Journal of Viral Hepatitis, 2014, 21, 491–498
doi:10.1111/jvh.12159
HLA-DQ polymorphisms with HBV infection: different outcomes upon infection and prognosis to lamivudine therapy X. Zhang,1 J. Jia,2 J. Dong,3 F. Yu,4 N. Ma,1 M. Li,1 X. Liu,1 W. Liu,1 T. Li1 and D. Liu1 1
Department of Epidemiology and Statistics, School of Public Health, Hebei Medical University, Shi Jiazhuang, China; 2Outpatient Clinic, Hebei
Medical University, Shi Jiazhuang, China; 3School of Biomedical Science, Chinese University of Hong Kong, Hong Kong, China; and 4Division of Gastroenterology, The Second Affiliated Hospital of Hebei Medical University, Shi Jiazhuang, China Received April 2013; accepted for publication July 2013
SUMMARY. Two recent genome-wide studies showed that
the single-nucleotide polymorphisms in the HLA-DQ region (rs2856718 and rs9275572) were associated with chronic hepatitis B virus infection and chronic hepatitis C virusassociated hepatocellular carcinoma in Japanese patients. We tested the effects of the two single-nucleotide polymorphisms for all major HBV outcomes and lamivudine treatment in Han Chinese. A total of 1649 samples were enrolled, and peripheral blood samples were collected in this study. The single-nucleotide polymorphisms in the HLA-DQ region were genotyped using matrix-assisted laser desorption/ionization time of flight mass spectrometry. Our study demonstrated the clear relevance of HLA-DQ rs2856718 and rs9275572 with HBV susceptibility, natural clearance and HBV-associated HCC. HLA-DQ rs2856718G and rs9275572A were strongly associated with decreased risk of chronic HBV infection (odds
INTRODUCTION Chronic hepatitis B virus (HBV) infection has become a major threat to human health owing to one-third of the population worldwide having a history of HBV infection and approximately 350 million living with chronic infection [1,2]. Data from epidemiological investigations showed
Abbreviations: ALT, alanine aminotransferase; CHB, chronic hepatitis B; CIB, HBV infection; CI, confidence interval; EDTA, ethylenediamine tetra-acetic acid; HBc, hepatitis B core; HBe, hepatitis B e; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; LAM, lamivudine; LC, liver cirrhosis; NC, natural clearance; OR, odds ratio. Correspondence: Dianwu Liu, PhD, Department of Epidemiology and Statistics, School of Public Health, Hebei Medical University, 361 Zhongshan East Road, Shi Jiazhuang, 050017, China. E-mail: [email protected] Xiaolin Zhang and Jinhai Jia contributed equally to this work.
© 2013 John Wiley & Sons Ltd
ratio = 0.641; P = 2.64 9 10 4; odds ratio = 0.627, P = 7.22 9 10 5) and HBV natural clearance (odds ratio = 0.610; P = 4.80 9 10 4; odds ratio = 0.714, P = 0.013). Moreover, rs9275572A was also associated with development of cirrhosis and hepatocellular carcinoma (odds ratio = 0.632, P = 0.008). In addition, we showed for the first time to our knowledge that rs9275572 was a predictor for lamivudine therapy (viral response: odds ratio = 2.599, P = 4.43 9 10 4; biochemical response: odds ratio = 2.279, P = 4.23 9 10 4). Our study suggested that HLA-DQ loci were associated with both HBV clearance and HBV-related diseases and outcomes of lamivudine treatment in Han Chinese. Keywords: HBV infection, HLA-DQ, lamivudine, polymorphisms.
a higher infection rate in Asia and Africa, with 5.3–12% of the hepatitis B surface antigen (HBsAg) carriers being Chinese, 8% living in Thailand and 10% in Africa [3]. Individuals infected with HBV present different responses: most recover spontaneously and 5% develop into chronic HBV infection. Similarly, different outcomes are observed among those suffering from chronic infection: 20% progress to liver cirrhosis (LC) and 5% into primary hepatocellular carcinoma (HCC). The complicated pathogenesis of HBV infection is attributable to the virus itself, immune state, genetic background of host and environmental condition. Among all the contributory factors, the host genetic background appears to serve as a major determinant of the different outcomes of HBV infection. Experimental results have revealed a series of genes potentially associated with HBV infection and natural clearance: including HLA, ESR1, TNF, TGFA, IL-18 and CXCL-10 [4–9]. The HLA complex is the first definitely
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identified genetic system showing strong association with HBV infection. A recent genome-wide association study performed by Japanese researchers indicated that the HLADQ polymorphism rs2856718 was significantly associated with chronic hepatitis B (CHB) [10], yet it remains unknown whether there is an association of HLA-DQ rs2856718 and prognosis of HBV infection in Chinese people. In addition, GWAS from Kumar et al. [11] in May 2011 suggested that the HLA-DQ polymorphism rs9275572 was relevant with HCV-associated HCC, but it is not completely understood whether HLA-DQ polymorphism rs9275572 is relevant to HBV infection and prognosis upon therapeutic intervention in Chinese people. This study was designed to explore the association of HLA-DQ polymorphisms rs2856718 and rs9275572 with HBV susceptibility, natural clearance and the development of HBV-associated diseases. In addition, further investigation was carried out for testing the relevance of the two HLA-DQ polymorphisms with prognosis after therapy with lamivudine (LAM).
PATIENTS AND METHODS Patients Patients were recruited from the Fifth Hospital of Shijiazhuang, the First and Second Affiliated Hospital of Hebei Medical University from January 2010 to January 2012. A total of 1649 samples were genotyped and analysed in this study. The samples included 507 cases of healthy controls, 350 cases of natural clearance, 484 cases of chronic HBV infection (225 cases of chronic hepatitis B and 259 cases of LC) and 308 cases of HBV-associated HCC. Those healthy people under investigation were selected according to the following criteria: no record of hepatitis B vaccine history was negative for HBsAg, hepatitis B e antibody (anti-HBe), hepatitis B core antibody (anti-HBc) and other HBV biomarkers, normal blood routine examination and biochemical parameters, no history of endocrine disorders, cardiovascular events, or liver- and kidney-associated diseases. HBV natural clearance (NC) was defined based on the following criteria: positive for hepatitis B surface antibody (anti-HBs) and anti-HBc definite absence of HBsAg accompanied by normal liver function and no history of HBV vaccination. Chronic hepatitis B (CHB) was divided into two groups according to HBeAg expression: HBeAgpositive and HBeAg-negative CHB. Cirrhosis was diagnosed based on clinical, biochemical, radiological or histological findings. HCC diagnosis was confirmed by a pathological examination and/or a-fetoprotein elevation (> 400 ng/mL) combined with imaging. The sequence of tests used to diagnose HCC depended on the size of the lesion: (i) those with a focal hepatic mass > 2 cm underwent with imaging wherein characteristic contrast enhancement features on the arterial phase with venous washout on an
MRI or CT could be demonstrated; (ii) those with a focal hepatic mass with atypical imaging findings, or a focal hepatic mass detected in a noncirrhotic liver, underwent a liver biopsy [12,13]. The requirements for LAM treatment were definite diagnosis of CHB or LC with a serum HBV DNA level of > 5 log copies/mL, a serum alanine aminotransferase (ALT) level two times higher than the upper limit of normal ALT range and LAM (100 mg/day) use for the first time. Those receiving LAM treatment were divided into two groups according to the virological response (DNA load < 3 log copies/mL or reduction in DNA load > 2 log copies/mL): viral response group and viral nonresponse group. Two other groups were obtained depending on the biochemical response (ALT concentration within normal levels): biochemical response group and biochemical nonresponsive group [14,15]. The information on all subjects under investigation included the following: baseline information (gender, age, history of smoking, drinking and HBV vaccine, etc.), historical disease background (condition and course of disease, with or without other disorders, family history), results of biochemical and serological testing (HBV markers, quantity of HBV DNA, ALT and AFP concentrations). The precise description of smoking and alcohol drinking was as follows: individuals who smoked one cigarette per day for over 1 year were defined as smokers, and those who consumed one or more alcohol drinks a week for over 6 months were considered alcohol drinkers. Informed consent was obtained from each subject, and the Institutional Review Board of Human Research of Hebei Medical University approved the study protocol.
Genotyping All participants were subjected to 2 mL venous blood collection under ethylenediamine tetra-acetic acid (EDTA) anticoagulation. A genomic DNA Purification Kit purchased from Promega was used for genomic DNA extraction and time of flight mass spectrometry technology from the American SEQUENOM company for all sample SNP genotyping. Primer design for the two SNP alleles was performed by the Hua Da Gene company with the aid of MassARRAYâ Assay Design 4.0 Software (Sequenom Inc., San Diego, CA, USA). Following quality control plan was employed for data reliability: (i) Negative control setting; (ii) Carrying out quality control criterion indoors; (iii) Genotyping in both replication sample sets had been performed in 5% of the total samples.
