AIM: To investigate the effect of human leukocyte antigen (HLA) DRB1 and DQB1 alleles on the inactive and advanced stages of chronic hepatitis B.

METHODS: Patient records at a single institution’s hepatology clinic were reviewed. Demographic data, laboratory results, endoscopy results, virological parameters, biopsy scores and treatment statuses were recorded. In total, 355 patients were eligible for the study, of whom 226 (63.7%) were male. Overall, 82 (23.1%) were hepatitis B early antigen (HBeAg) positive, 87 (24.5%) had cirrhosis, and 66 (18.6%) had inactive disease. The presence of DQB1 and DRB1 alleles was determined by polymerase chain reaction with sequence-specific primers. The distribution of the genotyped alleles among patients with cirrhosis and patients with chronic active hepatitis was analyzed.

RESULTS: The most frequent HLA DQB1 allele was DQB1*03:01 (48.2%), and the most frequent HLA DRB1 allele was DRB1*13/14 (51.8%). DQB1*05:01 was more frequent in patients with active disease than in inactive patients (27% vs 9.1%; P = 0.002, P_c = 0.026). DRB1*07 was rare in patients with cirrhosis compared with non-cirrhotics (3.4% vs 16%; P = 0.002, P_c = 0.022). Older age (P < 0.001) and male gender (P = 0.008) were the other factors that affected the presence of cirrhosis. In a multivariate logistic regression analysis, DRB1*07 remained a significant negative predictor of cirrhosis (P = 0.015). A bioinformatics analysis revealed that a polymorphic amino acid sequence in DRB1*07 may alter interaction with the T-cell recognition site.

CONCLUSION: This study demonstrates that HLA alleles may influence cirrhosis development and disease activity in Turkish chronic hepatitis B patients.

Hepatitis B virus (HBV) is observed all around the world, and nearly 350 million people have chronic HBV infections[1]. The estimated number of annual deaths due to the consequences of HBV infection is nearly 600000[2]. The virus itself is non-cytopathic, and liver damage during chronic infection is due to the host immune reaction against the virus. Several host immune mechanisms have been proposed to be involved in viral persistence, beyond viral factors[3]. Human leukocyte antigen (HLA)-restricted T lymphocytes, B lymphocytes in the humoral immunity system, dendritic cells, natural killer cells and numerous cytokines are required to generate an accurate immunologic response against the virus[4,5]. An inaccurate, nonselective cytolytic immune reaction to hepatocytes is believed to cause necroinflammation and further liver fibrosis, rather than eradicating the virus[3].

In the majority of chronic hepatitis B patients, an inactive state, with low DNA levels (HBV DNA levels less than 2000 IU/mL) and normal liver enzyme levels, is observed. Nonetheless, nearly 30% of these inactive carriers (hepatitis B early antigen (HBeAg) negative, normal serum liver enzyme level and low serum HBV DNA level) will develop active disease, and nearly 10% will develop cirrhosis[6,7]. To date, there is no defined test to predict which patient will remain in the inactive state without treatment and which patient will progress to chronic active hepatitis, which may culminate in cirrhosis unless specific antiviral treatment is introduced. It should be noted that when cirrhosis develops, the expected five-year survival decreases to 50% in untreated hepatitis B patients[8]. Additionally, for cirrhotics, the annual risk of developing hepatocellular carcinoma is 5 times higher than for non-cirrhotics[9]. Furthermore, in general, cirrhosis is the most common non-neoplastic cause of death related to the digestive system in the United States[10].

Studies investigating the effect of HLA polymorphism on the disease state of different populations with HBV infection have been performed. Studies in the Chinese population showed that HLA DRB1*03:01, DQB1*03:01, DQB1*03:03, DQB1*05:03, DRB1*06, DRB1*08 and DRB1*16 are associated with viral persistence[11-13]. In Taiwanese patients, DRB1*12:02 was shown to be related to viral persistence, whereas the DRB1*04:01 and DRB1*07:01 alleles were related to viral clearance[14]. In a study from the United States, the DQB1*03:01 allele was related to viral persistence in Afro-Americans[15]. A study from Turkey revealed that HLA DR7, DR13 and DQ3 alleles were related to susceptibility to chronic infection[16].

Recent genome-wide association studies revealed an association between the HLA class II gene region (DR, DQ and DP) and HBV chronicity[17-19]. Moreover, a recent study revealed the effect of DQB1 alleles on the treatment response to nucleoside/nucleotide analogs in chronic HBV infection[20]. In this study, we aimed to investigate the association between HLA DQB1 and DRB1 alleles and the outcome of chronic HBV disease (inactive state, active disease or cirrhosis).

In this study, we compared the allele distribution among chronic active hepatitis patients with the distribution among patients in the chronic inactive stage. We defined inactivity according to histology, persistently normal ALT levels and persistently low viremia. Chronic active hepatitis patients more frequently carried the HLA DQB1*05:01 allele than did inactive patients. This allele has been found to be related to a better response to the HBV vaccine[25] and to be a protective factor against chronic HBV infection[17]. Previous data have associated DQB1*05:01 with an appropriate immunologic response, but in the present study, DQB1*05:01 was related to active disease, thus revealing an inappropriate immune response. This finding could be a reflection of genetic divergence between different populations or a consequence of the genotype of the virus; only the D genotype is present in Turkey[26].

