The role of mitochondrial genomics in patients with non-alcoholic steatohepatitis (NASH)

Non-alcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease in the United States and worldwide [1, 2]. The prevalence rate of NAFLD is reported between 10 and 35 % in the general population with the prevalence rates being substantially higher in the obese and diabetic individuals [3].

NAFLD is characterized by accumulation of fat, mainly triglycerides, in the absence of excessive alcohol consumption, viral infection, hereditary disorders, or use of steatogenic medications [4, 5]. In its progressive form, NAFLD manifests as non-alcoholic steatohepatitis (NASH) defined as steatosis with ballooning degeneration and/or presence of Mallory-Denk bodies. NASH can ultimately lead to fibrosis followed by cirrhosis and hepatocellular carcinoma (HCC) [6‐8].

Both visceral obesity and metabolic syndrome are commonly associated with NASH [9]. However, not all patients with NASH are obese [10]. The progression of steatosis to NASH has been associated with a number of other metabolic factors, including the quality of nutrition and patient’s lifestyle [11] as well as genetic predisposition [12]. The mechanisms of genetic predisposition to the development of NASH remain unclear, but certainly involve the variation in individual susceptibility to NAFLD [12]. A number of nuclear loci have already been identified as potential contributors to development of NASH [13].

In 2008, a genome-wide association study in a population comprising Hispanic, African American and European American subjects showed that a common genetic variant in the patatin-like phospholipase domain containing 3 protein (PNPLA3) gene is associated with liver fat accumulation [14]. The isoleucine to methionine substitution at position 148 in the patatin-like phospholipase domain containing 3 protein (PNPLA3; I148M variant, rs738409) has since then been shown to be associated with liver fat accumulation and severity of NAFLD [15‐18]. The interaction between PNPLA3 and BMI has been supported by several population based studies [19‐21]. The studies have shown that body mass index interacts with PNPLA3 and increases susceptibility to NAFLD. However, PNPLA3 function and the effect of the I148M substitution are not yet completely understood.

PNPLA3 protein plays anabolic and catabolic role in lipid metabolism. Purified PNPLA3 protein has been shown to have both hydrolase and lysophosphatidic acid transacetylase activities, with the hydrolase activity predominating [22‐24]. However, its in-vivo function and physiological relevance remain controversial. In-vitro studies have shown the 148 M mutation to be a loss-of-function mutation abolishing the hydrolase activity [22, 24] while 148 M mutation is a gain of function mutation for lysophosphatidic acid transacetylase (LPAAT) activity [23]. Consistent with a hydrolase activity hypothesis, a relative reduction in hepatic very low-density lipoprotein secretion with the 148 M mutation has been shown in vitro and in humans [25]. Contrary to the hydrolase activity, wild-type PNPLA3 was also found to have lysophosphatidic acid transacetylase activity and the 148 M mutation was found to be a gain of function of the lipogenic activity [26]. In agreement, rats on a high-fat diet treated with pnpla3 antisense oligonucleotides showed decreased fatty acid esterification into hepatic triglycerides and reduced insulin resistance [27]. In a recent study of obese adolescents with hepatic steatosis, the I148M variant has been shown to modulate the association between oxidized metabolites derived from linoleic acid and cytokeratin 18 fragment, a robust biomarker of liver injury [28]. Recently, a cell specific PNPLA3 function linking PNPLA3 to liver fibrosis has been shown [29, 30]. The authors show retinyl-palmitate lipase activity of PNPLA3 in human hepatic stellate cells with I148M being a loss of function mutation. Thus, the genotypic effect of PNPLA3 protein is complex.

In this study, our central hypothesis is that mitochondrial haplotypes along with a susceptible nuclear genetic background may be involved in predisposing to NAFLD and NASH. With this in mind, we examined mitochondrial haplotypes along with patatin-like phospholipase domain containing 3 (PNPLA3) rs738409 genotype and determined their association with NAFLD and NASH.

The clinical, demographic and biochemical characteristics of study subjects (N = 341; BMI =48 ± 9.13, Age = 44 ± 11.2, 41.9 % Non-NASH NAFLD, 30.4 % NASH, 27.5 % controls) are summarized in Table 1.

Patients with histologic NASH when compared to non-NASH NAFLD had higher levels of serum aminotransferases compared to patients with non-NASH NAFLD (p < 0.001) (Table 2). In contrast, fasting serum HDL and platelets were lower in NASH patients compared to those with non-NASH NAFLD (p < 0.001) (Table 2). Fasting serum glucose, serum aminotransferases, serum triglycerides and total cholesterol was higher, while serum HDL and platelets were lower (p < 0.001) (Table 2) in patients with Non-NASH NAFLD compared to controls with normal liver histology. Among the NASH and non-NASH NAFLD subjects, there was no significant difference in the mean BMI, age, serum triglycerides, serum cholesterol, serum LDL and fasting glucose levels (p > 0.05) (Table 2). Similarly, subjects with normal liver histology (controls) had no significant difference in the mean BMI, WBC, platelets, serum LDL and HDL levels when compared to Non-NASH NAFLD subjects (p > 0.05) (Table 2). Notably, patients with normal liver histology were more likely to be younger and had higher platelets counts when compared to those with NASH (p < 0.001) (Table 2).