Statistical analysis Continuous variables of normal distribution were expressed as means SD (v s) and data of abnormal distribution expressed by median and interquartile range, M(Q). Cate© 2013 John Wiley & Sons Ltd
HLA-DQ polymorphisms with HBV infection
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gorical variables are presented as frequencies. Comparisons between continuous data were carried out using Student’s t-test and Wilcoxon test. The associations between categorical variables were evaluated using a Pearson chi-squared test. The chi-squared G test (goodness of fit) was employed to verify whether the proportions of the HLA-DQ rs2856718G and rs9275572A polymorphism genotypes were distributed in accordance with the Hardy–Weinberg equation (Table S1). Nonconditioned logistic regression was utilized to adjust involved factors such as age, gender, smoking, alcohol drinking, etc., and odds ratio (OR) and 95% confidence intervals (95% CI) were calculated. A Pvalue of 0.05 was considered significant in all analyses, and all P-values were two-sided. All analysis mentioned above were performed using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA).
(P > 0.05). There was a higher ratio of males in the CIB than in the healthy groups (67.7% vs 58.6%, P = 0.001), and the HCC group displayed a higher ratio of males than the CHB+LC group (79.8% vs 59.9%, P = 5.68 9 10 9). Concerning the ratio of females to males, there were no differences found between the NC and CIB group, LC and CHB group (P > 0.05). A similar ratio of smokers and drinkers was observed between the LC and CHB groups, with significance among the other three groups at P < 0.05 (Table 1). The viral response group had a lower ratio of males, smokers and drinkers than the viral nonresponse group (P < 0.05), along with similar levels of HBV DNA, ALT and AST before treatment (P > 0.05, Table S2). The biochemical response group presented with lower ratio of males, smokers and drinkers than the biochemical nonresponse group (Table S3).
RESULTS
Association of HLA-DQ rs2856718G polymorphism with HBV infection
Demographic and aetiological characteristics of the subjects The LC group had significantly higher mean age than the CHB group (51.11 11.12 vs 38.61 14.37, P = 2.33 9 10 23) and the HCC group higher than that of the CHB+LC group (55.96 9.06 vs 45.30 14.17, P = 9.52 9 10 35). There was no statistical significance between the healthy control and HBV infection (CIB) groups, NC group and CIB group with regard to age
The study of the healthy population and HBV infection cases indicated a strong association of rs2856718G with HBV susceptibility. In the dominant genetic model, GG genotype carriers and AG genotype carriers showed a lower risk of HBV infection than AA genotype carriers (OR: 0.636, P = 1.43 9 10 4), with the same relationship (OR: 0.52, P = 7.22 9 10 6) in the recessive genetic model. With adjustment for gender, age, smoking and alcohol, logistic regression analysis suggested an obvious relevance
Table 1 Demographic and aetiological characteristics of study subjects Gender (%) Characteristics HBV susceptibility CIB (n = 792) Healthy(n = 507) P OR(95% CI) HBV Clearance SR(n = 350) CIB (n = 792) P OR(95% CI) CHB?LC LC (n = 259) CHB (n = 225) P OR(95% CI) LC+CHB?HCC HCC (n = 308) LC+CHB(n = 484) P OR(95% CI)
Smokers (%)
Drinkers (%)
Age (mean SD)
Male
49.45 13.48 49.29 14.61 0.845
535(67.7) 256(32.3) 297(58.6) 210(41.4) 0.001 1.478(1.173–1.862)
355 (44.8) 437 (55.2) 141 (27.8) 366(72.2) 7.43 9 10 10 2.109(1.660–2.679)
395(49.9) 397(50.1) 171(33.7) 336(66.3) 1.03 9 10 8 1.955(1.552–2.463)
50.32 14.95 49.45 13.48 0.330
216(61.7) 134(38.3) 535(67.7) 256(32.4) 0.052 1.296(0.998,1.685)
101(28.9) 249(71.1) 355 (44.8) 437 (55.2) 3.79 9 10 7 2.003(1.528,2.624)
119 (34.0) 231(66.0) 395(49.9) 397(50.1) 6.66 9 10 7 1.931(1.487–2.508)
51.11 11.12 38.61 14.37 2.33 9 10 23
148(57.1) 111(42.9) 142(63.1) 83(36.9) 0.181 0.779(0.541,1.124)
84(32.4) 95(42.2)
101(39.0) 158(61.0) 103(45.8) 122(54.2) 0.132 0.757(0.527,1.088)
55.96 9.06 45.30 14.17 9.52 9 10 35
245(79.8) 62(20.2) 290(59.9) 194(40.1) 5.68 9 10 9 2.643(1.895,3.687)
176 (57.1) 132 (42.9) 179 (37.0) 305 63.0) 2.68 9 10 8 2.272(1.697,3.041)
191(62.0) 117(38.0) 204(42.1) 280(57.9) 5.02 9 10 8 2.241(1.673,3.002)
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Female
Yes
No
175(67.6) 130(57.8) 0.026 0.657(0.453,0.952)
Yes
No
0.032 0.099 0.730(0.547,0.974) 0.763(0.554,1.053)* 0.177 0.137 0.790(0.562,1.112)* 0.681(0.410,1.129)*
0.913 0.999 1.024(0.666,1.577)* 1.000(0.551,1.815)*
0.003 0.001 0.642(0.479,0.773)* 0.528(0.361,0.773)*
36 (11.7)
38 (14.7)
109 (13.8)
CHB+LC? HCC
CHB?LC
HBV natural clearance
HBV, hepatitis B virus; CIB, HBV infection; NC, HBV natural clearance; CHB, chronic hepatitis B; LC, liver cirrhosis; HCC, hepatocellular carcinoma; OR, odds ratio; CI, confidence interval. *OR and OR 95% CI adjusted for age, sex, smoking status and drinking status.
0.176 0.273 0.745(0.486,1.143) 0.766(0.475,1.234)*
0.786 0.962 0.933(0.567,1.537) 0.987(0.570,1.708)* 0.991(0.688,1.428) 1.018(0.679,1.528)*
0.962 0.931
4
0.545(0.416,0.714) 0.610(0.463,0.805)*
9.60 9 10 4.80 9 10
6
0.616(0.444,0.856) 0.688(0.492,0.962)*
0.004 0.029
5
0.520(0.390,0.694) 0.541(0.403,0.725)* 4
4
1.43 9 10 2.64 9 10 0.020 2.83 9 10 0.738(0.571,0.954)* 0.459(0.331,0.636)*
Healthy (n = 507) 163 225 119 (32.1) (44.4) (23.5) NC(n = 350) 101 177 72 (28.9) (50.6) (20.6) CHB (n = 225) 89 101 35 (39.6) (44.9) (15.6) CHB+LC(n = 484) 192 219 73 (39.7) (45.2) (15.1) 109 (13.8)
CIB (n = 792) 338 345 (42.7) (43.6) CIB(n = 792) 338 345 (42.7) (43.6) LC(n = 259) 103 118 (39.8) (45.6) HCC(n = 308) 146 126 (47.4) (40.9) HBV susceptibility
OR(95% CI) AG
GG
AG AA AA
GG
Codominant Controls, n (%)
Table 2 Association of HLA-DQ rs2856718 with hepatitis B virus outcomes
P
6
0.636(0.504,0.804) 0.641(0.505,0.814*
P OR(95% CI)
Dominant
Association of the HLA-DQ rs9275572A polymorphism with HBV infection There was relevance between rs9275572A and HBV susceptibility. With the healthy population as control group and HBV infection cases as case group, rs9275572A protects against HBV infection in the dominant and recessive genetic models (OR:0.596 and 0.436, P = 6.76 9 10 6; P = 4.31 9 10 5). In the codominant genetic models with adjustment for gender, age, smoking and alcohol, individuals carrying AA and AG alleles had a decreased risk of HBV infection with adjusted OR of 0.706 and 0.374 (P = 0.006; P = 6.30 9 10 6). The dominant and recessive genetic models were accompanied with 0.627 and 0.429 as adjusted OR (P = 7.22 9 10 5; P = 6.47 9 10 5). The study of HBV natural clearance cases as control group and HBV infection cases as case group indicated that rs9275572A was relevant to HBV natural clearance. In the dominant genetic model, rs9275572A was favourable in the course of HBV natural clearance (OR: 0.599, P = 7.23 9 10 5). Yet, there was no difference shown in the recessive genetic model with a P-value of 0.164 and OR value of 0.704. In the codominant genetic model subjected to adjustment for gender, age, smoking and alcohol, an adjusted OR value of 0.179 with a 0.02 P-value and 0.714 of adjusted OR value accompanied with a P-value of 0.013 in the dominant genetic model were obtained. In addition, rs9275572A association with progression of CHB and LC to HCC was found. Logistic regression analysis indicated lower risk of CHB and LC progression to HCC in individuals carrying AA and AG alleles when the CHB+LC was used as the control group and the HCC cases as the case group in the codominant genetic model, with adjusted OR values of 0.693 and 0.373 (P = 0.043; P = 0.011).
