Human leukocyte antigen-DP loci are associated with the persistent infection of hepatitis B virus in Chinese population

LING Yun, LIAO Xiang-Wei, LI Xin-Hua, HAN Yue, YANG Zhi-Tao, KONG Xiao-Fei, GU Lei-Lei, YU De-Ming, YAO Bi-Lian, ZHANG Dong-Hua, JIN Gen-Di, GONG Qi-Ming, ZHANG Xin-Xin

1. Department of Infectious Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; 2. Institute of Infectious and Respiratory Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; 3. Sino-French Laboratory of Life Science and Genomics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

Hepatitis B virus (HBV) infection is a major global health problem and the most serious type of viral hepatitis affects 350 to 400 million persons worldwide[1-3]. In China and other parts of Asia, 8% to 10% of the adult population, who were infected with HBV during childhood, are chronically infected[4]. About 90% of newborns infected perinatally will develop chronic HBV infection (CHB); 25% of adults infected during childhood become chronically infected; only 10% of healthy adults who infected with HBV will develop CHB[5]. HBV e antigen (HBeAg) status distinguishes two additional categories of CHB infection; HBeAg-positive and HBeAg-negative patients have different natural histories, outcomes, and need different treatments[6-8]. It can cause chronic liver disease and puts people at high risk of death from cirrhosis of the liver and liver cancer. The annual incidence of cirrhosis has been estimated to be 2% to 6% for HBeAg-positive and 8% to 10% for HBeAg-negative patients[9-11]. The study from Taiwan found the adjusted relative risk of hepatocellular carcinoma (HCC) was 6- to 7-fold higher among HBeAg-positive patients than in HBeAg-negative patients[12].

Host genetic factors play a major role in determining differential susceptibility to HBV infection[13,14]. A twin study in Taiwan indicated that host genetic background influences infection outcomes[15]. Especially, polymorphisms in cytokines and immune response-related genes could influence the outcomes of HBV infection; genetic variants in interferon γ (IFN-γ), tumor necrosis factor (TNF), vitamin D receptor (VDR), estrogen receptor α (ESR1) and several human leukocyte antigen (HLA) loci were shown to associate with CHB[16-19]. Interferon α/β receptor 2 (IFNAR2) may play an important role in determining IFN response and clinical phenotypes of HBV infection in the Han Chinese population[20]. Recently, a genome-wide association study (GWAS), which included 1 300 HBV-infected patients and 2 100 controls, identified that variants in the HLA-DP loci are strongly associated with a risk of persistent infection with HBV in Japanese and Thai individuals[21]. However, no data can be obtained in Chinese HBV-infected patients about these HLA-DP loci.

Another GWAS has confirmed that the rs12979860 variation, a polymorphism on chromosome 19, is associated with the response to anti-HCV infection treatment, among patients of European ancestry, African Americans[22]and Chinese (Antiviral Therapy, 2010). The rs12979860 variation is located 3 kb upstream of the interleukin 28B (IL-28B) gene, which encodes one type III interferon—IFN-λ3; IL-28A and IL-29 (also called IFN-λ2 and IFN-λ1, respectively) are the other two type III interferons and are clustered on human chromosome 19q13 with IL-28B[23]. It seems likely that the rs12979860 variation is related to the different expression levels of IFN-λs[24-27]. Replication of HBV in a human hepatoma cell line was reduced by approximately 30% following treatment with a high concentration of IFN-λ[28]. Recently, the data from Martin’s group showed that the C/C genotype of rs12979860 was not associated with HBV recovery[29]. However, no data are available to demonstrate the polymorphism of IL-28B in Chinese HBV-infected subjects.

The aim of this study was to investigate whether the HLA-DP loci and IL-28B were associated with different outcomes of HBV infection in the Han Chinese population.

