Association of human leukocyte antigen-DR-DQ-DP haplotypes with the risk of hepatitis B virus-related hepatocellular carcinoma

INTRODUCTION

Globally, primary liver cancer (PLC) is the sixth most common cancer and the third leading cause of cancer-related deaths, with an incidence-to-mortality ratio of nearly 1:1 (905,677 vs. 830,180 in 2020)^[1]. China contributes to more than half of global PLC cases. The age-standardized mortality rate of PLC is higher in the middle-aged (40-64 years) population than in the elderly (≥ 65 years) population, in contrast to the other major cancers in China^[2]. Hepatocellular carcinoma (HCC) accounts for 94.6% of PLC in eastern China. Although immunization of infants against hepatitis B virus (HBV) has reduced their risk of developing HCC, 87.5% of HCC is attributed to chronic HBV infection in eastern China^[3,4]. There are approximately 94 million subjects chronically infected with HBV in mainland China^[5]. We speculated that 32% male and 9% female subjects with chronic HBV infection might die of HCC before the average life (75 years old) in mainland China, based on the epidemiological data in Taiwan^[6]. The key issue to decrease HCC-caused premature death is to identify the HBV-infected subjects who are more likely to develop HCC or postoperative relapse, which is essential for specific prophylaxis of this deadly malignancy.

HBV-induced hepatocarcinogenesis approximately takes 40-50 years. During this process, HBV experiences evolution via accumulating mutations in its genome, especially in the core promoter and the preS regions^[7,8]. HBV is more apt to mutating than other DNA viruses due to the lack of proofreading function of the HBV genome during the reverse transcription process of pregenomic RNA, leading to a mutation rate of 2.2 × 10^-5 substitutions/site/month in viral replication^[9]. On the other hand, inflammatory molecules such as interleukin (IL)-6 trans-activate the expression of apoprotein B mRNA-editing enzyme catalytic polypeptides (APOBEC) 3B and inhibit the expression of uracil DNA glycosylasev, resulting in an increase in HCC-related viral mutations^[10,11]. These HBV mutations may change viral replication, pathogenicity, and viral epitope that affect immune response, thus promoting the development of HCC^[12-15]. Human leukocyte antigen (HLA) is mainly composed of HLA class I and class II molecules. HLA class I molecules may play a role in eliminating HBV-infected hepatocytes by presenting HBV peptides to CD8⁺ cytotoxic T lymphocytes. HLA class II molecules, which consist of three isotypes termed HLA-DR, HLA-DQ, and HLA-DP, can bind and present exogenous antigens to CD4⁺ T cells, thus initiating immune response. Several genome-wide association studies have shown that single nucleotide polymorphisms (SNPs) affecting the expression of HLA-II are associated with chronic HBV infection and HBV-related acute-on-chronic liver failure^[16-18]. Our previous studies demonstrated that the genetic polymorphisms of HLA-DP, HLA-DQ, and HLA-DR are closely associated with chronic HBV infection, and the progression of liver diseases especially HBV-related HCC^[19-21]. Several haplotypes of HLA class II genes were significantly associated with the response to an HBV vaccine^[22]. However, few studies mentioned the association of HLA haplotypes with the risks of HBV-induced liver diseases. Here, we carried out an epidemiological study to investigate the association of haplotypes composed of several important HLA class II functional SNPs with the outcome of chronic HBV infection.

METHODS

RESULTS

DISCUSSION

The outcomes of chronic HBV infection depend on two aspects: viral genotype and host immunity. Compared with genotype B HBV-infected subjects, genotype C was more apt to cause chronic infection^[28]. HLA-II whose genes encode both the alpha and beta chains has been proven to be the major immune factors affecting the outcomes of HBV infection^[16-26]. HLA-II molecules are normally expressed on antigen presenting cells including Kupffer cells, the resident macrophages in liver, and play key roles in the presentation of viral antigens to CD4⁺ T helper (Th) lymphocytes. Th1-mediated immune response facilitates HBV clearance and inhibits the development of HCC, while Th2-, Th22-, Th17-, and Treg-related inflammation may predispose HBV persistence, poor prognosis of HBV-related acute-on-chronic liver failure, and cirrhosis^[29-31]. Genetic predisposition of the three genes HLA-DR, HLA-DQ, and HLA-DP affects the host immunity and inflammation, mostly via affecting their expression. The genotypes that tend to upregulate their expression upregulate their immune responses^[19,32]. However, it remains a great challenge to know what kinds of expression patterns of HLA-DR, HLA-DQ, and HLA-DP contribute to the polarity towards Th1 or other Th isotypes.