OR(95% CI)
Recessive
P
under codominant, dominant and recessive models (adjusted OR: 0.738, 95% CI: 0.571-0.954, P = 0.020; adjusted OR: 0.459, 95% CI: 0.331- 0.636, P = 2.83 9 10 6). When HBV infection was used as case group and HBV natural clearance as control group, analysis of results revealed the beneficial action of rs2856718G on HBV natural clearance in the dominant (OR: 0.545, 95% CI: 0.416-0.714, P = 9.60 9 10 6) and recessive genetic models (OR: 0.616, 95% CI: 0.444–0.856, P = 0.004). After adjustment for gender, age, smoking and alcohol, a similar association still remained under the codominant, dominant and recessive genetic models (adjusted OR: 0.642,95% CI: 0.479–0.773, P = 0.003; adjusted OR = 0.528, 95% CI: 0.361–0.773, P = 0.001; adjusted OR: 0.610, 95% CI: 0.463–0.805, P = 4.80 9 10 4; OR: 0.688, 95% CI: 0.492–0.962, P = 0.029). There was no association detected between rs2856718G with LC development from CHB and HCC development from CHB and LC in the three genetic models (Table 2).
7.22 9 10 4.07 9 10
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Cases, n (%)
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© 2013 John Wiley & Sons Ltd
© 2013 John Wiley & Sons Ltd
0.002 0.008 0.616(0.456,0.833) 0.632(0.451,0.886)*
0.754 0.442 0.944(0.658,1.355) 0.853(0.570,1.278)*
0.043 0.011 0.693(0.486,0.988)* 0.373(0.175,0.796)*
0.320 0.696 0.807(0.528,1.232)* 1.183 (0.509,2.751)*
0.020 0.162 0.719(0.544,0.949)* 0.689(0.408,1.162)*
212 (68.8)
151 (58.3)
173 (49.4)
CHB+LC? HCC
CHB?LC
HBV natural clearance
495
HBV, hepatitis B virus; CIB, HBV infection; NC, HBV natural clearance; CHB, chronic hepatitis B; LC, liver cirrhosis; HCC, hepatocellular carcinoma; OR, odds ratio; CI, confidence interval. *OR and OR 95% CI adjusted for age, sex, smoking status and drinking status.
0.104 0.024 0.573(0.290,1.130) 0.420(0.198,0.890)*
0.292 0.549 1.485(0.709,3.110) 1.287(0.564,2.941)*
0.164 0.391 0.704(0.428,1.156) 0.801(0.482,1.331)* 5
7.23 9 10 0.013 0.599(0.465,0.772) 0.714(0.547,0.932)*
5
4.31 9 10 6.47 9 10 0.006 6.30 9 10 0.706(0.553,0.903)* 0.374(0.244,0.573)*
Healthy (n = 505) 60 196 249 (11.9) (38.8) (49.3) CIB(n = 792) 44 257 491 (5.6) (32.4) (62.0) CHB (n = 225) 12 85 128 (5.3) (37.8) (56.9) CHB+LC (n = 484) 32 173 279 (6.6) (35.7) (57.6) 491 (62.0)
CIB (n = 792) 44 257 (5.6) (32.4) NC(n = 350) 27 150 (7.7) (42.9) LC(n = 259) 20 88 (7.7) (34.0) HCC(n = 308) 12 84 (3.9) (27.3) HBV susceptibility
AG
GG
AG AA AA
GG
OR(95% CI)
P
6
0.596(0.476,0.747) 0.627(0.498,0.790)*
6.76 9 10 7.22 9 10
6
0.436(0.291,0.655) 0.429(0.283,0.650)*
P P OR(95% CI)
Dominant Codominant Controls, n (%) Cases, n (%)
Our study suggested that the rs9275572A allele polymorphism had relevance with the viral response to LAM therapy. In the dominant and recessive genetic models, individuals carrying the rs9275572A allele had a higher chance of responding to LAM in relation to viral load(OR: 2.585, 95% CI: 1.545–4.326, P = 2.32 9 10 4; OR: 3.311, 95% CI: 1.136–9.652, P = 0.021). After adjustment for gender, age, smoking and alcohol, there was an association of rs9275572A with the viral response in the codominant genetic models (adjusted OR: 2.062, 95% CI: 1.153–3.687, P = 0.015; adjusted OR: 5.553, 95% CI: 1.827–16.877, P = 0.003). In the dominant and recessive genetic models, rs9275572AA and rs9275572AG provided increased chances of responding to LAM therapy in relation to the viral response (adjusted OR: 2.599, 95% CI: 1.525–4.428, P = 4.43 9 10 4; adjusted OR: 4.684, 95% CI: 1.552–14.133, P = 0.006). In addition, the rs9275572 polymorphism showed diverse relevance to the biochemical response: rs9275572AA+AG carriers having a 2.342 higher chance than rs9275572GG carriers with regard to the biochemical response in the dominant genetic model (95% CI: 1.510–3.632, P = 1.26 9 10 4) and in the recessive genetic models rs9275572AA carriers displayed a 2.430 higher than rs9275572AG+GG carriers (95% CI:
Table 3 Association of HLA-DQ rs9275572 with hepatitis B virus outcomes
Association of the HLA-DQ rs9275572A polymorphism with viral and biochemical responses
Recessive
In the dominant and recessive genetic models, carrying the HLA-DQ rs2856718G polymorphism, there was no relevance between viral response and LAM intervention, even when gender, age, smoking and alcohol were adjusted. There was, however, rs2856718G association with the biochemical response, and in the dominant genetic model, the rs2856718GG+AG carriers had a 1.753 higher chance than individuals carrying the rs2856718AA (OR95% CI: 1.145–2.684, P = 0.010), and rs2856718GG carriers had an increased possibility of an ALT response than rs2856718 AG + AA carriers (OR=1.913, 95% CI: 1.061– 3.450, P = 0.010). Logistic regression analysis suggested a definite rs2856718G relevance to biochemical response: rs2856718GG individuals having a 2.458 higher chance than rs2856718AA individuals (95% CI: 1.277–4.730, P = 0.007) in the dominant genetic model with adjusted OR values of 1.691 (95% CI: 1.093–6.615, P = 0.018) and 2.040 (95% CI: 1.104–3.772, P = 0.020) in the dominant and recessive genetic models (Table 4).
OR(95% CI)
Association of HLA-DQ rs2856718G polymorphism with viral and biochemical responses
5
The dominant and recessive models displayed adjusted OR values of 0.632 (P = 0.008) and 0.632(P = 0.008), respectively, and no rs9275572A association with the progression of CHB into LC was observed (Table 3).
5
HLA-DQ polymorphisms with HBV infection
0.018
0.020
0.020 4
4
1.26 9 10
4.23 9 10
0.018
0.010
1.913 (1.061,3.450) 2.040 (1.104,3.772)* 2.430 (1.126,5.246) 2.678 (1.182,6.068)*
0.029
1.126–5.246, P = 0.020). There was still rs9275572 polymorphism relevance with biochemical response after adjustment for gender, age, smoking and alcohol: in the codominant genetic model carrying rs9275572AG and rs9275572AA alleles had an increased possibility of a biochemical response (adjusted OR: 2.037, 95% CI: 1.231– 3.369, P = 0.006; adjusted OR: 3.245, 95% CI: 1.415– 7.440, P = 0.005) with an adjusted OR of 2.279 (95% CI: 1.442–3.602, P = 4.23 9 10 4) and 2.678(95% CI: 1.182–6.068, P = 0.018) in the dominant and recessive genetic models, respectively(Table 4).
DISCUSSION
(70.8) (23.4) (5.8) (36.1) (13.1)
(50.8)
121 40 10 93 66 24 rs9275572
(11.7) (39.2) (49.1) (20.2) (44.3) (35.5)
*OR and OR 95% CI adjusted for age, sex, smoking status and drinking status.