1 Methods and patients

1.1 Patients

A total of 378 Han Chinese subjects were enrolled from Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. Among them, 185 had persistent HBV infection before treatment and 193 had previous self-limited HBV (SLHBV) infection with no family history. The diagnostic criteria for persistent HBV infection were based on the presence of HBV surface antigen (HBsAg) and anti-core IgG-antibody (anti-HBc), and the absence of anti-HBs antibody for more than 6 months. The mean time from the presumed onset of HBV infection was defined as the first documented seropositivity for HBsAg with or without elevated serum liver enzyme. An HBeAg-positive patient is characterized by the presence of HBeAg, high levels of HBV DNA, with normal alanine aminotransferase (ALT) or not. An HBeAg-negative patient is characterized by absence of HBeAg, presence of anti-HBV e antibody (anti-HBe), with detectable or undetectable HBV DNA in serum. SLHBV infection was defined as being positive for anti-HBs and anti-HBc, without HBV vaccination. Serum HBV DNA was analyzed to exclude patients with occult HBV infection. Subjects negative for all HBV markers were not included in the study as these subjects were unlikely to have been exposed to HBV. If they had been exposed to the virus, it would be impossible to predict their outcomes. Patients with concurrent hepatitis A, C, D, E or human immunodeficiency virus (HIV) infection were excluded from the study. Patients with liver disease caused by other factors, such as excess alcohol consumption and autoimmune hepatitis, were also excluded from the study. Our study conforms to the ethical guidelines of the 2004 Declaration of Helsinki.

Fig.1 Positions of analyzed SNPs and primers

1.2 Methods

1.2.1 Genomic DNA extraction Whole blood was taken from patients and genomic DNA was extracted using the QIAGEN DNA extraction kit (Hilden, Germany) according to the manufacturer’s instructions. The concentration of genomic DNA in each sample was determined by an ultraviolet spectrophotometer (Eppendorf, Germany) and samples were stored at-80 ℃ until genetic polymorphism analyses were performed.

1.2.2 Genotyping of gene polymorphism The rs9277535 near HLA-DPB1，rs3077 near HLA-DPA1 and rs12979860 near IL-28B were determined by DNA sequencing. First, polymerase chain reaction (PCR) was performed to amplify the DNA fragment encompassing single nucleotide polymorphisms (SNPs) using the primers (Tab.1). For the high mutation in HLA loci[30], we designed a forward consensus primer and two reverse primers for rs92775353, one of which is specific for the wild type and the other for the mutated alleles. For the same reason, we designed two forward primers and a reverse consensus primer for rs3077. Amplification was performed using GoTaq DNA polymerase (Promega, Madison, WI, USA) in 50 μl reaction mixtures. Samples were cycled under the following conditions: 95 ℃ for 2 min, then 40 cycles of 95 ℃ for 30 s, 56 ℃ for 45 s, and 72 ℃ for 1 min, followed by 72 ℃ for 10 min to generate 297-bp amplicons. PCR products were sequenced using the sequencing primers (Tab.1) using Big Dye Terminator chemistry on an ABI 3730 sequencer (Applied Biosystems, Foster City, CA, USA). Samples were genotyped according to the sequencing results.

1.2.3 Serological test Five milliliters of whole blood samples were collected from each subject, and the sera were stored at -20 ℃. HBsAg, anti-HBs, HBeAg, anti-HBe, total anti-HBc or anti-HBc IgM were monitored using commercially available kits (AxSYM, Abbott, Abbott Park, IL, USA), and HBV DNA was detected using TaqMan real-time PCR (PiJi Bio-Tech Company, Shenzhen, China).

1.3 Statistical analysis

Differences between observed and expected counts according to genotype were determined using the chi-squared test or Fisher’s exact test if needed. Odds ratio (OR) and 95% confidence interval (95%CI) were calculated. All analyses were carried out using statistical analysis system version 8.0 software (SAS Institute Inc., Cary, NC, USA). In all tests, a value ofP<0.05 was considered statistically significant.

2 Results

2.1 Demographic and clinical features

Demographic and clinical features of all patients are shown in Tab.2. 193 SLHBV subjects had normal ALT and undetectable HBV DNA levels. 185 patients with persistent HBV infection were with abnormal ALT (318.8±419.8), and detectable levels of HBV DNA in serum. Among 193 SLHBV subjects and CHB patients, the female to male ratio was 1.14∶1 (103∶90) and 0.53∶1 (64∶121). Among SLHBV individuals, the rs9277535 AG genotype was the main genotype (AA: 24.4%; AG: 45.6%; GG: 30.1%). The rs3077 CT and CC genotypes were the main genotypes (CC: 43.5%; CT: 46.6%; TT: 9.8%). The rs12979860 CC genotype was the main genotype (CC: 90.2%; CT: 9.3%; TT: 0.5%). In CHB patients, the rs9277535 GG (AA: 10.8%; AG: 42.1%; GG: 47.2%), rs3077 CC (CC: 54.1%; CT: 41.1%; TT: 4.9%), rs12979860 CC (CC: 86.5%; CT: 13.5%; TT: 0%) were the main genotypes.
Fig.2 General characteristics of 378 subjects (n, %)

