Polymorphisms of microsomal triglyceride transfer protein gene and manganese superoxide dismutase gene in non-alcoholic steatohepatitis

Abstract

Background/Aims

The pathogenesis of non-alcoholic steatohepatitis (NASH) is poorly understood. The aim of this study was to examine genetic influences on NASH pathogenesis.

Methods

Blood samples from 63 patients with biopsy-proven NASH and 150 healthy controls were analyzed by the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). Two functional polymorphisms were studied: the −493 G/T polymorphism in the promoter of microsomal triglyceride transfer protein (MTP) and the 1183 T/C polymorphism in the mitochondrial targeting sequence of manganese superoxide dismutase (MnSOD).

Results

NASH patients had a much higher incidence of the MTP gene G allele (P=0.001) and of the G/G genotype (P=0.002) compared to the controls. Fat occupied more area in liver lobules and the stage of NASH was advanced in patients with the G/G-genotype than in patients with G/T-genotype (P=0.04). NASH patients also had a higher incidence of the MnSOD T/T genotype (P=0.016).

Conclusions

The G allele in the MTP promoter leads to decreased MTP transcription, less export of triglyceride from hepatocytes, and greater intracellular triglyceride accumulation. The T allele in MnSOD mitochondrial targeting sequence leads to less transport of MnSOD to the mitochondria. Therefore, functional polymorphisms in MTP and MnSOD may be involved in determining susceptibility of NASH.

Introduction

Non-alcoholic steatohepatitis (NASH) is a frequent syndrome that has only recently become widely recognized as a source of liver injury. The prevalence of NASH is known to be rising worldwide, particularly in the United States, where NASH is now the most frequent liver disorder seen in primary care and in liver referral centers [1]. Because of its increasing importance, considerable efforts are being made to study NASH and elucidate the pathophysiologic mechanisms behind the disease. One model under discussion is the ‘two-hit’ hypothesis [2]. In this model, the normal liver becomes steatosis after prolonged central obesity. This ‘first hit’ then sensitizes the liver to further damage from several different mechanisms currently under study. Examples of these mechanisms include oxidative stress, insulin resistance, excessive tumor necrosis factor-α, antitoxin, and adenosine 5′-triphosphate (ATP) depletion. These ‘second hits’ then lead to the hepatitis seen in NASH and to subsequent fibrosis [3].

Genetic variation in lipid metabolism may produce differences in the speed and extent of hepatocyte lipid accumulation, the first hit. One enzyme that has attracted some interest is the microsomal triglyceride transfer protein (MTP). This protein transfers triglycerides to nascent apolipoprotein B, producing very low-density lipoprotein (VLDL) and removing lipid from the hepatocyte. Murine models have shown that the steatosis observed in hepatitis C infection results from inhibition of MTP by the hepatitis C viral core protein [4]. Patients with abetalipoproteinemia, an autosomal recessive disease caused by mutations in the MTP coding region, develop marked hepatic steatosis early in life [5], [6]. MTP mRNA is down regulated in human hepatocytes by insulin [7]. A common polymorphism in the MTP gene promoter, −493 G/T, has been described, with the G allele producing less gene transcription than the T allele [8], [9].

There is considerable evidence for the role of oxidative damage in NASH, one of the second hits. Lipid peroxidation likely damages plasma and intracellular membranes, leading to apoptosis and necrosis of hepatocytes. Lipid peroxidation end products (malondialdehyde and 4-hydroxy-2-nonenal (HNE)) may also trigger inflammatory and immune-mediated mechanisms of hepatocyte injury [3]. Animal models of non-alcoholic fatty liver disease [10] and biopsies of human patients with alcoholic liver disease [11] have shown that the degree of lipid peroxidation in the liver correlates with the extent of steatosis. It is suspected that free fatty acids may generate increased amounts of reactive oxygen species (ROS) by three different mechanisms. In a steatotic liver, free fatty acids are diverted to the mitochondria, where they are oxidized by beta-oxidation [3].

One enzyme that is important in detoxifying mitochondrial ROS is manganese superoxide dismutase (MnSOD). This enzyme is synthesized in the cytosol, then post-transcriptionally modified for transport to the mitochondria [12]. A limited number of polymorphisms have been described for MnSOD, including a T/C polymorphism in the mitochondrial targeting sequence. This polymorphism leads to a valine-to-alanine amino acid change in the mitochondrial targeting sequence [13]. In turn, this amino acid substitution may alter the helical structure of the mitochondrial targeting sequence, enhancing transport of MnSOD into the mitochondrial matrix [14].

We hypothesized that both MTP and MnSOD have an important role in determining an individual's susceptibility to NASH. The aim of our study was to examine the frequency of the MTP gene G/T polymorphism, and the MnSOD gene T/C polymorphism in Japanese NASH patients, and to compare these frequencies to those of healthy controls.