0.005
0.006
0.007
1.753 (1.145,2.684) 1.691 (1.093,2.615)* 2.342 (1.510,3.632) 2.279 (1.442,3.602)* 0.107
1.472 (0.920,2.355)* 2.458 (1.277,4.730)* 2.037 (1.231,3.369)* 3.245 (1.415,7.440)* 20 67 84 37
4.43 9 10 0.003 (75.5) (20.6) (3.9) (33.7) (11.9)
(54.4)
77 21 4 137 85 30 rs9275572
81
4
2.32 9 10 0.015
0.279 (13.7) (42.2) (44.1) (17.1) (41.7) (41.3)
Biochemical Response rs2856718 65
0.006
0.233
0.021 4
1.293 (0.673,2.483) 1.513 (0.766,2.991)* 3.311 (1.136,9.652) 4.684 (1.552,14.133)* 0.623
1.123 (0.706,1.787) 1.091 (0.677,1.756)* 2.585 (1.545,4.326) 2.599 (1.525,4.428)* 0.910
0.971 (0.582,1.620)* 1.492 (0.723,3.078)* 2.062 (1.153,3.687)* 5.553 (1.827,16.877)* 14 43 45 43 105 104 Viral Response rs2856718
GG AG
0.721
OR(95% CI)
Recessive
P OR(95% CI)
Dominant
P OR(95% CI)
Codominant
GG AA
AG
Nonresponse, n (%)
AA
Response, n (%)
Table 4 Association of HLA-DQ rs2856718 and rs9275572 with lamivudine therapy
0.439
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P
496
The present study for the first time demonstrated the obvious relevance of the HLA-DQ rs9275572 and rs2856718 polymorphisms with HBV susceptibility, spontaneous recovery and development of HBV-related diseases, as well as the viral and biochemical responses to LAM therapy. A GWAS reported by Kumar et al. [11] in April 2011 showed that rs9275572 had relevance with HCV infection and rs9275572A allele acted as a risk factor for HCV infection. Our study indicated that rs9275572A protected against HBV infection, benefited HBV spontaneous recovery, delayed the progression of CHB and LC to HCC and promoted viral and biochemical responses to LAM therapy. Our results are contrary to those of Kumar et al. [11], which perhaps resulted from the different pathogenetic mechanisms between HBV and HCV and subjects from different ethnic groups. Similar reports are available concerning HLA and HBV and HCV. HLA is protective for HCV infection in Japanese, whereas it is a susceptibility factor for chronic HBV in Koreans and Chinese [16]. In addition, in our study, we first revealed the clear relationship of rs9275572 with viral and biochemical responses to LAM intervention. Our analysed data showed that patients with CHB and LC carrying the rs9275572A allele had an increased possibility of responding to LAM treatment. In all respects, including HBV susceptibility, HBV natural clearance, LC and CHB progression to HCC and biochemical responses to HBV, HLADQ rs9275572A protected against HBV infection. The first evidence concerning the relationship between rs2856718 and HBV susceptibility was proposed by GWAS research. In this study, rs2856718A was indicated as a risk factor in HBV infection: a 0.47-allele frequency in healthy controls and a 0.59-allele frequency in patients infected with HBV. Our study suggested a 0.54-allele frequency for rs2856718A in the healthy population, whereas a 0.64-allele frequency was observed in the HBV patients. Our conclusion is consistent with the results in Japanese subjects: rs2856718G is beneficial against HBV infection and rs2856718A serves as a risk factor in HBV infection. In addition, the positive contribution to HBV natural clearance and slight but significant prevention against HCC development of rs2856718G were also evident in our © 2013 John Wiley & Sons Ltd
HLA-DQ polymorphisms with HBV infection study, and the results accord with another investigation which was conducted at the same time as ours in China. The investigation was also concerned with rs2856718 relevance to HBV natural clearance and HCC progression. On the basis of our conclusion about the protective effects of rs2856718G against HBV infection, we also found that it promoted a good response to LAM treatment. Many studies [17,18] have reported that host genetic factors play a major role in the persistence of HBV infection. Gene expression in host immune responses that actively interact with HBV and result in subsequent clinical outcome of the infection may be affected by both epigenetic changes and polymorphic variations [19,20]. HLA loci involved in the immune response are among the most numerous and diverse in the human genome. Classical HLA loci spanning 4 Mb on the short arm of chromosome 6p2124 include the class I and class II molecules presenting antigen to CD8+ and CD4+ T cells, respectively. Human HLA class II molecules are classified in three isotypes: HLADR, HLA-DQ, and HLA–DP, which are expressed as cell surface glycoproteins that bind and present short peptide epitopes to CD4+ T cells. The T cell helper response is crucial in HBV clearance, as evidenced by the vigorous humoral immune response observed in patients achieving spontaneous clearance [21,22]. Variability in the immune response against HBV infection is also associated with HLA polymorphisms in Chinese CHB [16]. Different HLA-DR polymorphisms have been detected to be significantly associated with either chronic carriage or rates of viral clearance. It was also reported that both HLA-DR and HLA-DQB heterozygosity were beneficial to clear hepatitis B virus
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[23]. Alternatively, homozygosity at these loci would limit the variability of antigenic peptide presentation for interactions between HLA molecules and T lymphocytes [24]. Our findings suggest that variations in HLA-DQ molecules would perhaps regulate virus clearance and chronic hepatitis B pathogenesis. Further study should focus on how these variants impact gene expression and function. Taken together, our study suggests that HLA-DQ loci were candidate susceptibility regions that had marker SNPs for both HBV clearance, HBV-related diseases and outcomes of LAM treatment in Han Chinese. Our study supports the notion that carriage of the HLA-DQ rs2856718G or rs9275572A allele was a protective factor for chronic HBV infection. In addition, our findings suggest that antigen presentation on the HLA-DQ molecules might be critical for virus elimination and play an important role in LAM treatment. These findings might provide further evidence to support the importance of the HLA’s role in HBV infection and provide potential implications for therapeutic guidance. Further study should focus on how these variants impact gene expression and function to validate the biological plausibility of these two SNPs in hepatitis B.
ACKNOWLEDGEMENT This study was supported by National Basic Research Program of China (30972516).
CONFLICT OF INTEREST The authors declare that they have no competing interests.
REFERENCES 1 Ocama P, Opio CK, Lee WM. Hepatitis B virus infection: current status. Am J Med 2005; 118(12): 1413. 2 Ott JJ, Stevens GA, Groeger J et al. Global epidemiology of hepatitis B virus infection: new estimates of age-specific HBsAg seroprevalence and endemicity. Vaccine 2012; 30 (12): 2212–2219. 3 Custer B, Sullivan SD, Hazlet TK et al. Global epidemiology of hepatitis B virus. J Clin Gastroenterol 2004; 38(10 Suppl. 3): S158–S168. 4 Godkin A, Davenport M, Hill AV. Molecular analysis of HLA class II associations with hepatitis B virus clearance and vaccine nonresponsiveness. Hepatology 2005; 41(6): 1383–1390. 5 Ben-Ari Z, Mor E, Papo O et al. Cytokine gene polymorphisms in patients infected with hepatitis B
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virus. Am J Gastroenterol 2003; 98 (1): 144–150. Zheng MH, Xiao DD, Lin XF et al. The tumour necrosis factor-alpha-238A allele increases the risk of chronic HBV infection in European populations. J Viral Hepat 2012; 19(2): e11–e17. Deng G, Zhou G, Zhai Y et al. Association of estrogen receptor alpha polymorphisms with susceptibility to chronic hepatitis B virus infection. Hepatology 2004; 40(2): 318–326. Kim YJ, Kim HY, Kim JS et al. Putative association of transforming growth factor-alpha polymorphisms with clearance of hepatitis B virus and occurrence of hepatocellular carcinoma in patients with chronic hepatitis B virus infection. J Viral Hepat 2010; 17(7): 518–526. Deng G, Zhou G, Zhang R et al. Regulatory polymorphisms in the pro-
moter of CXCL10 gene and disease progression in male hepatitis B virus carriers. Gastroenterology 2008; 134 (3): 716–726. 10 Mbarek H, Ochi H, Urabe Y et al. A genome-wide association study of chronic hepatitis B identified novel risk locus in a Japanese population. Hum Mol Genet 2011; 20(19): 3884–3892. 11 Kumar V, Kato N, Urabe Y et al. Genome-wide association study identifies a susceptibility locus for HCVinduced hepatocellular carcinoma. Nat Genet 2011; 43(5): 455–458. 12 El-Serag HB, Marrero JA, Rudolph L et al. Diagnosis and treatment of hepatocellular carcinoma. Gastroenterology 2008; 134(6): 1752–1763. 13 Bruix J, Sherman M. Management of hepatocellular carcinoma. Hepatology 2005; 42(5): 1208–1236.
