INTRODUCTION
The human leukocyte antigen (HLA) complex on the short arm of chromosome 6 comprises HLAs A, B, C, and D. HLAs A, B, and C encode class I molecules. The HLA-D region consists of three primary sub-regions designated as DP (DPA1 and DPB1), DQ (DQA1 and DQB1), and DR (DRB1) loci, which encode class II molecules. HLA class I molecules, expressed in many somatic nucleated cells, are largely responsible for presenting pathogen-derived peptides from the cytosol to CD8+ cytotoxic T lymphocytes. HLA class II molecules are expressed on the surface of antigen-presenting cells and target cells. They bind to antigen peptides, which can be recognized by CD4+ T cells, produce an immune reaction, stimulate cytokine production to modulate CD8+ cytotoxic T cells, and promote antibody production [Reference Zhu1].
Hepatitis B virus (HBV) infections have various clinical outcomes, such as spontaneous recovery (SR), acute self-limited hepatitis (ASH), and chronic hepatitis (CH). CH is associated with a high lifetime risk of developing cirrhosis and hepatocellular cell carcinoma (HCC) [Reference Shepard2, Reference Elgouhari, Abu-Rajab Tamimi and Carey3]. Vigorous CD4+ and CD8+ T lymphocyte responses to various HBV antigens are associated with HBV self-elimination, whereas insufficient CD4+ T-cell activity and defective CD8+ T cells in early stages of infection are associated with viral persistence [Reference Jung and Pape4, Reference Urbani5]. However, factors that determine HBV clearance and disease persistence and progress remain unclear. Potential mechanisms may involve interaction between viruses and host immune systems [Reference Valsamakis6, Reference Niu7].
Studies have analysed the association between clinical outcomes of HBV infection and HLA polymorphism and haplotypes [Reference Cho8, Reference Nishida9]. Currently, HBV is classified into at least eight genotypes (A–H), based on a nucleotide sequence divergence in strains >8% [Reference Zhao10, Reference Aranz-Ruiz11]; HBV genotypes are further divided into 34 sub-genotypes. We have previously described the association between sub-genotype distribution and clinical outcomes of HBV infection [Reference Wang12]. We have also previously analysed the association between clinical outcomes of HBV infection and HLA polymorphism combined with HBV sub-genotypes [Reference Tatematsu13, Reference Li14]. The present study demonstrates the association of HLA haplotypes, combined with HBV sub-genotypes, with HBV infection outcomes in a northeastern Chinese population.
METHODS
Subjects
Overall, 440 subjects (230 HBV-infected and 210 healthy) were enrolled from February 2006 to October 2010. HBV-infected individuals were divided into three groups (ASH, CH, and SR groups). In total, 54 individuals had ASH with C2 sub-genotype [ASH-C2, hepatitis B surface antigen (HBsAg+) with high-titre IgM of anti-HBc, and HBV DNA ⩾1·0 × 103 copies/ml, and detectable HBV C2 sub-genotype]. The ASH group was characterized by self-recovery from liver disease, negative HBsAg, and positive anti-HB scores for ⩽24 weeks after treatment following initial symptom presentation. Overall, 144 individuals had CH with C2 and B2 sub-genotypes (117 CH-C2 and 27 CH-B2 infections). Individuals in the CH group who visited the Department of Infectious Diseases in the Second Affiliated Hospital of Harbin Medical University and had received antiviral therapy showed persistent HBsAg for >6 months, HBV DNA ⩾1·0 × 103 copies/ml, and detectable HBV B2 or C2 sub-genotypes; 32 individuals spontaneously recovered (SR, negative for both HBsAg and HBV DNA, but positive for both anti-HBc and anti-HBs, in a routine physical examination without any clinical symptoms) without sub-genotype results. Individuals in the control group were negative for HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc, and HBV DNA; no familial relationship was identified among them. Subjects were diagnosed in accordance with the Virus Hepatitis Diagnosis Standard of China (2000). None of the subjects was positive for hepatitis viruses A, C, and D (HAV, HCV, and HDV) or human immunodeficiency virus (HIV), based on antibody testing. The study protocol conformed to the 1975 Declaration of Helsinki regulations. The procedures were approved by our ethics committee, and informed consent was obtained from all participants.
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008.
METHODS
Laboratory assays, including HBV serology, serum HBV DNA extraction, HBV sub-genotype analysis, and HLA typing (HLA-A, HLA-B, HLA-DRB1), have been described previously [Reference Li14]; demographics and detailed descriptions of serology tests are listed in Table 1.
