Hepatitis B virus (HBV) infection is a major public health problem. Two billion people are expected to be infected with HBV during their lifetime and about 350 million are estimated to be chronic carriers [
Human leukocyte antigen (HLA) is an integral component of the immune response on which majority of host genetic studies have focused. Numerous reports described the associations of highly polymorphic HLA gene with the outcome of a wide range of infectious diseases. It is because antiviral cytotoxic T lymphocytes (CTLs) are believed to play a major role in eradication of infection by virtue of their capacity to identify and kill virus-infected cells through recognition of viral peptides presented by HLA class I molecules [
The genes of the HLA locus are located on the short arm of chromosome 6. They are arranged in three clusters: class I, class II, and class III. Many previous studies have shown the associations of certain HLA class I genes with the course of HBV infection, but these associations are inconsistent even within the same population [
The protocol was approved by the Ethics Committee of the Qidong Liver Cancer Institute, and all patients provided written, informed consent before enrollment.
A total of 728 unrelated male individuals with hepatitis B surface antigen (HBsAg) positive in sera were enrolled in the Institute of Liver Cancer of Qidong in 1995. They were followed up for 10 years and tested for HBV infection again. In 2005, 588 individuals were included in this cohort because of loss to follow-up and exclusion of the patients who were treated with anti-HBV therapy. Among these 588 persons, 448 remained HBsAg positive (named HBV persistent infection group), while 140 were negative for HBsAg but positive for both anti-hepatitis B virus core antigen (HBc) and anti-HBsAg (named HBsAg clearance group). None of the subjects were positive for hepatitis C virus (HCV), hepatitis D virus (HDV), and human immunodeficiency virus (HIV) antibody. All of them are Han population.
Plasma samples were obtained from all the subjects and stored at −20°C. Their serologic status with regard to HBsAg, anti-HBs, anti-HBc, hepatitis B virus e antigen (HBeAg), and anti-HBe was determined by enzyme-linked immunosorbent assay according to the manufacturer’s instructions (Boehringer-Mannheim, Munich, Germany).
Genomic DNA for each individual was extracted from peripheral blood mononuclear cells using the QIAamp DNA blood kit (Qiagen Inc., Chatsworth, CA). All DNA samples (100–200 ng/mL) were stored at 4°C (short term) or −20°C (long term) in TE buffer (10 mM Tris-HCl, pH 8.0, 2 mM ethylene diamine tetraacetic acid) and diluted to 10–12 ng/mL in PCR.
Genomic DNA was amplified by the standard HLA locus-specific primers [
The allele frequency was calculated as the number of the allele divided by the double numbers of the total samples. The significance of allelic associations was determined either by chi-square test or by Fisher’s exact test (when there were less than five subjects in a cell). In order to reduce the number of comparisons with inadequate power, only alleles with frequency not less than 3% (29 alleles in this population) were analyzed. The level of significance was adjusted for multiple testing using the Bonferroni correction. According to the Bonferroni correction, the frequency of HLA allele was considered to be statistically different only when uncorrected
588 Qidong Han persons who were tested as HBsAg positive in 1995 were included in this study. 448 were classified as HBV persistent infection group as they remained HBsAg positive 10 years of later. The HBsAg clearance group includes the remaining 140 individuals who were negative for HBsAg but positive for both anti-HBc and anti-HBs after 10 years follow-up. Age distribution showed no statistical difference between the two groups.
A total of 31 HLA-A alleles, 54 HLA-B alleles, and 35 HLA-C alleles were detected in the two study groups (Tables
Frequencies of HLA-A alleles in each study group.