Another finding of this study was the more frequent presence of HLA DRB1*07 in the non-cirrhotic group compared with the cirrhotics. Liver histology scores were better in patients with the DRB1*07 allele, and the mean fibrosis score and HAI score of these patients were lower (P = 0.001 and P = 0.008, respectively). Chronic HBV infection is an immunologically driven disease, and HBV is a non-cytopathic virus. Host genetic factors and viral and environmental factors have been proposed to determine disease progression or regression[3]. Immune action against the virus involves HLA-restricted T lymphocytes, antibody-secreting B lymphocytes, natural killer cells and numerous cytokines. The immune recognition of viruses requires the presentation of viral antigens to CD4-positive and CD8-positive lymphocytes by HLA class II and class I molecules, after which viral clearance or persistence occurs[27,28]. When an adequate vigorous polyclonal, multispecific response to HBV antigens is not produced, CD4-positive and CD8-positive T cells that are capable of terminating HBV infection do not accumulate in the liver[4,5]. If HBV-specific CD8-positive and CD4-positive T cell responses are absent, the immune response then becomes nonselective and results in chronic necroinflammation and fibrosis. This long-lasting cytolytic and inappropriate immune response against infected hepatocytes is responsible for liver damage. Previous reports have suggested an association between cirrhosis and HLA DRB1; for instance, two studies from China have demonstrated an association between cirrhosis and HLA DRB1*12[29,30]. Another study, from Brazil, revealed the protective effect of HLA DRB1*11 against severe liver damage in HCV patients[31]. Other studies and a recent meta-analysis implicated DRB1*07 as a risk factor for persistent HBV infection[16,32,33]. Studies from Turkey[34], the United Kingdom[35], Belgium[25], Spain[36], Germany[37] and China[38,39] revealed that the presence of DRB1*07 (or DR7) causes unresponsiveness to the HBV vaccine. Furthermore, in a Turkish study exploring HLA alleles in renal transplant candidates, panel-reactive antibodies were less frequently detected in the patient group with the DRB1*07 allele, despite the fact that this group had a higher predisposition to developing these antibodies[40]. This finding indicates a hypoimmune profile associated with DRB1*07. In the current study, the DRB1*07 allele was less frequent in cirrhotic patients compared with non-cirrhotics. Together with previous reports, the results of the present study may indicate that patients with the DRB1*07 allele are not able to mount a specific immune reaction to HBV and are predisposed to chronic infection. This hypoimmune response also yields less hepatocyte damage and less cirrhosis. The puzzling immune response observed in patients carrying the DRB1*07 allele may be attributable to this allele’s interaction with the T-cell recognition site. The available crystal structures of HLAs reveal that these proteins carry allele-specific motifs[41]. The antigenic peptide specificity of HLA class II proteins comes from the determined polymorphic pockets in the peptide-binding cleft. T-cell receptor recognition is accomplished by both the peptide and HLAs[41]. Certain HLA DRB1 types show polymorphisms specifically at the T-cell recognition site, without significantly altering peptide binding[42,43]. Interestingly, our bioinformatics analysis revealed that DRB1*07 contains four substitutions at the T-cell interaction site, which are located at β30, β67, β71 and β86, according to ClustalW analysis (Figure 1). Two of these residues, β67 and β71, have been determined to be important for peptide binding and T-cell receptor recognition in mutagenesis studies[42,43]. These residues are solvent exposed and are predicted to be critical for direct T-cell receptor binding, so any alteration at these sites would change T-cell receptor recognition. Additionally, the buried residues β30 and β86 have been shown to be important for T-cell recognition in mutagenesis studies[41,42]. Although these residues cannot have a direct effect on T-cell recognition, they might have an indirect conformational influence that can be communicated to the T-cell recognition site (Figure 2). Due to changes in the T-cell recognition residues in the HLA DRB1*07 variant, the immune response can be weakened, resulting in chronic infection without cirrhosis.

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Figure 2 Possible model illustrating the single-nucleotide polymorphic regions of major histocompatibility complex class II that affect T-cell receptor recognition. The yellow asterisks show mutational sites that are buried in the peptide-binding pocket, whereas the white asterisks indicate mutations in the solvent-exposed T-cell recognition regions. This model was built according to the available crystal structures, which were published previously[41].

This study has certain limitations. A larger sample size could be used to identify further associations between HLA subgroups and disease outcomes. This is a cohort study, so it is not possible to conclude that there is a direct causative relationship between our findings and the disease state. Molecular studies are necessary to investigate HBV-specific antigenic peptides and the specific structure of the rearranged T-cell receptor. However, the well-defined patient groups, the long duration of the follow-up period and the medium- to high-resolution genotyping of HLA alleles are the strengths of this study.

In conclusion, the natural course of HBV disease is not easily predictable; viral factors, environmental factors and host factors may affect the disease course. This study suggests that the HLA DQB1*05:01 allele is associated with chronic persistent disease and that the DRB1*07 allele is a protective factor against cirrhosis.

We are grateful to Asli Kumbasar for critically editing the manuscript.