Non-alcoholic fatty-liver disease (NAFLD) is poised to become a predominant cause of chronic liver disease worldwide (Loomba and Sanyal, 2013). If obesity and diabetes mellitus (DM) stabilize in the future, it is predicted that there will be a modest growth in total NAFLD cases (0–30%) between 2016 and 2030, with the highest growth expected to be seen in China, due to the effects of urbanization (Estes et al., 2018). Chronic hepatitis B (CHB) is a disease caused by hepatitis B virus (HBV) infection and affects more than 257 million individuals worldwide. The rising prevalence of obesity and metabolic syndrome has resulted in an increase in the number of patients with concurrent NAFLD and CHB (NAFLD + CHB).

Both CHB and NAFLD are leading causes of liver-related morbidity and mortality. NAFLD comprises a spectrum of liver diseases that includes simple steatosis, non-alcoholic steatohepatitis, fibrosis, and cirrhosis, and ultimately develops into hepatocellular carcinoma (HCC) (Younossi et al., 2015). CHB contributes substantially to the global disease burden owing to its high prevalence and probability of progression to cirrhosis and HCC (Shen et al., 2012; Lok et al., 2017). Positive hepatitis B core antibody (anti-HBc) have been associated with cirrhosis and possibly HCC in Chinese patients with NAFLD (Chan et al., 2020). In a follow-up study, patients with NAFLD + CHB were found to have a 7.3-fold increased risk of HCC compared to patients with CHB alone (Chan et al., 2017). These studies highlight the challenge of preventing and treating NAFLD + CHB. However, the underlying mechanisms of this compound disease remain unclear.

DNA methylation is the best-known and most studied epigenetic modification, and it refers to heritable changes in gene expression associated with modifications of DNA that are not due to any alteration in the DNA sequence. DNA methylation plays a key role in transcriptional regulation by silencing genes through hypermethylation or activating genes through hypomethylation (Shen et al., 2012; Murphy et al., 2013; Zhang et al., 2013; Zeybel et al., 2016). In addition to gene-specific DNA methylation, the loss of global DNA methylation in sequences that are normally methylated, such as the repetitive sequences LINE-1 and satellite-2 and inter-spread cytosine-phosphate-guanine (CpG) islands, can lead to chromosomal abnormalities, chromosomal instability, chromosome fragility, and ultimately the development of disease (Amir et al., 2012; Nishida et al., 2013; Patchsung et al., 2018).

DNA methylation accounts for the impacts of environmental factors on liver disease. Marked decrease in the global DNA methylation level was detected in the livers of NAFLD and HCC mice model fed with high fat diet and methyl donor deficient diet (choline, methionine, folic acid, and vitamin B12 deficiency) (Tryndyak et al., 2011; Wang et al., 2014). Similar change also occurs in chemically induced NAFLD and HCC (Chen et al., 2004; Tao et al., 2005; Komatsu et al., 2012). In addition, HBV X protein (HBx) encoding by HBV X gene induces global hypomethylation of satellite-2 repeating sequences (Park et al., 2007). HBx is required for the virus infection and has been shown to induce demethylation of distal regulatory regions to facilitate HCC tumorigenesis (Lee et al., 2014).

Global DNA hypomethylation has also been implicated in HBV exposed HCC patients (Zhang et al., 2013). Furthermore, reduced global DNA methylation of LINE-1 in white blood cells has been associated with a twofold higher risk of HCC in hepatitis B surface antigen (HBsAg) carriers (Wu et al., 2012). These studies indicate that HBV exposure contributes to a decrease in global DNA methylation and a subsequent increase in the risk of developing HCC. Findings from our laboratory and others have shown that the NAFLD and HCC patients have lower levels of global DNA methylation than corresponding controls (Wu et al., 2012; Nishida et al., 2013; Wang et al., 2014; Lai et al., 2020). However, whether the co-occurrence of CHB with NAFLD further aggravates global DNA hypomethylation in NAFLD patients has not been evaluated.

