A population-based study to investigate host genetic factors associated with hepatitis B infection and pathogenesis in the Chinese population

Hepatitis B virus (HBV) infection is a significant public health problem that may lead to chronic liver disease, cirrhosis, and hepatocellular carcinoma (HCC) [1]. Approximately 30% of the world's population has been infected with HBV and approximately 350 million (5–6%) are persistent carriers. Infants infected perinatally by vertical transmission from e antigen positive mothers have a 90% risk of becoming persistent carriers. Approximately 90% of preschool children infected with HBV will fail to achieve clearance and develop persistent HBV infection. For adults, the majority of HBV-infected individuals achieve clearance with only 5–10% becoming persistent carriers of HBV. HBV accounts for 80% of all liver cancer and is an important carcinogen [2]. Of individuals persistently infected with HBV, 10–30% will develop liver cirrhosis (LC) and HCC [2]. These highly variable outcomes in both clearance rates and disease outcomes in persistently infected individuals cannot be fully explained by differences in viral or environmental factors. Thus, differences in host genetic factors may affect hepatitis B natural history.

Viral factors that may influence HBV outcomes include HBV DNA levels, HBV genotypes, HBV genetic variants, and co-infection with other hepatitis viruses. HBV DNA levels are correlated with T-cell hyporesponsiveness to HBV antigens [3] and are a risk predictor for HCC development [4, 5]. Treatment with lamivudine [6] and interferon-alpha (IFN-α) [7, 8] decreases viral load [3] and reduces occurrence of HCC. Of the eight HBV genotypes (A-H), HBV-A has been associated with persistence [9], HBV-C with severe liver disease [10, 11], and HBV-B with more benign disease [11]; however, HBV-B was found to be a predictor for HCC [10]. A double mutation in the base core promoter of the HBV genome reported to aggravate chronic hepatitis is more frequent in HBV-C isolates than in HBV-B isolates [12]. Amino acid replacements in the "α" determinant of the HBs protein, the proposed coformational epitope essential for recognition and neutralization by anti-HBs antibodies, have been reported [13, 14]. A precore stop codon mutation (1896G to A) [15] and two mutations within the core promoter region (1762A to T and 1764G to A) [16, 17] have been associated with fulminant hepatitis B. Both variants show a defect in hepatitis e antigen (HBeAg) expression [18, 19], which may modify the immune response of the host [20, 21]. However, in many cases of fulminant hepatitis B, particularly those from nonendemic areas [22, 23], neither of these mutations was observed. These investigations indicate that HBV viral burden, genotype, and genetic polymorphism are important contributors to the natural history of HBV disease and may explain, in part, the observed heterogeneity in outcomes of infection.

Environmental factors are clearly implicated in HBV pathogenesis. Alcohol and aflatoxin are two important factors that affect the progression of chronic hepatitis B. Alcohol consumption increases the severity of liver disease [24, 25] and increases the risk of developing liver decompensation from cirrhosis [26]. Patients with chronic hepatitis B exposure to aflatoxins are at an increased risk of HCC [27], especially in the Fusui County of Guangxi Zhuan Autonomous Region [27, 28] and the Qidong district of Jiangsu Province [29, 30] in China, where the highest rates of HCC are found and aflatoxins levels are high in many local foods and grains. In Fusui County of China, the rate of HCC is 120 per 100,000 persons/year among men [31], a rate 35 times higher than that in United States. The tumor suppressor gene p53 294^ser mutation is a 'hotspot' mutation in HCC from patients in regions with dietary aflatoxins exposure [32, 33].

The role of host genetic factors on HBV persistence and pathogenesis is less well understood. CD4+ T cell proliferative responses in acute HBV infection are significantly more vigorous than those seen in persistent HBV infection, suggesting that MHC class II polymorphisms influence susceptibility to persistent infection. Several MHC class II alleles have been identified in association with clearance or persistence of HBV infection [34‐37]. Polyclonal and multispecific CD8+ T cells are readily detectable in the peripheral blood of patients with acute HBV infection or HBV clearance, but are rarely detectable in patients with persistent HBV infection. This suggests that HLA alleles may be key determinants of HBV clearance; however, there have been few comprehensive studies of HLA class I alleles and the results for class II alleles have been inconsistent [34‐37]. In a comprehensive case-control study, Thio et al recently showed that the class I allele, A*301 and class II allele DRB1*1302 are associated with clearance and two class I alleles, B*08 and B*44, are associated with persistence, thus confirming an earlier studies implicating DRB1*1302 in HBV clearance [38].