SLHBV(n=193)Persistent HBV infectionTotal (n=185)HBeAg(+) (n=91)HBeAg(-) (n=94)HBeAg(-)DNA(+)(n=33)DNA(-) (n=61)Sex (female/male)103/9064/12128/6336/5813/2023/38Age (mean±SD)51.6±16.438.0±13.034.7±12.041.1±13.742.8±11.740.1±13.8ALT (×ULN)(mean±SD)Normal318.8±419.8370.7±522.5257.7±235.5300.7±291.5228.6±187.9HBV DNA (Log10)(mean±SD)Undetectable4.63±2.045.4±2.03.78±1.665.42±1.57Undetectablers9277535(HLA-DPB1)rs3077(HLA-DPA1)rs361527(HLA-DPA1)rs12979860(IL-28B)AA47(24.4)20(10.8)8 (8.8)12 (12.8)6(18.2)6(9.8)AG88(45.6)78(42.1)41 (45.1)37 (39.4)12(36.4)25(41.0)GG58(30.1)87(47.2)42 (46.2)45 (47.9)15(45.5)30(49.2)CC84(43.5)100(54.1)51 (56.0)49 (52.1)19(57.6)30(49.2)CT90(46.6)76(41.1)38 (41.8)38 (40.4)14(42.4)24(39.3)TT19(9.8)9(4.9)2 (2.2)7 (7.4)0(0.0)7(11.5)?P/P84(43.5)98(53.0)51 (56.0)47 (50.0)18(54.5)29(47.5)P/N92(47.7)80(43.2)38 (41.8)42 (44.7)15(45.5)27(44.3)N/N17(8.8)7(3.8)2 (2.2)5 (5.3)0(0.0)5(8.2)CC174(90.2)160(86.5)78 (85.7)82 (87.2)29(87.9)53(86.9)CT18(9.3)25(13.5)13 (14.3)12 (12.8)4(12.1)8(13.1)TT1(0.5)0(0.0)0 (0.0)0 (0.0)0(0.0)0(0.0)

*P=ATAAATGTTGA, N=without insertion.

In 185 CHB patients, 91 patients were HBeAg-positive, and 94 patients were HBeAg-negative. In 94 HBeAg-negative patients, 33 patients were HBV DNA-positive in serum, and 61 patients with undetectable HBV DNA.

2.2 Association of SNP allele with outcomes of HBV infection

The association of SNP alleles and outcomes of HBV infection are shown in Tab.3 and Fig.1. The rs9277535 near HLA-DPB1 G was strongly associated with CHB (P=0.000 018 1,OR=1.905); not only in HBeAg-positive CHB patients (P=0.000 4,OR=1.956), but also in HBeAg-negative CHB patients (P=0.000 9,OR=1.857), and HBeAg-negative CHB individuals without detectable levels of HBV DNA in serum (P=0.001 1,OR=2.05). The rs3077 near HLA-DPA1 C was associated with CHB (P=0.020 6,OR=0.686 5) and HBeAg-positive CHB (P=0.014 3,OR=0.604 7) infection status. Meanwhile, a genetic variation of insertion-deletion (INDEL) polymorphism near HLA-DPA1 (rs361527, -/ATAAATGTTGA) was associated with CHB (P=0.030 7,OR=0.702 8) and HBeAg-positive CHB (P=0.023 3,OR=0.619) infection status. 84 (43.5%) of SLHBV subjects and 98 (53.0%) of CHB patients had the INDEL sequence in both strands from double-stranded DNA. 92 (47.7%) of SLHBV and 80 (43.2%) of CHB had the INDEL sequence in one strand of double-stranded DNA. The rest of the subjects had no INDEL sequences. The rs12979860 genotype near IL-28B had no correlation with CHB, whether HBeAg-positive or HBeAg-negative. Patients with rs9277535 allele A, rs3077 allele T, and the lack of ATAAATGTTGA were easier to get rid of virus after HBV infection.