Demographic and serological description of 198 HBV infections
HBV, Hepatitis B virus; ASH, acute self-limited hepatitis; SR, spontaneous recovery from hepatitis B without clinical syndrome; CH, chronic hepatitis; ALT, alanine aminotransferase; n.d., not detected.
Positive scores for HBsAg, anti-HBs anti-HBc, HBeAg, anti-HBe and anti-HBcIgM are given as number of patients (%).
* ASH vs. CH.
Statistical analysis
Arlequin v. 3.5.1.2 (http://cmpg.unibe.ch/software/) [Reference Kummee15], including Hardy–Weinberg equilibrium (HWE) tests, multilocus haplotype inferences, and pairwise global linkage disequilibrium (LD), was used to analyse data. Maximum-likelihood haplotype frequencies were estimated using the expectation maximization (EM) algorithm in Arlequin v. 3.5.1.2. Classic coefficients of LD (Δ) and normalized LD (Δrel) were computed for each individual haplotype, as described previously [Reference Huang and Chang16, Reference Fletcher17].
HLA haplotype frequencies were analysed with a two-tailed Fisher's exact test in SPSS v. 17.0 (SPSS Inc., USA). P < 0·05 was considered statistically significant. The odds ratio (OR), indicating the likelihood of carrying a specific HLA haplotype, and 95% confidence intervals (95% CI) were also calculated.
RESULTS
HLA haplotype frequency (HF)
Calculated haplotypes with HF >0·05 are listed in Table 2. A*02-B*15, A*02-B*40, A*30-B*13, A*02-DRB1*09, and B*13-DRB1*07 (HF = 0·058, 0·071, 0·059, 0·062, and 0·052, respectively) were the most frequent haplotypes. Of these haplotypes, A*02-B*40 had the highest HF (0·071) in this population.
Frequency of HLA-A, -B, and DRB1 haplotypes in the study group
ASH, Acute self-limited hepatitis; SR, spontaneous recovery from hepatitis B without clinical syndrome; RH, recovery from hepatitis B; CH-B2, CH-C2, chronic hepatitis with sub-genotypes B2 and C2.
Haplotype frequencies <0·05 are not shown.
HLA haplotypes associated with HBV infection
As shown in Table 3, the frequencies of A*02-DRB1*08, A*02-DRB1*09, A*02-DRB1*12, A*11-DRB1*15, A*24-DRB1*09, A*33-DRB1*07, B*13-DRB1*12, B*15-DRB1*15, B*40-DRB1*08, B*40-DRB1*09, B*51-DRB1*09, B*51-DRB1*15, and A*02-B*15-DRB1*09 were significantly higher in the infection group (including 32 SR, 54 ASH, and 144 CH) than in the control group. However, frequencies of A*24-B*13, A*02-DRB1*04, A*02-DRB1*07, A*33-DRB1*13, A*33-DRB1*15, B*13-DRB1*09 and A*02-B*40-DRB1*15 were significantly lower in the infection group than in the control group.
Comparison of frequency of HLA-A, B, and DRB1 haplotypes between infection and control groups
OR, Odds ratio; CI, confidence interval; n.a., not available.
P(Fisher) values <0·05 are shown in the Table; values ⩾0·05 have been omitted.
HLA haplotypes associated with HBV infection recovery or persistence
In the 230 HBV-infected participants, 86 RH (recovery from hepatitis B) (including ASH and SR) recovered from the disease within 6 months. Frequencies of A*24-DRB1*14, B*15-DRB1*04, B*46-DRB1*09, and A*02-B*15-DRB1*09 were significantly higher than those in the CH group (including CH-C2 and CH-B2, n = 117; Table 4). By contrast, frequencies of A*02-DRB1*12 and B*40-DRB1*12 were significantly lower than those in the CH group (including CH-C2 and CH-B2, n = 117).
Comparison of frequency of HLA-A, B, and DRB1 haplotypes in infection groups
OR, Odds ratio; CI, confidence interval; RH, recovery from hepatitis B; CH, chronic hepatitis; ASH, acute self-limited hepatitis; C2, sub-genotype B2; SR, spontaneous recovery from hepatitis B without clinical syndrome.
P(Fisher) values <0·05 are shown in the Table; values ⩾0·05 have been omitted.