Oxidative stress is an important player in development of progressive NAFLD or NASH. In fact, oxidative stress related cellular damage, reflected as hepatic necrosis or apoptosis, is a mitochondrial dependent factor that potentially influences the progression of NAFLD [38‐40]. Given that mitochondria are critical players in maintaining balance of energy input and expenditure [41] as well as the pathogenesis of NASH, it is enticing to explore mitochondrial genome variation as a potential genetic factor that could contribute to the pathogenesis of NASH.

Mitochondria have their own circular DNA that can be categorized into various haplogroups based on variants within its non-coding hypervariable region. Notably, the non-coding region is indispensable for replication and transcription of the mitochondrial genome. Thus variations in this region are expected to influence the ability of mitochondria to produce ATP and ROS. In turn, availability of ATP and the production of ROS are known to affect tissue responses to extrinsic or intrinsic stressors and energy demands. Consequently, mitochondrial DNA variations have been the focus of studies in patients with altered metabolism [42‐44]. Additionally, in a recent study, there is evidence that mitochondrial DNA haplogroups may influence expression of nuclear genes that are induced under oxidative stress [45].

NAFLD is a complex disease with genetic and environmental factors interacting to influence the development and progression of the disease. The relative importance of these factors may vary between populations depending on genetic background and lifestyle. This study is unique as we show for the first time the differential prevalence of mitochondrial haplogroups in obese patients (BMI > 35) with varying severity of NAFLD. We also examine the interaction between mitochondrial genome variation and nuclear PNPLA3 genotype in this patient cohort.

In this study, we present evidence that mitochondrial haplogroup L is less prevalent in subjects with NASH and may thus be protective against NASH (Table 3). Haplogroup L has the highest sequence divergence from other haplogroups [46]. A recent study demonstrated that L haplogroup is also functionally different [45]. When compared to the cybrids with H haplogroup, those with L haplogroup demonstrated lower levels of the ROS production but similar levels of ATP generation, indicative of more efficient oxidative phosphorylation with lower electron leakage and higher efficiency of aerobic respiration [45]. These observations support the hypothesis that the patients with L haplogroup may be intrinsically protected from oxidative stress and thus, potential liver damage by ROS.

It is also important to note that the mitochondrial haplogroups are commonly used as a proxy to ethnic origin of individual. According to the population-based studies, NAFLD and NASH are more prevalent in individuals of Hispanic origin [3, 47], while African Americans are somewhat protected against high prevalence of NAFLD and NASH [48]. The high prevalence of L haplogroup in subjects of African origin may serve as a physiological explanation for this phenomenon, although further studies will be needed to determine the causality. In our study cohort, 88 % (N = 44) of the self-reported African Americans had L haplotype and 66 % (N = 33) of them had normal liver biopsy, 22 % (N = 11) had steatosis and 12 % (N = 6) had NASH. Additional studies with a larger African American cohort will be needed to further validate this trend. Notably, the haplotype results of our study are consistent with the haplogroup proportions reported in a recent large population based study in United States [37].

In addition to mitochondrial haplotypes, we also measured the well-described gene PNPLA3 variant rs738409 associated with NAFLD. The rs738409 variant is located in coding region of PNPLA3 gene. This variant corresponds to Isoleucine to Methionine substitution (p.I148M) and has previously been identified as risk factor for steatohepatitis, NAFLD-cirrhosis [14, 49] and hepatocellular carcinoma [15, 20, 50‐52]. The 148 M variant has also been linked to elevated aminotransferase levels [53] and liver fibrosis [54]. However, the effect of the I148M substitution on the functioning of PNPLA3 is still enigmatic [15]. The 148 M variation is reported to result in loss of hydrolase activity (loss-of-function model) [22, 24, 55] and thus cause accumulation of triglyceride in the liver. However, studies in knock-out mice show contrary results [26, 56]. These studies show that loss of PNPLA3 hydrolase activity does not alter hepatic triglycerides. PNPLA3 variant has also been shown to have a gain of lysophosphatidic acid transacetylase (LPAAT) activity. The 148 M substitution increases LPAAT activity and results in increased TAG synthesis (gain-of-function model) [15, 23]. Thus, PNPLA3 148 M variant may increase hepatic triglyceride levels by multiple changes in triglyceride metabolism.

Our results with regards to PNPLA3 are in agreement with the previously reported studies. The alleles of PNPLA3 locus showed differential distribution in cohorts with NAFLD, NASH (Table 2) and pericellular fibrosis. Moreover, multivariate analysis showed that heterozygosity in this locus is independently associated with higher risk of having NASH as well as with pericellular fibrosis. Overall, carrying G allele in position rs738409 appears to increase the odds of developing NASH and pericellular fibrosis. Importantly, in the final multivariate model of NAFLD (Table 4), no interaction between the nuclear rs738409 variant and mitochondrial haplogroup was detected. This indicates that the somatic genotype and mitochondrial genotype can have independent and important impact in the pathogenesis of NASH.

In conclusion, our data shows that the presence of haplogroup L exhibits certain degree of protection against the development of NASH and pericellular fibrosis. This suggests that, besides nuclear genome variants and environmental factors, the mitochondrial genetics may play an important role in NAFLD probably by modulation of oxidative stress and the efficiency of oxidative phosphorylation. Further studies will be needed to confirm the causality. Moreover, United States cohorts are substantially admixed, resulting in ambiguity in the questionnaire-based ethnicity assessments [57]. Thus, ethnicity verifying mitochondrial haplogroup data should be coupled with the other biomarkers to serve as a valuable resource for future studies investigating NAFLD and NASH in such populations.