HLA-A alleles | Persistent infection group ( |
HBsAg clearance group ( |
||
---|---|---|---|---|
Allele frequency (%) | Count | Allele frequency (%) | Count | |
|
0.11 | 1 | 0.00 | 0 |
|
2.00 | 18 | 2.86 | 8 |
|
0.00 | 0 | 0.36 | 1 |
|
1.34 | 12 | 1.43 | 4 |
|
6.58 | 59 | 5.71 | 16 |
|
9.93 | 89 | 9.64 | 27 |
|
0.33 | 3 | 0.00 | 0 |
|
0.22 | 2 | 0.36 | 1 |
|
0.11 | 1 | 0.00 | 0 |
|
15.29 | 137 | 16.79 | 47 |
|
0.33 | 3 | 0.71 | 2 |
|
18.30 | 164 | 21.07 | 59 |
|
4.35 | 39 | 5.71 | 16 |
|
0.00 | 0 | 0.36 | 1 |
|
0.11 | 1 | 0.36 | 1 |
|
0.33 | 3 | 0.00 | 0 |
|
0.11 | 1 | 0.00 | 0 |
|
0.11 | 1 | 0.00 | 0 |
|
0.33 | 3 | 0.71 | 2 |
|
0.00 | 0 | 0.36 | 1 |
|
22.32 | 200 | 20.36 | 57 |
|
2.57 | 23 | 2.14 | 6 |
|
0.22 | 2 | 0.00 | 0 |
|
2.23 | 20 | 1.43 | 4 |
|
5.25 | 47 | 5.00 | 14 |
|
0.22 | 2 | 0.36 | 1 |
|
0.11 | 1 | 0.71 | 2 |
|
6.36 | 57 | 2.86 | 8 |
|
0.11 | 1 | 0.00 | 0 |
|
0.45 | 4 | 0.00 | 0 |
|
0.22 | 2 | 0.71 | 2 |
Frequencies of HLA-B alleles in each study group.
HLA-B alleles | Persistent infection group ( |
HBsAg clearance group ( |
||
---|---|---|---|---|
Allele frequency (%) | Count | Allele frequency (%) | Count | |
|
0.11 | 1 | 0.00 | 0 |
|
0.22 | 2 | 0.00 | 0 |
|
0.11 | 1 | 0.00 | 0 |
|
3.46 | 31 | 8.57 | 24 |
|
3.46 | 31 | 2.86 | 8 |
|
0.11 | 1 | 0.00 | 0 |
|
2.90 | 26 | 2.5 | 7 |
|
0.11 | 1 | 0.00 | 0 |
|
0.22 | 2 | 0.36 | 1 |
|
3.24 | 29 | 4.26 | 12 |
|
0.47 | 4 | 0.00 | 0 |
|
0.00 | 0 | 0.36 | 1 |
|
1.34 | 12 | 1.43 | 4 |
|
0.22 | 2 | 0.71 | 2 |
|
2.01 | 18 | 2.14 | 6 |
|
0.45 | 4 | 0.00 | 0 |
|
12.05 | 108 | 10 | 28 |
|
0.11 | 1 | 0.00 | 0 |
|
0.22 | 2 | 0.00 | 0 |
|
4.35 | 39 | 4.29 | 12 |
|
0.59 | 5 | 0.71 | 2 |
|
0.11 | 1 | 0.00 | 0 |
|
0.11 | 1 | 0.00 | 0 |
|
0.11 | 1 | 0.00 | 0 |
|
0.59 | 5 | 0.71 | 2 |
|
0.33 | 3 | 1.79 | 5 |
|
0.45 | 4 | 0.00 | 0 |
|
0.33 | 3 | 1.07 | 3 |
|
3.57 | 32 | 3.57 | 10 |
|
0.33 | 3 | 0.00 | 0 |
|
3.01 | 27 | 3.57 | 10 |
|
0.33 | 3 | 0.00 | 0 |
|
0.11 | 1 | 0.00 | 0 |
|
0.11 | 1 | 0.00 | 0 |
|
1.11 | 10 | 0.00 | 0 |
|
16.52 | 148 | 15.36 | 43 |
|
0.00 | 0 | 0.36 | 1 |
|
0.33 | 3 | 0.00 | 0 |
|
0.11 | 1 | 0.00 | 0 |
|
18.30 | 164 | 16.43 | 46 |
|
0.33 | 3 | 0.71 | 2 |
|
0.33 | 3 | 0.00 | 0 |
|
1.00 | 9 | 0.36 | 1 |
|
0.11 | 1 | 1.07 | 3 |
|
0.11 | 1 | 0.00 | 0 |
|
6.03 | 54 | 7.14 | 20 |
|
5.58 | 50 | 4.64 | 13 |
|
0.22 | 2 | 0.36 | 1 |
|
0.78 | 7 | 0.36 | 1 |
|
0.22 | 2 | 0.00 | 0 |
|
2.23 | 20 | 2.86 | 8 |
|
0.59 | 5 | 0.36 | 1 |
|
0.70 | 6 | 1.07 | 3 |
|
0.22 | 2 | 0.00 | 0 |
Frequencies of HLA-C alleles in each study group.