HLA haplotypes associated with acute or occult clinical course of HBV infection recovery
ASH and SR infections were characterized by self-recovery from liver disease. ASH infections had an obvious clinical course, whereas SR infections showed no clinical symptoms. The two groups were compared, and the results showed that the frequency of A*02-B*46 was significantly lower, whereas that of A*02-B*15 was significantly higher in the ASH group than in the SR group (Table 4).
HLA haplotypes associated with outcomes of HBV-C2 sub-genotype infections
Overall, 171 HBV patients were infected with HBV-C2 sub-genotype, including 54 ASH and 117 CH. Frequencies of A*02-B*15, A*02-DRB1*15, A*24-DRB1*14, B*15-DRB1*04, and A*02-B*15-DRB1*09 (Table 4) were significantly higher in the ASH-C2 group than in the CH-C2 group. B2 sub-genotype was also detected in some CH infections; however, low numbers prevented further analysis.
DISCUSSION
The relationship between HLA polymorphisms and HBV infection outcomes has been explored [Reference Zhang18–Reference Brouwer20]. However, the relationship between HLA haplotypes and HBV infection outcomes is rarely studied [Reference Cho21, Reference Lu22]. Furthermore, the influence of HLA-A, HLA-B, DRB1, and HBV sub-genotypes on HBV infection outcomes has not been reported.
Hwang et al. [Reference Hwang23] reported that the HLA haplotypes A*33-B*44, B*44-DRB1*07, B*46-DRB1*08, A*33-DRB1*07, A*02-DRB1*14, and A*24-DRB1*12 are more frequent in infected than control groups. Moreover, HFs of A*33-B*44-DRB1*07 and A*02-B*46-DRB1*08 are higher in infected than control groups. By contrast, B*44-DRB1*13, B*62-DRB1*04, A*24-DRB1*04, and A*33-B*44-DRB1*14 are more common in control groups. However, the data presented here differ. A*02-DRB1*08, A*02-DRB1*09, A*02-DRB1*12, A*11-DRB1*15, A*24-DRB1*09, A*33-DRB1*07, B*13-DRB1*12, B*15-DRB1*15, B*40-DRB1*08, B*40-DRB1*09, B*51-DRB1*09, B*51-DRB1*15, and A*02-B*15-DRB1*09 haplotypes may pose a higher risk of HBV infection compared to that posed by controls. By contrast, A*24-B*13, B*13-DRB1*09, A*02-DRB1*07, A*33-DRB1*13, A*33-DRB1*15, and A*02-B*40-DRB1*15 may protect subjects from HBV infection.
Cho et al. [Reference Cho8] reported that HLA haplotypes DRB1*1302-DQB1*0609, DQB1*0609-DPB1*0201, and DRB1*1302-DQB1*0609-DPB1*0201 are strongly associated with viral clearance, which differs from our study. Our previous [Reference Li14] and current studies have found that DRB1*13 and A*33-DRB1*13 carriers are not susceptible to HBV infection, whereas no association with DRB1*13 and A*33-DRB1*13 infections and clearance and persistence was found. HLA-DQB1 and DPB1 were not investigated in our study, and if LD with HLA-DRB1*13–DQB1*06 and HLA-DRB1*13–DQB1*06-DPB1*02 is associated with HBV clearance, this aspect has not been reported previously and, hence, needs to be investigated. Thio et al. [Reference Thio24] reported that A*01-B*08-DRB1*03, B*44-Cw*1601, and B*44-Cw*0501 haplotypes are associated with viral persistence. Zhu et al. [Reference Zhu1] reported that HLA-A*1101-B*4601-C*0102 and DQA1*0302-DQB1*0303-DRB1*09 haplotypes have lower HFs in patients who may be responsive to interferon-α treatment than those in the group non-responsive to chronic HBV infection. Pan et al. [Reference Pan25] have reported that A*110 101G-C*070201G, A*110101G-C*140201G, A*110101G-B*510101G-C*140201G, A*330301G-B*580101G, B*580101G-C*030201G, A*330301G-B*580101G-C*030201G, and B*510101G-C*140201 G haplotypes are positively associated with HCC, whereas A*020701G-B*460101G, B*460101G-C*010201G, and A*020701G-B*460101G-C*010201 G are negatively associated with HCC. However, these results focus on the influence of either HLA class I or HLA class II haplotypes on HBV infection outcomes. No result has been reported from the combined study of HLA class I and class II alleles. In our study, A*02-DRB1*12 and B*40-DRB1*12 haplotypes may be associated with viral persistence. Furthermore, A*24-DRB1*14, B*15-DRB1*04, B*46-DRB1*09, and A*02-B*15-DRB1*09 haplotypes may be associated with viral clearance. However, A*02-B*15 and A*02-DRB1*15 carriers may easily clear the virus with acute clinical onset. B*46-DRB1*09 was no longer associated with viral clearance when HBV-C2 infection was observed. When infected with HBV-C2, A*02-DRB1*12 and B*40-DRB1*12 were no longer associated with HBV persistence. This study is the first to report such findings. In a previous study, class I alleles were most strongly associated with HBV infection, suggesting that CD8+ cytotoxic T lymphocytes are important to viral clearance or persistence [Reference Thio24]. Viral peptides differ in their ability to bind to particular HLA glycoproteins; this suggests an internal mechanism underlying the association of HLA class II allele polymorphisms with HBV infection outcomes and response to vaccination [Reference Mert26]. Therefore, further investigation of the influence of combined HLA class I and class II alleles on HBV infection outcomes is required.