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14 Kwak MS, Choi JW, Lee JS et al. Long-term efficacy of entecavir therapy in chronic hepatitis B patients with antiviral resistance to lamivudine and adefovir. J Viral Hepat 2011; 18(10): e432–e438. 15 Rapti I, Dimou E, Mitsoula P et al. Adding-on versus switching-to adefovir therapy in lamivudine-resistant HBeAg-negative chronic hepatitis B. Hepatology 2007; 45(2): 307–313. 16 Singh R, Kaul R, Kaul A et al. A comparative review of HLA associations with hepatitis B and C viral infections across global populations. World J Gastroenterol 2007; 13(12): 1770–1787. 17 Thursz M. Genetic susceptibility in chronic viral hepatitis. Antiviral Res 2001; 52(2): 113–116.
18 Wang FS. Current status and prospects of studies on human genetic alleles associated with hepatitis B virus infection. World J Gastroenterol 2003; 9(4): 641–644. 19 Duan ZP, Zhao XY, Huang DZ et al. RANTES gene single nucleotide polymorphisms and expression in patients with chronic hepatitis B virus infection. Chin Med J (Engl) 2005; 118(11): 909–914. 20 He Y, Zhao Y, Zhang S et al. Not polymorphism but methylation of class II transactivator gene promoter IV associated with persistent HBV infection. J Clin Virol 2006; 37 (4): 282–286. 21 An P, Winkler C, Guan L et al. A common HLA-DPA1 variant is a major determinant of hepatitis B
virus clearance in Han Chinese. J Infect Dis 2011; 203(7): 943–947. 22 Zhang M, Zhou J, Zhao T et al. Dissection of a circulating and intrahepatic CD4(+)Foxp3(+) T-cell subpopulation in chronic hepatitis B virus (HBV) infection: a highly informative strategy for distinguishing chronic HBV infection states. J Infect Dis 2012; 205(7): 1111–1120. 23 Thursz MR, Thomas HC, Greenwood BM et al. Heterozygote advantage for HLA class-II type in hepatitis B virus infection. Nat Genet 1997; 17(1): 11–12. 24 Zinkernagel RM. Immunology taught by viruses. Science 1996; 271(5246): 173–178.
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Table S1. Hardy-Weinberg test of the 2 SNPs in healthy and spontaneously recovered subjects.
Table S2. Baseline clinical characteristics of viral response and nonresponse subjects. Table S3. Baseline clinical characteristics of biochemical response and nonresponse subjects.
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doi:10.1111/jvh.12159
HLA-DQ polymorphisms with HBV infection: different outcomes upon infection and prognosis to lamivudine therapy X. Zhang,1 J. Jia,2 J. Dong,3 F. Yu,4 N. Ma,1 M. Li,1 X. Liu,1 W. Liu,1 T. Li1 and D. Liu1 1
Department of Epidemiology and Statistics, School of Public Health, Hebei Medical University, Shi Jiazhuang, China; 2Outpatient Clinic, Hebei
Medical University, Shi Jiazhuang, China; 3School of Biomedical Science, Chinese University of Hong Kong, Hong Kong, China; and 4Division of Gastroenterology, The Second Affiliated Hospital of Hebei Medical University, Shi Jiazhuang, China Received April 2013; accepted for publication July 2013
SUMMARY. Two recent genome-wide studies showed that
the single-nucleotide polymorphisms in the HLA-DQ region (rs2856718 and rs9275572) were associated with chronic hepatitis B virus infection and chronic hepatitis C virusassociated hepatocellular carcinoma in Japanese patients. We tested the effects of the two single-nucleotide polymorphisms for all major HBV outcomes and lamivudine treatment in Han Chinese. A total of 1649 samples were enrolled, and peripheral blood samples were collected in this study. The single-nucleotide polymorphisms in the HLA-DQ region were genotyped using matrix-assisted laser desorption/ionization time of flight mass spectrometry. Our study demonstrated the clear relevance of HLA-DQ rs2856718 and rs9275572 with HBV susceptibility, natural clearance and HBV-associated HCC. HLA-DQ rs2856718G and rs9275572A were strongly associated with decreased risk of chronic HBV infection (odds
INTRODUCTION Chronic hepatitis B virus (HBV) infection has become a major threat to human health owing to one-third of the population worldwide having a history of HBV infection and approximately 350 million living with chronic infection [1,2]. Data from epidemiological investigations showed
Abbreviations: ALT, alanine aminotransferase; CHB, chronic hepatitis B; CIB, HBV infection; CI, confidence interval; EDTA, ethylenediamine tetra-acetic acid; HBc, hepatitis B core; HBe, hepatitis B e; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; LAM, lamivudine; LC, liver cirrhosis; NC, natural clearance; OR, odds ratio. Correspondence: Dianwu Liu, PhD, Department of Epidemiology and Statistics, School of Public Health, Hebei Medical University, 361 Zhongshan East Road, Shi Jiazhuang, 050017, China. E-mail: [email protected] Xiaolin Zhang and Jinhai Jia contributed equally to this work.
© 2013 John Wiley & Sons Ltd
ratio = 0.641; P = 2.64 9 10 4; odds ratio = 0.627, P = 7.22 9 10 5) and HBV natural clearance (odds ratio = 0.610; P = 4.80 9 10 4; odds ratio = 0.714, P = 0.013). Moreover, rs9275572A was also associated with development of cirrhosis and hepatocellular carcinoma (odds ratio = 0.632, P = 0.008). In addition, we showed for the first time to our knowledge that rs9275572 was a predictor for lamivudine therapy (viral response: odds ratio = 2.599, P = 4.43 9 10 4; biochemical response: odds ratio = 2.279, P = 4.23 9 10 4). Our study suggested that HLA-DQ loci were associated with both HBV clearance and HBV-related diseases and outcomes of lamivudine treatment in Han Chinese. Keywords: HBV infection, HLA-DQ, lamivudine, polymorphisms.
a higher infection rate in Asia and Africa, with 5.3–12% of the hepatitis B surface antigen (HBsAg) carriers being Chinese, 8% living in Thailand and 10% in Africa [3]. Individuals infected with HBV present different responses: most recover spontaneously and 5% develop into chronic HBV infection. Similarly, different outcomes are observed among those suffering from chronic infection: 20% progress to liver cirrhosis (LC) and 5% into primary hepatocellular carcinoma (HCC). The complicated pathogenesis of HBV infection is attributable to the virus itself, immune state, genetic background of host and environmental condition. Among all the contributory factors, the host genetic background appears to serve as a major determinant of the different outcomes of HBV infection. Experimental results have revealed a series of genes potentially associated with HBV infection and natural clearance: including HLA, ESR1, TNF, TGFA, IL-18 and CXCL-10 [4–9]. The HLA complex is the first definitely
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identified genetic system showing strong association with HBV infection. A recent genome-wide association study performed by Japanese researchers indicated that the HLADQ polymorphism rs2856718 was significantly associated with chronic hepatitis B (CHB) [10], yet it remains unknown whether there is an association of HLA-DQ rs2856718 and prognosis of HBV infection in Chinese people. In addition, GWAS from Kumar et al. [11] in May 2011 suggested that the HLA-DQ polymorphism rs9275572 was relevant with HCV-associated HCC, but it is not completely understood whether HLA-DQ polymorphism rs9275572 is relevant to HBV infection and prognosis upon therapeutic intervention in Chinese people. This study was designed to explore the association of HLA-DQ polymorphisms rs2856718 and rs9275572 with HBV susceptibility, natural clearance and the development of HBV-associated diseases. In addition, further investigation was carried out for testing the relevance of the two HLA-DQ polymorphisms with prognosis after therapy with lamivudine (LAM).