HLA-C alleles | Persistent infection group ( |
HBsAg clearance group ( |
||
---|---|---|---|---|
Allele frequency (%) | Count | Allele frequency (%) | Count | |
|
26.00 | 233 | 26.07 | 73 |
|
0.11 | 1 | 0.36 | 1 |
|
1.00 | 9 | 1.07 | 3 |
|
4.58 | 41 | 3.21 | 9 |
|
11.83 | 106 | 14.29 | 40 |
|
0.00 | 0 | 0.71 | 2 |
|
0.33 | 3 | 0.00 | 0 |
|
0.11 | 1 | 0.00 | 0 |
|
8.26 | 74 | 9.29 | 26 |
|
0.22 | 2 | 0.36 | 1 |
|
6.92 | 62 | 3.93 | 11 |
|
4.47 | 40 | 4.29 | 12 |
|
0.11 | 1 | 0.00 | 0 |
|
0.11 | 1 | 0.00 | 0 |
|
0.89 | 8 | 1.07 | 3 |
|
0.22 | 2 | 0.00 | 0 |
|
0.22 | 2 | 0.00 | 0 |
|
0.33 | 3 | 0.00 | 0 |
|
0.11 | 1 | 0.00 | 0 |
|
0.11 | 1 | 0.36 | 1 |
|
0.11 | 1 | 0.00 | 0 |
|
12.72 | 114 | 13.93 | 39 |
|
0.22 | 2 | 0.36 | 1 |
|
6.47 | 58 | 7.86 | 22 |
|
4.91 | 44 | 5.00 | 14 |
|
0.56 | 5 | 0.00 | 0 |
|
7.14 | 64 | 4.29 | 12 |
|
0.11 | 1 | 0.00 | 0 |
|
1.34 | 12 | 2.14 | 6 |
|
0.11 | 1 | 0.00 | 0 |
|
0.00 | 0 | 0.36 | 1 |
|
0.22 | 2 | 0.00 | 0 |
|
0.11 | 1 | 0.71 | 2 |
|
0.00 | 0 | 0.36 | 1 |
To maintain statistical power, only those alleles with the frequencies not less than 3% were compared between the persistent infection and HBsAg clearance groups. The frequency of
It is doubtless that host genetic factors are associated with the clinical outcome of different infectious diseases. The most-studied host factors that might affect the susceptibility to viral infection are HLA genes [
The relationship between HLA class I polymorphisms and the outcome of HBV infection has been explored previously, but this relationship does not appear to be universal since the investigated populations are different. In Taiwanese Aborigines,
In this study, we investigated a cohort of Qidong Han population who lived in an area of high prevalence of HBV infection. The samples were made of unrelated males and were followed up for 10 years. No previous prospective cohort study has been conducted to clarify the relationship between the polymorphisms of HLA alleles and the outcome of HBV infection in such a population. We found that
The frequency of
In summary, we found that
The authors have declared that there is no conflict of interests.
The authors thank the Qidong Liver Cancer Institute for providing the samples. They would like to thank Dr. Youji He for critical reading of the paper. This work is funded by the National Natural Science Foundation (30571703), National Natural Science Foundation for Young Scholars (81201601), National Science Foundation for Distinguished Young Scholars (30325017) of China, Science Foundation of Department of Health of Jiangsu Province H200745), and Science Foundation of Science and Technology Bureau of Nanjing (200702067). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the paper.