HLA haplotype distribution has been investigated in many Chinese regions [Reference Mert26–Reference Wu28]. For instance, A*02-B*46 (HF = 0·071) and A*11-B*15 (HF = 0·051) are the predominant haplotypes in the Han population. A*02-B*46 and A*30-B*13 have significantly strong LD, whereas A*02-B*15 and A*02-B*40 have low LD [Reference Mert26]. However, A*30-B*13-DRB1*07 is the most frequent haplotype in the northern Chinese Han population. In addition, A*30-B*13, A*33-B*58, A*01-B*37, A*30-DRB1*07, A*33-DRB1*13, A*01-DRB1*10, B*37-DRB1*10, B*08-DRB1*17, B*13-DRB1*07, and B*58-DRB*17 exhibit a significant positive LD [Reference Yuan27]. In this study, the A*02-B*15, A*02-B*40, A*30-B*13, A*02-DRB1*09, and B*13-DRB1*07 haplotypes were the most frequent HLA-A, HLA-B, and DRB1 haplotypes. Of these haplotypes, A*02-B*40 exhibited the highest HF in the northeastern Chinese Han population. By contrast to the haplotypes described in other studies, A*30-B*13 and B*13-DRB1*07 (not A*02-B*15 and A*02-B*40) showed a significantly high LD in this study. This discrepancy may be due to regional differences. Various HLA class I and class II alleles are related to HBV infection outcomes in different ethnic populations. However, finding consistent associations in different studies is difficult due to racial diversity, variations in study design, methodology, and complex immune-regulatory mechanisms [Reference Pan25]. Furthermore, the sample size involved in each study may be insufficient and findings may be incidental [Reference Zhu1]. Therefore, further validation studies considering other ethnic groups and a larger sample size are required.
In conclusion, our results revealed that B*46-DRB1*09 is probably associated with viral clearance, whereas B*40-DRB1*12 may be related to viral persistence. A*02-DRB1*12 and A*02-B*15-DRB1*09 haplotype carriers were susceptible to HBV infection, whereas the presence of A*02-DRB1*12 may indicate HBV persistence, and the presence of A*02-B*15-DRB1*09 may indicate HBV clearance. Additionally, HBV infections, including ASH-C2 and SR without the detected subtypes, were associated with A*02-B*15-DRB1*09. A*24-DRB1*14 and B*15-DRB1*04 carriers may be associated with viral clearance and recovery from the disease with acute clinical onset, when infected with HBV-C2. In patients infected with the HBV-C2 sub-genotype, A*02-B*15 and A*02 -DRB1*15 carriers also showed acute clinical onset before recovery. HLA haplotypes in ASH and SR infections were compared, and the results showed that A*02-B*46 carriers were more likely to recover from a disease with an occult clinical course. The data obtained in these and previous studies suggest a link between HLA haplotypes and HBV pathogenesis, thereby providing potential therapeutic targets to treat HBV infections.
ACKNOWLEDGEMENTS
The authors thank Dr Zhenzi Jin, Dr Jingqiu Zhang, and Dr Ping Shi of the Harbin Infectious Diseases Hospital, Heilungkiang Province, China for their assistance in sample collection. This study was supported by the Scientific Research Foundation of Youth Innovation Talents of Harbin (grant no. 2012RFQYS062).
DECLARATION OF INTEREST
None.