Cytokines, chemokines and their receptors may also have a role in HBV persistence and disease. A number of associations with cytokines have been reported. Tumor necrosis factor (TNF)-α was associated with HBV persistence [39] and HCC [40, 41]. Interleukin(IL)-10 was associated with high risk of HCC [42, 43]. Genetic associations have also been observed for the mannose binding protein (MBP) [44‐47] and the vitamin receptor D [48] with persistence, and hormonal markers with HCC [49, 50]. Many of the genetic associations have been inconsistent among studies, possibly due to population substructure, small sample size, or differences in study design, or have not yet been replicated in duplicate studies.

These studies strongly suggest that genetic factors influence HBV disease; however, these influences are likely interactive with viral and environmental factors. The primary objective of this study is to localize and identify genes that influence HBV persistence and adverse outcomes of HBV infection by employing a population-based genetic association strategy with both candidate-gene and genome-wide association approaches. We have therefore implemented a HBV genetic study powered to detect genetic factors and their interactions in individuals representing the different stages of HBV disease: clearance, persistent infection, chronic hepatitis B, cirrhosis, and HCC, as well as normal healthy controls from the Han Chinese population in the north and west region of China (Table 1). Specific hypotheses and comparison groups are listed in Table 2.

Chronic HBV infection is a major contributor to morbidity and mortality worldwide despite the availability of efficient vaccines and vaccination programs in China and elsewhere [52]. The underlying environment factors contributing to HBV chronic infection are well known, but the pathophysiology of HBV-related cirrhosis and HCC are incompletely understood and the interactions between environmental and genetic factors have not been systematically explored. The susceptibility genes leading to chronic infection and subsequent disease likely affect many different pathways ranging from mediators of immune response and inflammation to oncogenic pathways. These genes may have main effects or be interactive with environmental or other genetic factors. The discovery of either main effect or interactive genes will help us to better understand HBV pathogenesis. This study is unique in that we have enrolled participants representing the spectrum of HBV exposure and pathogenesis ranging from spontaneous clearance to persistent infection leading to decompensated cirrhosis and liver cancer in a population from north and west of China (Table 1).

Heterogeneity in HBV pathogenesis and disease outcomes may be influenced by environmental factors that differ over time or geographically. In an effort to reduce bias due to changes in preventive medicine and public health measures that have impacted nutrition, HBV exposure, vaccination, and medical treatment, this study is limited to persons greater than 40 years old, except for cirrhosis and HCC. A particular problem with many genetic studies is population substructure where cases are genetically different from controls. If undetected, population substructure may lead to false discovery, even within populations considered to be genetically homogeneous [53‐56]. We have attempted to minimize substructure by enrolling only Han Chinese whose primary residence is in north and west of China.

The strongest association with HCC is dietary aflatoxin exposure, a known carcinogen with high levels documented in southeast China, and particularly in Fusui County, Guangxi Province, in the Qidong district of Jiangsu Province. After 1986, food-handling practices were modified to eliminate aflatoxin contamination and food product inspections were mandated by the China government; however, aflatoxin exposure continues to be a major food contaminate in southeast China and, to lesser extent, throughout China. Limiting recruitment to hospitals in regions with lower risks for aflatoxin exposures has reduced the confounding impact of environmental dietary factors, and particularly aflatoxin exposure. Enrollment is also limited to probands and controls who are not co-infected with HCV or HDV viruses to avoid confounding by these hepatitis viruses known to interact with HBV. These inclusion and exclusion criteria are expected to increase specificity by reducing heterogeneity due to non-genetic factors.

Patients were stratified according to their infection status and the severity of their disease using stringent clinical and laboratory criteria. We anticipate that the identification of genetic risk predictors for different specific outcomes will inform clinical management of persons with chronic infection and lead to the development of better therapeutic agents. The identification of genes implicated in pathways leading to chronic HBV replication, liver inflammation, fibrosis, and the carcinogenic process will hopefully lead to improved diagnosis, risk prediction, and clinical care.