interact to produce disease phenotype and determine disease progression [5], [6], [7]. Basic science is helping to elucidate the mechanisms perpetuating liver cell injury and fibrosis in NAFLD through in vitro and in vivo studies. Attention is currently focused on the role of increased free fatty acid (FFA) flux on a background of insulin resistance as key drivers of pathogenesis through hepatocellular oxidative stress secondary to reactive oxygen species (ROS) production during β- and ω-FFA oxidation, direct lipotoxicity, cytokine release and endoplasmic reticulum stress. Consequent cellular damage triggers a mixture of immune mediated hepatocellular injury and both necrotic and apoptotic cell death pathways [8], [9], [10], [11]. Persistence of these processes culminates in hepatic fibrosis [12].
Whilst the reasons for the apparent variation in individual susceptibility to progressive disease are incompletely understood, family/ethnic studies suggest that genetic factors play a significant role [13], [14]. Accordingly, genes encoding proteins influencing the magnitude of these different forms of cellular stress are obvious candidates as genetic factors contributing to susceptibility to progressive NAFLD [5]. Single nucleotide polymorphisms (SNPs) in several candidate genes involved in inflammation, oxidative stress and fibrogenesis have been associated with the severity of liver damage in NAFLD (reviewed [5]). As yet, only the association of PNPLA3 with advanced NASH has been consistently replicated in large studies [5].
The mitochondrial enzyme manganese-dependent superoxide dismutase (MnSOD), encoded by the nuclear SOD2 gene, plays an important role in protecting cells from superoxide radicals [15]. SOD2 is subject to a common polymorphism (C47T, rs4880) which results in an amino acid substitution in the signal sequence targeting the enzyme to the mitochondrion (Ala16Val) and may induce a conformational change in the protein tertiary structure [16]. The presence of alanine at position −9 in this sequence (C47 allele) has been demonstrated to be associated with more efficient protein import than valine (T47 allele) by in vitro expression studies and is predicted to result in higher enzyme activity [17], [18]. This SOD2 polymorphism (rs4880) has been investigated as a possible susceptibility factor in NASH and several other diseases where oxidative stress is considered to play a role in pathogenesis including hereditary hemochromatosis [19] and drug induced liver injury [20]. A small study in 63 Japanese patients found a significantly increased prevalence of the lower activity homozygous T genotype among cases [21]. More recently, the homozygous T genotype was reported to be associated with an increased incidence of diabetic nephropathy in large studies of type 1 diabetics from Sweden [22] and Denmark [23] and with susceptibility to pancreatic cancer in a US-based study [24]. SOD2 has also been investigated in relation to susceptibility to alcoholic liver disease but the results have been inconsistent and inconclusive [25], [26].
To further examine this biologically plausible association between SOD2 genotype and susceptibility to fibrosing steatohepatitis, we have used two complementary approaches. Firstly, we have carried out a family study analyzing trios consisting of children with fibrotic NAFLD and their two parents in which we have performed transmission disequilibrium test (TDT) analysis to determine whether there is preferential transmission of a particular parental allele to the affected children [27]. Secondly, we performed a classical case-control allelic association study in unrelated patients with NAFLD of varying severity including five times more patients than that reported previously [21].
Section snippets
Patients
A large patient cohort was recruited across centers in UK and Italy. The study had all necessary ethical approvals in both countries and all participants (or their parents) gave informed consent.
The Italian family study, collected DNA from 71 Italian family ‘trios’ in Rome (210 individuals) each comprising two living parents and an index child with biopsy-proven fibrotic NAFLD. Other causes of liver disease were excluded, including increased alcohol intake, chronic viral hepatitis, autoimmune
The SOD2 C47T allele is preferentially transmitted to children with fibrosing steatohepatitis
In the Italian family study, 61 out of the 71 children had fibrosing steatohepatitis (59 patients exhibited stage 1 fibrosis and 2 patients had stage 2 disease). Transmission disequilibrium testing (TDT) [27] was used to seek preferential transmission of either SOD2 allele to affected children in the family study. For SOD2 rs4880, 55 out of the 71 families were informative in that one or both parents were heterozygous for this SNP. In these families, the T allele was transmitted on 47/76 (62%)
Discussion
Using both case-control and intra-familial association methodologies, we have shown a consistent association between a functional SNP in the mitochondrial targeting sequence of SOD2 and fibrosis severity in NAFLD. These results provide persuasive genetic evidence that mitochondria-derived oxidative stress is important in the pathogenesis of advanced NAFLD. In particular, use of TDT analysis in a young NAFLD population is a novel feature. In addition, we have also demonstrated a strong
Conflict of interest
The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
Acknowledgements
Q.M.A. is the recipient of a Clinical Senior Lectureship Award from the Higher Education Funding Council for England (HEFCE). Q.M.A., A.K.D. and C.P.D. are recipients of an MRC Programme Grant to identify and study novel genetic modifiers of liver disease and are members of the FLIP research consortium funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement Health-F2-2009-241762.
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