PATIENTS AND METHODS Patients Patients were recruited from the Fifth Hospital of Shijiazhuang, the First and Second Affiliated Hospital of Hebei Medical University from January 2010 to January 2012. A total of 1649 samples were genotyped and analysed in this study. The samples included 507 cases of healthy controls, 350 cases of natural clearance, 484 cases of chronic HBV infection (225 cases of chronic hepatitis B and 259 cases of LC) and 308 cases of HBV-associated HCC. Those healthy people under investigation were selected according to the following criteria: no record of hepatitis B vaccine history was negative for HBsAg, hepatitis B e antibody (anti-HBe), hepatitis B core antibody (anti-HBc) and other HBV biomarkers, normal blood routine examination and biochemical parameters, no history of endocrine disorders, cardiovascular events, or liver- and kidney-associated diseases. HBV natural clearance (NC) was defined based on the following criteria: positive for hepatitis B surface antibody (anti-HBs) and anti-HBc definite absence of HBsAg accompanied by normal liver function and no history of HBV vaccination. Chronic hepatitis B (CHB) was divided into two groups according to HBeAg expression: HBeAgpositive and HBeAg-negative CHB. Cirrhosis was diagnosed based on clinical, biochemical, radiological or histological findings. HCC diagnosis was confirmed by a pathological examination and/or a-fetoprotein elevation (> 400 ng/mL) combined with imaging. The sequence of tests used to diagnose HCC depended on the size of the lesion: (i) those with a focal hepatic mass > 2 cm underwent with imaging wherein characteristic contrast enhancement features on the arterial phase with venous washout on an
MRI or CT could be demonstrated; (ii) those with a focal hepatic mass with atypical imaging findings, or a focal hepatic mass detected in a noncirrhotic liver, underwent a liver biopsy [12,13]. The requirements for LAM treatment were definite diagnosis of CHB or LC with a serum HBV DNA level of > 5 log copies/mL, a serum alanine aminotransferase (ALT) level two times higher than the upper limit of normal ALT range and LAM (100 mg/day) use for the first time. Those receiving LAM treatment were divided into two groups according to the virological response (DNA load < 3 log copies/mL or reduction in DNA load > 2 log copies/mL): viral response group and viral nonresponse group. Two other groups were obtained depending on the biochemical response (ALT concentration within normal levels): biochemical response group and biochemical nonresponsive group [14,15]. The information on all subjects under investigation included the following: baseline information (gender, age, history of smoking, drinking and HBV vaccine, etc.), historical disease background (condition and course of disease, with or without other disorders, family history), results of biochemical and serological testing (HBV markers, quantity of HBV DNA, ALT and AFP concentrations). The precise description of smoking and alcohol drinking was as follows: individuals who smoked one cigarette per day for over 1 year were defined as smokers, and those who consumed one or more alcohol drinks a week for over 6 months were considered alcohol drinkers. Informed consent was obtained from each subject, and the Institutional Review Board of Human Research of Hebei Medical University approved the study protocol.
Genotyping All participants were subjected to 2 mL venous blood collection under ethylenediamine tetra-acetic acid (EDTA) anticoagulation. A genomic DNA Purification Kit purchased from Promega was used for genomic DNA extraction and time of flight mass spectrometry technology from the American SEQUENOM company for all sample SNP genotyping. Primer design for the two SNP alleles was performed by the Hua Da Gene company with the aid of MassARRAYâ Assay Design 4.0 Software (Sequenom Inc., San Diego, CA, USA). Following quality control plan was employed for data reliability: (i) Negative control setting; (ii) Carrying out quality control criterion indoors; (iii) Genotyping in both replication sample sets had been performed in 5% of the total samples.
Statistical analysis Continuous variables of normal distribution were expressed as means SD (v s) and data of abnormal distribution expressed by median and interquartile range, M(Q). Cate© 2013 John Wiley & Sons Ltd
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gorical variables are presented as frequencies. Comparisons between continuous data were carried out using Student’s t-test and Wilcoxon test. The associations between categorical variables were evaluated using a Pearson chi-squared test. The chi-squared G test (goodness of fit) was employed to verify whether the proportions of the HLA-DQ rs2856718G and rs9275572A polymorphism genotypes were distributed in accordance with the Hardy–Weinberg equation (Table S1). Nonconditioned logistic regression was utilized to adjust involved factors such as age, gender, smoking, alcohol drinking, etc., and odds ratio (OR) and 95% confidence intervals (95% CI) were calculated. A Pvalue of 0.05 was considered significant in all analyses, and all P-values were two-sided. All analysis mentioned above were performed using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA).
(P > 0.05). There was a higher ratio of males in the CIB than in the healthy groups (67.7% vs 58.6%, P = 0.001), and the HCC group displayed a higher ratio of males than the CHB+LC group (79.8% vs 59.9%, P = 5.68 9 10 9). Concerning the ratio of females to males, there were no differences found between the NC and CIB group, LC and CHB group (P > 0.05). A similar ratio of smokers and drinkers was observed between the LC and CHB groups, with significance among the other three groups at P < 0.05 (Table 1). The viral response group had a lower ratio of males, smokers and drinkers than the viral nonresponse group (P < 0.05), along with similar levels of HBV DNA, ALT and AST before treatment (P > 0.05, Table S2). The biochemical response group presented with lower ratio of males, smokers and drinkers than the biochemical nonresponse group (Table S3).
RESULTS
Association of HLA-DQ rs2856718G polymorphism with HBV infection
Demographic and aetiological characteristics of the subjects The LC group had significantly higher mean age than the CHB group (51.11 11.12 vs 38.61 14.37, P = 2.33 9 10 23) and the HCC group higher than that of the CHB+LC group (55.96 9.06 vs 45.30 14.17, P = 9.52 9 10 35). There was no statistical significance between the healthy control and HBV infection (CIB) groups, NC group and CIB group with regard to age
The study of the healthy population and HBV infection cases indicated a strong association of rs2856718G with HBV susceptibility. In the dominant genetic model, GG genotype carriers and AG genotype carriers showed a lower risk of HBV infection than AA genotype carriers (OR: 0.636, P = 1.43 9 10 4), with the same relationship (OR: 0.52, P = 7.22 9 10 6) in the recessive genetic model. With adjustment for gender, age, smoking and alcohol, logistic regression analysis suggested an obvious relevance
Table 1 Demographic and aetiological characteristics of study subjects Gender (%) Characteristics HBV susceptibility CIB (n = 792) Healthy(n = 507) P OR(95% CI) HBV Clearance SR(n = 350) CIB (n = 792) P OR(95% CI) CHB?LC LC (n = 259) CHB (n = 225) P OR(95% CI) LC+CHB?HCC HCC (n = 308) LC+CHB(n = 484) P OR(95% CI)
Smokers (%)
Drinkers (%)
Age (mean SD)
Male
49.45 13.48 49.29 14.61 0.845
535(67.7) 256(32.3) 297(58.6) 210(41.4) 0.001 1.478(1.173–1.862)
355 (44.8) 437 (55.2) 141 (27.8) 366(72.2) 7.43 9 10 10 2.109(1.660–2.679)
395(49.9) 397(50.1) 171(33.7) 336(66.3) 1.03 9 10 8 1.955(1.552–2.463)
50.32 14.95 49.45 13.48 0.330
216(61.7) 134(38.3) 535(67.7) 256(32.4) 0.052 1.296(0.998,1.685)
101(28.9) 249(71.1) 355 (44.8) 437 (55.2) 3.79 9 10 7 2.003(1.528,2.624)
119 (34.0) 231(66.0) 395(49.9) 397(50.1) 6.66 9 10 7 1.931(1.487–2.508)
51.11 11.12 38.61 14.37 2.33 9 10 23
148(57.1) 111(42.9) 142(63.1) 83(36.9) 0.181 0.779(0.541,1.124)
84(32.4) 95(42.2)
101(39.0) 158(61.0) 103(45.8) 122(54.2) 0.132 0.757(0.527,1.088)
55.96 9.06 45.30 14.17 9.52 9 10 35
245(79.8) 62(20.2) 290(59.9) 194(40.1) 5.68 9 10 9 2.643(1.895,3.687)
176 (57.1) 132 (42.9) 179 (37.0) 305 63.0) 2.68 9 10 8 2.272(1.697,3.041)
191(62.0) 117(38.0) 204(42.1) 280(57.9) 5.02 9 10 8 2.241(1.673,3.002)
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Female
Yes
No
175(67.6) 130(57.8) 0.026 0.657(0.453,0.952)
Yes
No
0.032 0.099 0.730(0.547,0.974) 0.763(0.554,1.053)* 0.177 0.137 0.790(0.562,1.112)* 0.681(0.410,1.129)*
0.913 0.999 1.024(0.666,1.577)* 1.000(0.551,1.815)*
0.003 0.001 0.642(0.479,0.773)* 0.528(0.361,0.773)*
36 (11.7)
38 (14.7)
109 (13.8)
CHB+LC? HCC
CHB?LC
HBV natural clearance
HBV, hepatitis B virus; CIB, HBV infection; NC, HBV natural clearance; CHB, chronic hepatitis B; LC, liver cirrhosis; HCC, hepatocellular carcinoma; OR, odds ratio; CI, confidence interval. *OR and OR 95% CI adjusted for age, sex, smoking status and drinking status.
