| Hepatocellular carcinoma(HCC) is the second leading cause of cancer-related deaths worldwide, with the incidence on the rise both in developed and developing countries. HCC development is influenced by complex factors including viral infection, environmental factors, and genetic makeup, with most studies having identified susceptibility loci at the human leukocyte antigen(HLA) class II region at 6p21.The hepatitis B virus(HBV) infection is a mainly risk factor for HCC. HBV has been classified into different genotypes according to a sequence divergence of > 8% in the entire genome, and the genotypes are further separated into subgenotypes if the divergence in the nucleotide sequence is between 4 and 8%. HBV genotypes and subgenotypes have distinct geographic distributions and have been implicated to differ with regard to clinical liver diseases, disease outcomes, and responses to interferon treatment. In East Asia, although HBV genotypes B and C are endemic, the HBV genotype frequency varies in these areas. Numerous studies have found that HBV genotype C related to the development of HCC. Because of the relatively small study sample sizes, the low success rates of HBV typing and the different study designs, the effect of HBV genotype on the outcomes of HBV persistent infection also varied greatly. The basal core promoter, which is regulated by the enhancer II to a great extent, controls the transcription of precore m RNA. The precore protein is processed to produce the secreted hepatitis B e antigen(HBe Ag), which indicates active viral replication and is associated with an increased risk of HCC. Longitudinal studies with less than 50 cases demonstrated that some of the mutations in the enhancer II/basal core promoter/precore(Enh II/BCP/PC) region would occur years before a diagnosis of HCC is made and gradually accumulate during the development of HCC.Host genetic factors also play an important role in the development of HCC. Currently, three genome-wide association studies(GWAS), all from China, have been conducted on HBV-related HCC, two of which consistently identified HLA-DQ/DR as susceptibility loci. Of the two independent GWAS, one study with 1,538 cases and 1,465 controls for GWA scan identified the single nucleotide polymorphism(SNP) rs9272105(located between HLA-DQA1 and HLA-DRB1), while the other study with 1,161 HCC cases and 1,353 controls for GWA scan identified the SNP rs9275319 at HLA-DQ. These findings highlight the importance of HLA-DQ/DR molecules in the development of HBV-related HCC.HBV mutations are most likely selected via virus-immune interactions in the inflammatory microenvironment. Therefore, we performed a large multi-centre study to evaluate the effects of HBV genotype, mutations in the Enh II/BCP/PC region, GWAS-identified HLA-DQ/DR single nucleotide polymorphisms(SNPs)(rs9272105 and rs9275319) and their interactions on HCC risk. Furthermore, we evaluated the risk prediction effects of these factors in HBV-related HCC.The cases in this study were HBV-related HCC patients. The diagnosis of HCC was confirmed by a pathological examination and/or an alpha-fetoprotein elevation(> 400 ng/ml) combined with an imaging examination(i.e., magnetic resonance imaging and/or computerised tomography). The controls were HBV persistent carriers, defined as positive for hepatitis B surface antigen(HBs Ag) and HBV core antibody(HBc Ab). All study subjects were negative for the hepatitis C virus(HCV) antibody(anti-HCV). This large multi-centre case-control study included 1,507 HBV-related HCC cases and 1,560 HBV persistent carriers from central and southern Jiangsu Province, matched by area, age and gender. The HBV-related HCC cases included 535 cases from Nanjing, 522 cases from Nantong and 450 cases from Qidong, and the HBV persistent carriers included 510 controls from Zhangjiagang, 500 controls from Taixing and 550 controls from Danyan.We used nested multiplex PCR to detect HBV genotype A-F, subgenotypes B1, B2, C1 and C2, and used nested PCR and direct DNA sequencing to detect HBV mutations of Enh II/BCP/PC region. Moreover, we genotyped HLA SNPs rs9272105 and rs9275319 using Taq Man allelic discrimination assay. Then we constructed a risk prediction model to classify the HCC cases and controls. HBV genotype, the independent HCC-related mutations, the HLA SNPs, the HCC-related multiplicative interactions and the main effect of the interactions were considered to be the predictive factors by conducting a stepwise logistic regression model. The model performance was evaluated by receiver-operator characteristic(ROC) curves.We found the frequencies of the HBV genotypes B and BC(coinfection) were varied among the studied areas, but the frequency of genotype C was consistently higher in HBV-related HCC subjects(80.6% among HCC vs. 34.4% among HBV persistent carriers). Compared with subjects infected with HBV B-related genotypes(B and BC), the subjects infected with non-B genotypes(C and D) had a 7.18-fold increased HCC risk(adjusted OR = 8.18, 95% CI = 6.91-9.68). Moreover, 11 HBV hotspot mutations in the Enh II/BCP/PC region(C1653T, C1673 T, T1674C/G, C1730 G, A1752 G, T1753 C, A1762 T, G1764 A, G1899 A, G1915A/C and C1969T) were independently and significantly associated with HCC risk.The logistic regression analyses in the additive genetic model showed that a variant allele of rs9272105 increased the host HCC risk compared to the HBV persistent carriers(adjusted OR = 1.31, 95% CI = 1.18-1.45), whereas a variant allele of rs9275319 was associated with a decreased HCC risk(adjusted OR = 0.66, 95% CI = 0.56-0.78). Through interaction analysis, we detected a significant interaction between rs9272105 and HBV genotype on HCC risk. The crossover analysis suggested that the subjects infected with HBV non-B genotypes and carrying the rs9272105 AA genotype had a more prominent risk effect(adjusted OR = 17.70, 95% CI = 12.21-25.64) compared to those infected with HBV B-related genotypes and carrying the rs9272105 GG genotype. We also detected significant interactions between rs9272105 and the HBV mutations C1673 T, G1719 T, A1726 C, C1730 G, A1752 G and G1799 C on HCC risk. However, we did not observe any significant interaction between rs9275319 and HBV genotype or mutations on HCC risk.Through a stepwise regression analysis, the HBV genotype, the 11 mutations, HLA-DQ/DR rs9272105 and rs9275319, and the interaction of the mutation A1752 G with rs9272105 were all entered into the prediction model of HCC, and the area under the curve(AUC) for the panel including the HLA-DQ/DR SNPs, HBV genotype and mutations was 0.840(sensitivity = 81.3%, specificity = 74.8%).In this large multi-centre study, we found that HBV genotype C and subgenotype C2 were the risk factors for HCC, and 11 HBV mutations were found to be significantly associated with HCC risk. We also detected significant interactions of HLA-DQ/DR rs9272105 with both HBV genotype and mutations, which may imply a potential biological significance for rs9272105. Excitingly, the panel that combined the HLA-DQ/DR SNPs, HBV genotypes and mutations provided a high sensitivity and specificity to discriminate the HCC patients from the controls. The HBV genotype, the mutations and the HLA-DQ/DR SNPs may serve as biomarkers for the surveillance of HBV persistent carriers. Large studies with prospective designs are warranted to further evaluate our results. |
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