The histological progression of liver diseases is associated with a decrease in global DNA methylation levels, especially in the case of fibrosis. Studies of animal liver fibrosis models induced by a methionine-choline-deficient diet have reported that the global DNA methylation level in the liver is reduced in these animals (Tryndyak et al., 2011; Page et al., 2016). Mouse models with early stage liver fibrosis also display hypomethylation (Komatsu et al., 2012). In advanced biliary atresia patients, DNA hypomethylation in blood was observed with severe fibrosis compared with mild fibrosis (Udomsinprasert et al., 2016). However, it is not clear whether the superposition of fibrosis on CHB is associated with the decrease in levels of global DNA methylation in NAFLD patients.

Given this uncertainty, we analyzed liver biopsies from NAFLD patients to test whether the co-occurrence of CHB with NAFLD aggravates global DNA hypomethylation, and whether the presence of concurrent CHB and liver fibrosis in NAFLD patients is also associated with a decrease in global DNA methylation levels.

This is the first study to report that patients with concurrent NAFLD and CHB show significantly lower levels of hepatic global DNA methylation than patients with NAFLD alone. The presence of both CHB and fibrosis was associated with decreased hepatic global DNA methylation levels, compared to those of patients who had neither of these conditions.

DNA methylation is one kind of epigenetic modifications that connect environment and disease. Feeding a methyl donor deficient diet caused loss of global DNA methylation in the livers of rodents and induced hepatic fibrosis and HCC (Tryndyak et al., 2011). DNA hypomethylation was also found in the livers of rodents with fatty liver induce by high fat diet and alcohol (Lu et al., 2000; Wang et al., 2014). On the contrary, methyl donor supplementation could restore the global DNA methylation level and reversed liver injury (Medici et al., 2013). In addition to diet, global DNA hypomethylation can be induced by many carcinogenic chemicals in the cells or livers such as arsenic, chromium (Takiguchi et al., 2003; Chen et al., 2004). Hepatitis infection is another risk factor to induce DNA hypomethylation. In vivo studies on human hepatocyte chimeric mouse models have shown that the levels of LINE-1 methylation in long-term HBV- and HCV-infected mice are lower than those in uninfected control mice (Okamoto et al., 2014). Another in vivo study demonstrated that the HBV X protein induces global hypomethylation of satellite-2 repeating sequences (Park et al., 2007). Furthermore, global DNA hypomethylation has been implicated in HBV-exposed HCC patients (Zhang et al., 2013). Similarly, as we observed that the levels of global DNA methylation in NAFLD + CHB patients were lower than those in NAFLD patients.

However, the mechanisms for the decrease in global DNA methylation levels upon the co-occurrence of CHB is not very clear. DNA methylation is a process that transforms 5′-cytosine into 5′-methylcytosine with the adding methyl group from the universal methyl donor-S-adenosylmethionine (SAM). This reaction is catalyzed by a family of enzymes known as DNA methyltransferases (DNMTs). Several possibilities that may contribute to the development of DNA hypomethylation have been proposed. One underlying mechanism is the reduction of methylation capacity because of intracellular depletion of SAM. SAM is an essential and critical methyl donor for cellular transmethylation reactions including DNA, RNA, and histone methylation (Ouyang et al., 2020). Diet low in source of methyl donors, high in fat can lead to global DNA hypomethylation in the livers by impairing synthesis of SAM, which could be reversed by methyl donor supplementation (Medici et al., 2013; Wang et al., 2014; Bakir et al., 2019). However, the change of one-carbon metabolism in HBV is still unclear. The other mechanism is the changes in DNMTs expression and/or activity. HBV-induced aberrant global DNA hypomethylation is associated with the reduced expression of genes DNMT1 and DNMT3b (Park et al., 2007; Huang et al., 2010). Aside from regulating the transcription of DNMT genes, HBV also affects the capacity of these genes to bind their corresponding regulatory elements. A study using chromatin immunoprecipitation assays showed that HBV partially or completely abrogates DNMT3a binding to target gene promoters, contaminant with a decreased DNA methylation in regulatory elements (Zheng et al., 2009). Therefore, the global DNA hypomethylation of NAFLD patients with CHB may be associated with downregulated expression and activity of DNMTs. The DNA integrity is another critical factor that affects the normal status of DNA methylation (Pogribny and Beland, 2009). Diminished methylation capacity of DNA methyltransferases, leading to DNA hypomethylation can be induced by the presence of unrepaired lesions in DNA, such as 8-oxoguanine and 5-hydroxymethylcytosine (Pogribny and Beland, 2009). It has been reported that diet-induced NAFLD is associated with revisable 5-hydroxymethylcytosine change in the liver (Lyall et al., 2020). While the effect of HBV on genome integrity has not been reported yet.