0.176 0.273 0.745(0.486,1.143) 0.766(0.475,1.234)*
0.786 0.962 0.933(0.567,1.537) 0.987(0.570,1.708)* 0.991(0.688,1.428) 1.018(0.679,1.528)*
0.962 0.931
4
0.545(0.416,0.714) 0.610(0.463,0.805)*
9.60 9 10 4.80 9 10
6
0.616(0.444,0.856) 0.688(0.492,0.962)*
0.004 0.029
5
0.520(0.390,0.694) 0.541(0.403,0.725)* 4
4
1.43 9 10 2.64 9 10 0.020 2.83 9 10 0.738(0.571,0.954)* 0.459(0.331,0.636)*
Healthy (n = 507) 163 225 119 (32.1) (44.4) (23.5) NC(n = 350) 101 177 72 (28.9) (50.6) (20.6) CHB (n = 225) 89 101 35 (39.6) (44.9) (15.6) CHB+LC(n = 484) 192 219 73 (39.7) (45.2) (15.1) 109 (13.8)
CIB (n = 792) 338 345 (42.7) (43.6) CIB(n = 792) 338 345 (42.7) (43.6) LC(n = 259) 103 118 (39.8) (45.6) HCC(n = 308) 146 126 (47.4) (40.9) HBV susceptibility
OR(95% CI) AG
GG
AG AA AA
GG
Codominant Controls, n (%)
Table 2 Association of HLA-DQ rs2856718 with hepatitis B virus outcomes
P
6
0.636(0.504,0.804) 0.641(0.505,0.814*
P OR(95% CI)
Dominant
Association of the HLA-DQ rs9275572A polymorphism with HBV infection There was relevance between rs9275572A and HBV susceptibility. With the healthy population as control group and HBV infection cases as case group, rs9275572A protects against HBV infection in the dominant and recessive genetic models (OR:0.596 and 0.436, P = 6.76 9 10 6; P = 4.31 9 10 5). In the codominant genetic models with adjustment for gender, age, smoking and alcohol, individuals carrying AA and AG alleles had a decreased risk of HBV infection with adjusted OR of 0.706 and 0.374 (P = 0.006; P = 6.30 9 10 6). The dominant and recessive genetic models were accompanied with 0.627 and 0.429 as adjusted OR (P = 7.22 9 10 5; P = 6.47 9 10 5). The study of HBV natural clearance cases as control group and HBV infection cases as case group indicated that rs9275572A was relevant to HBV natural clearance. In the dominant genetic model, rs9275572A was favourable in the course of HBV natural clearance (OR: 0.599, P = 7.23 9 10 5). Yet, there was no difference shown in the recessive genetic model with a P-value of 0.164 and OR value of 0.704. In the codominant genetic model subjected to adjustment for gender, age, smoking and alcohol, an adjusted OR value of 0.179 with a 0.02 P-value and 0.714 of adjusted OR value accompanied with a P-value of 0.013 in the dominant genetic model were obtained. In addition, rs9275572A association with progression of CHB and LC to HCC was found. Logistic regression analysis indicated lower risk of CHB and LC progression to HCC in individuals carrying AA and AG alleles when the CHB+LC was used as the control group and the HCC cases as the case group in the codominant genetic model, with adjusted OR values of 0.693 and 0.373 (P = 0.043; P = 0.011).
OR(95% CI)
Recessive
P
under codominant, dominant and recessive models (adjusted OR: 0.738, 95% CI: 0.571-0.954, P = 0.020; adjusted OR: 0.459, 95% CI: 0.331- 0.636, P = 2.83 9 10 6). When HBV infection was used as case group and HBV natural clearance as control group, analysis of results revealed the beneficial action of rs2856718G on HBV natural clearance in the dominant (OR: 0.545, 95% CI: 0.416-0.714, P = 9.60 9 10 6) and recessive genetic models (OR: 0.616, 95% CI: 0.444–0.856, P = 0.004). After adjustment for gender, age, smoking and alcohol, a similar association still remained under the codominant, dominant and recessive genetic models (adjusted OR: 0.642,95% CI: 0.479–0.773, P = 0.003; adjusted OR = 0.528, 95% CI: 0.361–0.773, P = 0.001; adjusted OR: 0.610, 95% CI: 0.463–0.805, P = 4.80 9 10 4; OR: 0.688, 95% CI: 0.492–0.962, P = 0.029). There was no association detected between rs2856718G with LC development from CHB and HCC development from CHB and LC in the three genetic models (Table 2).
7.22 9 10 4.07 9 10
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Cases, n (%)
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0.002 0.008 0.616(0.456,0.833) 0.632(0.451,0.886)*
0.754 0.442 0.944(0.658,1.355) 0.853(0.570,1.278)*
0.043 0.011 0.693(0.486,0.988)* 0.373(0.175,0.796)*
0.320 0.696 0.807(0.528,1.232)* 1.183 (0.509,2.751)*
0.020 0.162 0.719(0.544,0.949)* 0.689(0.408,1.162)*
212 (68.8)
151 (58.3)
173 (49.4)
CHB+LC? HCC
CHB?LC
HBV natural clearance
495
HBV, hepatitis B virus; CIB, HBV infection; NC, HBV natural clearance; CHB, chronic hepatitis B; LC, liver cirrhosis; HCC, hepatocellular carcinoma; OR, odds ratio; CI, confidence interval. *OR and OR 95% CI adjusted for age, sex, smoking status and drinking status.
0.104 0.024 0.573(0.290,1.130) 0.420(0.198,0.890)*
0.292 0.549 1.485(0.709,3.110) 1.287(0.564,2.941)*
0.164 0.391 0.704(0.428,1.156) 0.801(0.482,1.331)* 5
7.23 9 10 0.013 0.599(0.465,0.772) 0.714(0.547,0.932)*
5
4.31 9 10 6.47 9 10 0.006 6.30 9 10 0.706(0.553,0.903)* 0.374(0.244,0.573)*
Healthy (n = 505) 60 196 249 (11.9) (38.8) (49.3) CIB(n = 792) 44 257 491 (5.6) (32.4) (62.0) CHB (n = 225) 12 85 128 (5.3) (37.8) (56.9) CHB+LC (n = 484) 32 173 279 (6.6) (35.7) (57.6) 491 (62.0)
CIB (n = 792) 44 257 (5.6) (32.4) NC(n = 350) 27 150 (7.7) (42.9) LC(n = 259) 20 88 (7.7) (34.0) HCC(n = 308) 12 84 (3.9) (27.3) HBV susceptibility
AG
GG
AG AA AA
GG
OR(95% CI)
P
6
0.596(0.476,0.747) 0.627(0.498,0.790)*
6.76 9 10 7.22 9 10
6
0.436(0.291,0.655) 0.429(0.283,0.650)*
P P OR(95% CI)
Dominant Codominant Controls, n (%) Cases, n (%)
Our study suggested that the rs9275572A allele polymorphism had relevance with the viral response to LAM therapy. In the dominant and recessive genetic models, individuals carrying the rs9275572A allele had a higher chance of responding to LAM in relation to viral load(OR: 2.585, 95% CI: 1.545–4.326, P = 2.32 9 10 4; OR: 3.311, 95% CI: 1.136–9.652, P = 0.021). After adjustment for gender, age, smoking and alcohol, there was an association of rs9275572A with the viral response in the codominant genetic models (adjusted OR: 2.062, 95% CI: 1.153–3.687, P = 0.015; adjusted OR: 5.553, 95% CI: 1.827–16.877, P = 0.003). In the dominant and recessive genetic models, rs9275572AA and rs9275572AG provided increased chances of responding to LAM therapy in relation to the viral response (adjusted OR: 2.599, 95% CI: 1.525–4.428, P = 4.43 9 10 4; adjusted OR: 4.684, 95% CI: 1.552–14.133, P = 0.006). In addition, the rs9275572 polymorphism showed diverse relevance to the biochemical response: rs9275572AA+AG carriers having a 2.342 higher chance than rs9275572GG carriers with regard to the biochemical response in the dominant genetic model (95% CI: 1.510–3.632, P = 1.26 9 10 4) and in the recessive genetic models rs9275572AA carriers displayed a 2.430 higher than rs9275572AG+GG carriers (95% CI:
Table 3 Association of HLA-DQ rs9275572 with hepatitis B virus outcomes
Association of the HLA-DQ rs9275572A polymorphism with viral and biochemical responses
Recessive
In the dominant and recessive genetic models, carrying the HLA-DQ rs2856718G polymorphism, there was no relevance between viral response and LAM intervention, even when gender, age, smoking and alcohol were adjusted. There was, however, rs2856718G association with the biochemical response, and in the dominant genetic model, the rs2856718GG+AG carriers had a 1.753 higher chance than individuals carrying the rs2856718AA (OR95% CI: 1.145–2.684, P = 0.010), and rs2856718GG carriers had an increased possibility of an ALT response than rs2856718 AG + AA carriers (OR=1.913, 95% CI: 1.061– 3.450, P = 0.010). Logistic regression analysis suggested a definite rs2856718G relevance to biochemical response: rs2856718GG individuals having a 2.458 higher chance than rs2856718AA individuals (95% CI: 1.277–4.730, P = 0.007) in the dominant genetic model with adjusted OR values of 1.691 (95% CI: 1.093–6.615, P = 0.018) and 2.040 (95% CI: 1.104–3.772, P = 0.020) in the dominant and recessive genetic models (Table 4).