In this study, we found that the haplotype CCAACG with a “mutation” at HLA-DR rs3135338 and “wild-types” at other five SNPs was significantly associated with a decreased risk of HCC, compared to HBV-free subjects and HBV-infected subjects without HCC, indicating the upregulation of HLA-DRA might predispose the Th1 response to reduce the risk of HCC. Similarly, TCAATA with two “mutations” at HLA-DP functional SNPs rs3077 and rs9277535 was significantly associated with a decreased risk of HCC compared to HBV-free subjects, indicating an increased transcription of HLA-DPA1 and HLA-DPB1 in normal liver might predispose the Th1 immune response too. Interestingly, TCAACA with one “mutation” at HLA-DP rs9277535 was associated with an increased risk of benign liver diseases (CHB + LC) compared to ASCs, possibly because HLA-DPB1 in normal liver predisposes to Th2-, Th22-, Th17-, and Treg-related inflammation. TTAACG with one “mutation” at rs477515 was associated with an increased risk of LC compared to ASCs and CHB patients, indicating that high expression of HLA-DRB1 alone might be sufficient to induce the polarity towards higher Th17/Treg ratio, which is related to HBV-caused liver fibrosis^[19,31,33]. Interestingly, TCGATA with one “mutation” at HLA-DQ rs2856718 and two “mutations” at HLA-DP rs3077 and rs9277535 was significantly associated with a decreased risk of HCC and an increased risk of LC [Tables 2 and 5], possibly because the co-expression of HLA-DQ, HLA-DPA1, and HLA-DPB1 might contribute to the polarity towards higher Th9- and Th17-mediated immune responses in liver. Th17 cells differentiate from Th0 naïve T cells in response to transforming growth factor β1 and interleukin-6, which drive the development of liver fibrosis^[34]. HBV-specific Th9 cells are decreased in HBV-related HCC patients^[35]. Thus, the haplotypes of HLA-DR, HLA-DQ, and HLA-DP might predispose the polarity of immune response upon HBV infection and even the consequences of chronic HBV infection.

An insufficient and persistent antiviral immune reaction can contribute to the generation of the HBV mutations during HBV evolution. We hypothesize that different expression profiles of HLA-DR, HLA-DQ, and HLA-DP might affect the development of HCC or LC via influencing the generation of HCC- or LC-related HBV mutations. In this study, the haplotypes promoting HBV persistence were found to be associated with a higher prevalence of HCC-risk HBV mutations, indicating that the effect of the haplotypes on the development of HCC might be indirectly influenced by the generation of HCC-risk HBV mutations. The immune polarity regulated by the expression profile of HLA isotypes might maintain the proinflammatory microenvironment for the generation and immune selection of HCC-risk HBV mutations. APOBECs whose expressions are trans-activated by IL-6 and other inflammatory factors can cause APOBEC-signature mutations in the HBV genome^[10]. APOBEC-signature mutations can promote the development of HCC^[11]. Thus, APOBECs can bridge chronic hepatitis and HCC. However, the effect of the haplotypes with the development of LC is unlikely to be influenced by the generation of LC-risk HBV mutations. The haplotype TTAACG was associated with an increased risk of LC, but it was positively associated with T1674C/G and T48A, whereas T1674C/G was associated with a decreased risk of LC compared to ASCs and CHB patients [Supplementary Table 5]. Thus, the effect of TTAACG on the development of LC should be determined by other pathways such as Th17-related signaling pathways.

The haplotype analysis provides an important option to know the effects of one or more SNPs of the three HLA-II isotypes on the development of HBV-related diseases especially HCC. It also provides an excellent opportunity to know which one or two of the HLA-DR, HLA-DQ, and HLA-DP expressions contribute to the polarity towards Th1 or other T helper isotypes. However, our study has several limitations. First, cases and controls were not matched on age and gender. HCC is more frequent in males than in females^[1,6]. Second, the sample size was small, especially for those with HBV-related LC and HBV natural clearance subjects, resulting in the uncertainty of some associations. Third, the sample size of the HBV-infected subjects whose HBV fragments have been both amplified and sequenced was small, resulting in loss of data concerning HBV mutations. Further studies with larger sample sizes are needed to validate our findings. Fourth, although we excluded people who received antiviral treatment, other potential confounders might also influence the results.

In summary, the present study suggests that the haplotypes CCAACG, CCGACG, TCAATA, and TCGATA at rs3135338, rs477515, rs2856718, rs9275319, rs3077, and rs9277535 are significantly associated with a decreased risk of HCC. These haplotypes are also associated with decreased frequencies of most HCC-risk HBV mutations including the APOBEC-signature mutations in the core promoter and preS regions of the HBV genome. These data provide epidemiological evidence regarding the effect of haplotypes of HLA class II alleles in chronic HBV infection, viral evolution, and HCC development, which help provide genetic clues for choosing proper prophylactic and therapeutic options for HBV-infected subjects.