Our study found that the presence of both CHB and fibrosis in NAFLD patients significantly reduced global DNA methylation levels compared to those in the absence of both conditions. After adjusting for anthropometric and biochemical characteristics, the presence of CHB and fibrosis was negatively associated with global DNA methylation levels in the liver. Hepatic global DNA methylation level is reduced in animal liver fibrosis models induced by a methionine-choline-deficient diet and intraperitoneal injection of CCl₄ (Tryndyak et al., 2011; Komatsu et al., 2012; Page et al., 2016). In addition, DNA hypomethylation has been observed in the blood of advanced biliary atresia patients with severe fibrosis, compared to those with mild fibrosis (Udomsinprasert et al., 2016). The above data from our study and others indicated that DNA hypomethylation may be involved in the pathological progress of liver diseases. While there is no consensus on how DNA hypomethylation promotes the development of disease. Generally, molecular mechanisms of global hypomethylation on adverse outcome may be attributed to the dysregulation of chromosomal abnormalities, and genomic instability. First, global DNA hypomethylation may lead to chromosomal abnormalities. Demethylation of repetitive sequences located at centromeric, pericentromeric, and subtelomeric chromosomal regions may cause the induction of chromosomal abnormalities (Pogribny and Beland, 2009). It has been reported that DNA hypomethylation in HCCs is clearly associated with the amount of chromosomal alterations (Nishida et al., 2013). Second, global DNA hypomethylation may promote chromosomal instability (CIN), which has been proved to be mediated by DNA damage. The increase of DNA strand breaks precedes DNA hypomethylation (James et al., 2003), and DNA damage is a precursor of mutation (Patchsung et al., 2018) and can lead to related pathological findings. In addition, global DNA hypomethylation can also lead to chromosome fragility, which in turn leads to CIN in HCC (Nishida et al., 2013). For example, global DNA hypomethylation promotes early liver tumor formation by leading to aneuploidy, chromosome translocation and copy number changes in HCC mouse models, human hepatoma cell lines (Yamada et al., 2005; Tryndyak et al., 2011; Komatsu et al., 2012; Udomsinprasert et al., 2016).

There are several limitations to the present study. First, the number of subjects was relatively small, leading to lack of power to determine the statistical differences in global DNA methylation levels between different groups. Second, the data were obtained from cross-sectional analyses, and we therefore could not detect the causal relationship between global DNA methylation and HBV infection or pathological changes. Third, this is an association study, so we cannot sum up the causes, mechanisms and consequences by itself. Whether or not there is an association between changes in DNA-methylation and gene expression remains unclear and should be investigated in following studies.

Despite the limitations of this study, we have assessed the level of global DNA methylation in patients who are affected by both NAFLD and CHB, which are rarely involved in previous studies. We also evaluated the association between global DNA hypomethylation and liver pathology. As the overexpression of oncogenes caused by DNA hypomethylation may play an important role in tumorigenesis (Good et al., 2018), global DNA methylation could be used to characterize the epigenetic characteristics of these patients. Our data also provide a theoretical basis for further investigations into the epigenetics of chronic liver disease.

In conclusion, our study shows that patients with concurrent NAFLD and CHB exhibited lower levels of global DNA methylation than patients with NAFLD alone. The co-occurrence of CHB and liver fibrosis in NAFLD patients was associated with a decrease in global DNA methylation levels. Our findings provide new insights into the epigenetic events underpinning NAFLD + CHB and may provide the basis for new research into specific epigenetic modifications mediated by virus.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by the Medical Ethics Committee of the School of Public Health, Sun Yat-sen University (SYSU) approved the study protocol (Project identification code: [2012] No. 17). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

FYL and LJW designed the research study, analyzed the data, and wrote the manuscript. QO, ZWL, LZP, XYC, LRW, LQS, XPL, YYW, HX, JW, and FW performed the research and collected liver biopsy and other biological samples. HLZ was acting as the submission’s guarantor and revised the manuscript. All authors approved the final version of the article, including the authorship list.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.