OR(95% CI)
Association of HLA-DQ rs2856718G polymorphism with viral and biochemical responses
5
The dominant and recessive models displayed adjusted OR values of 0.632 (P = 0.008) and 0.632(P = 0.008), respectively, and no rs9275572A association with the progression of CHB into LC was observed (Table 3).
5
HLA-DQ polymorphisms with HBV infection
0.018
0.020
0.020 4
4
1.26 9 10
4.23 9 10
0.018
0.010
1.913 (1.061,3.450) 2.040 (1.104,3.772)* 2.430 (1.126,5.246) 2.678 (1.182,6.068)*
0.029
1.126–5.246, P = 0.020). There was still rs9275572 polymorphism relevance with biochemical response after adjustment for gender, age, smoking and alcohol: in the codominant genetic model carrying rs9275572AG and rs9275572AA alleles had an increased possibility of a biochemical response (adjusted OR: 2.037, 95% CI: 1.231– 3.369, P = 0.006; adjusted OR: 3.245, 95% CI: 1.415– 7.440, P = 0.005) with an adjusted OR of 2.279 (95% CI: 1.442–3.602, P = 4.23 9 10 4) and 2.678(95% CI: 1.182–6.068, P = 0.018) in the dominant and recessive genetic models, respectively(Table 4).
DISCUSSION
(70.8) (23.4) (5.8) (36.1) (13.1)
(50.8)
121 40 10 93 66 24 rs9275572
(11.7) (39.2) (49.1) (20.2) (44.3) (35.5)
*OR and OR 95% CI adjusted for age, sex, smoking status and drinking status.
0.005
0.006
0.007
1.753 (1.145,2.684) 1.691 (1.093,2.615)* 2.342 (1.510,3.632) 2.279 (1.442,3.602)* 0.107
1.472 (0.920,2.355)* 2.458 (1.277,4.730)* 2.037 (1.231,3.369)* 3.245 (1.415,7.440)* 20 67 84 37
4.43 9 10 0.003 (75.5) (20.6) (3.9) (33.7) (11.9)
(54.4)
77 21 4 137 85 30 rs9275572
81
4
2.32 9 10 0.015
0.279 (13.7) (42.2) (44.1) (17.1) (41.7) (41.3)
Biochemical Response rs2856718 65
0.006
0.233
0.021 4
1.293 (0.673,2.483) 1.513 (0.766,2.991)* 3.311 (1.136,9.652) 4.684 (1.552,14.133)* 0.623
1.123 (0.706,1.787) 1.091 (0.677,1.756)* 2.585 (1.545,4.326) 2.599 (1.525,4.428)* 0.910
0.971 (0.582,1.620)* 1.492 (0.723,3.078)* 2.062 (1.153,3.687)* 5.553 (1.827,16.877)* 14 43 45 43 105 104 Viral Response rs2856718
GG AG
0.721
OR(95% CI)
Recessive
P OR(95% CI)
Dominant
P OR(95% CI)
Codominant
GG AA
AG
Nonresponse, n (%)
AA
Response, n (%)
Table 4 Association of HLA-DQ rs2856718 and rs9275572 with lamivudine therapy
0.439
X. Zhang et al.
P
496
The present study for the first time demonstrated the obvious relevance of the HLA-DQ rs9275572 and rs2856718 polymorphisms with HBV susceptibility, spontaneous recovery and development of HBV-related diseases, as well as the viral and biochemical responses to LAM therapy. A GWAS reported by Kumar et al. [11] in April 2011 showed that rs9275572 had relevance with HCV infection and rs9275572A allele acted as a risk factor for HCV infection. Our study indicated that rs9275572A protected against HBV infection, benefited HBV spontaneous recovery, delayed the progression of CHB and LC to HCC and promoted viral and biochemical responses to LAM therapy. Our results are contrary to those of Kumar et al. [11], which perhaps resulted from the different pathogenetic mechanisms between HBV and HCV and subjects from different ethnic groups. Similar reports are available concerning HLA and HBV and HCV. HLA is protective for HCV infection in Japanese, whereas it is a susceptibility factor for chronic HBV in Koreans and Chinese [16]. In addition, in our study, we first revealed the clear relationship of rs9275572 with viral and biochemical responses to LAM intervention. Our analysed data showed that patients with CHB and LC carrying the rs9275572A allele had an increased possibility of responding to LAM treatment. In all respects, including HBV susceptibility, HBV natural clearance, LC and CHB progression to HCC and biochemical responses to HBV, HLADQ rs9275572A protected against HBV infection. The first evidence concerning the relationship between rs2856718 and HBV susceptibility was proposed by GWAS research. In this study, rs2856718A was indicated as a risk factor in HBV infection: a 0.47-allele frequency in healthy controls and a 0.59-allele frequency in patients infected with HBV. Our study suggested a 0.54-allele frequency for rs2856718A in the healthy population, whereas a 0.64-allele frequency was observed in the HBV patients. Our conclusion is consistent with the results in Japanese subjects: rs2856718G is beneficial against HBV infection and rs2856718A serves as a risk factor in HBV infection. In addition, the positive contribution to HBV natural clearance and slight but significant prevention against HCC development of rs2856718G were also evident in our © 2013 John Wiley & Sons Ltd
HLA-DQ polymorphisms with HBV infection study, and the results accord with another investigation which was conducted at the same time as ours in China. The investigation was also concerned with rs2856718 relevance to HBV natural clearance and HCC progression. On the basis of our conclusion about the protective effects of rs2856718G against HBV infection, we also found that it promoted a good response to LAM treatment. Many studies [17,18] have reported that host genetic factors play a major role in the persistence of HBV infection. Gene expression in host immune responses that actively interact with HBV and result in subsequent clinical outcome of the infection may be affected by both epigenetic changes and polymorphic variations [19,20]. HLA loci involved in the immune response are among the most numerous and diverse in the human genome. Classical HLA loci spanning 4 Mb on the short arm of chromosome 6p2124 include the class I and class II molecules presenting antigen to CD8+ and CD4+ T cells, respectively. Human HLA class II molecules are classified in three isotypes: HLADR, HLA-DQ, and HLA–DP, which are expressed as cell surface glycoproteins that bind and present short peptide epitopes to CD4+ T cells. The T cell helper response is crucial in HBV clearance, as evidenced by the vigorous humoral immune response observed in patients achieving spontaneous clearance [21,22]. Variability in the immune response against HBV infection is also associated with HLA polymorphisms in Chinese CHB [16]. Different HLA-DR polymorphisms have been detected to be significantly associated with either chronic carriage or rates of viral clearance. It was also reported that both HLA-DR and HLA-DQB heterozygosity were beneficial to clear hepatitis B virus
497
[23]. Alternatively, homozygosity at these loci would limit the variability of antigenic peptide presentation for interactions between HLA molecules and T lymphocytes [24]. Our findings suggest that variations in HLA-DQ molecules would perhaps regulate virus clearance and chronic hepatitis B pathogenesis. Further study should focus on how these variants impact gene expression and function. Taken together, our study suggests that HLA-DQ loci were candidate susceptibility regions that had marker SNPs for both HBV clearance, HBV-related diseases and outcomes of LAM treatment in Han Chinese. Our study supports the notion that carriage of the HLA-DQ rs2856718G or rs9275572A allele was a protective factor for chronic HBV infection. In addition, our findings suggest that antigen presentation on the HLA-DQ molecules might be critical for virus elimination and play an important role in LAM treatment. These findings might provide further evidence to support the importance of the HLA’s role in HBV infection and provide potential implications for therapeutic guidance. Further study should focus on how these variants impact gene expression and function to validate the biological plausibility of these two SNPs in hepatitis B.
ACKNOWLEDGEMENT This study was supported by National Basic Research Program of China (30972516).
CONFLICT OF INTEREST The authors declare that they have no competing interests.
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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Table S1. Hardy-Weinberg test of the 2 SNPs in healthy and spontaneously recovered subjects.
Table S2. Baseline clinical characteristics of viral response and nonresponse subjects. Table S3. Baseline clinical characteristics of biochemical response and nonresponse subjects.
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