2.3 Linkage disequilibrium testing of rs361527 and rs3077 near HLA-DPA1

For SNPs determined by DNA sequencing and highly variance in HLA loci, there are several SNP loci in the sequenced fragment. We found that the INDEL of rs361527 (-/ATAAATGTTGA) was also associated with CHB and HBeAg-positive CHB infection status. Moreover, the correlation of SNPs were studied in CHB patients and SLHBV subjects (Tab.4). The rs361527 was in accordance with rs3077 genotypes (r2=0.949,D′=0.981). In our data, we found 181 rs3077CC subjects had the INDEL sequence of ATA-AATGTTGA in double-stranded DNA. 165 rs3077 CT subjects were with the INDEL sequence in one strand of double-stranded DNA.

A: The rs9277535 near HLA-DPB1 G was strongly associated with CHB, HBeAg-positive CHB, HBeAg-negative CHB, and HBeAg-negative CHB with undetectable HBV DNA infection status. B: The rs3077 near HLA-DPA1 C was associated with CHB and HBeAg-positive CHB infection status. C: The INDEL polymorphism (rs361527, -/ATAAATGTTGA) was associated with CHB and HBeAg-positive CHB infection status (P=ATAAATGTTGA, N=without insertion).

Fig.3 Allele distributions of rs9277535, rs3077 and IL-28B in patients with self-limited or persistent HBV infection (n, %)

*P=ATAAATGTTGA, **N=without insertion.

Fig.4 Linkage disequilibrium testing of SNPs/INDEL

24 rs3077 TT subjects showed no INDEL sequence. Only 8 subjects showed no accordance between rs3077 and the INDEL sequence. rs3077 and rs361527 had a low accordance with rs9277535 genotypes (r2=0.317,D′=0.708;r2=0.309,D′=0.703). rs12979860 nearly IL-28B had no correlation with other SNPs.

2.4 Association study of all possible gene combinations of the two independent SNPs

In order to determine the combined contribution to the risk of persistent HBV infection, logistic regression analysis was conducted (Tab.5). The rs9277535 GG genotype and rs3077 CC genotype, which correlated with persistent HBV infection, were set as the reference. The AA+TT, AA+CT and AG+CC combination showed no correlation with CHB (P=0.000 2,OR=0.117 6;P=0.001 9,OR=0.277 8; andP=0.029 3,OR=0.466 7). So the patients with genotype combinations of AA+TT, AA+CT and AG+CC are easier to get rid of virus when suffered from HBV infection.

Fig.5 Association study of all possible gene combinations of the two independent HBV-associated SNPs

3 Discussion

Kamatani’s study demonstrated that genetic variants in HLA-DP genes are strongly associated with CHB in Japanese and Thai patients. Control groups consisted of individuals with diseases other than chronic hepatitis[21]. However, there was no direct evidence that these controls ever suffered from HBV and cleared the virus. In our study, we detected two SNPs in CHB patients and SLHBV subjects. SLHBV individuals were those who had recovered from HBV infection but had evidence of previous HBV infection, without previous HBV vaccination. The control cohort was more reasonable for studying the differences in different outcomes after HBV infection. Therefore, it is reasonable that theORof rs9277535 in our study (1.905) is higher than in Yoichiro’s study (0.56). Furthermore, we compared the differences among different types of CHB by Mantel-Haenszel test.

HBV infection is an immune-based disorder in which the extent of disease as well as the frequency and quality of virologic response are profoundly influenced by the depth of the host immunologic response[31,32]. CHB has different outcomes, including HBeAg-positive and HBeAg-negative. HBeAg-positive CHB is accompanied by high-level HBV replication and spontaneous sero-conversion from HBeAg-positive to anti-HBe-positive infection which coincides with a reduction in HBV replication and clinical improvement[1,6-8]. Patients with HBeAg-negative CHB trend to have progressive liver injury, fluctuating ALT activity, and lower levels of HBV DNA than patients with HBeAg-reactive HBV infection. HBeAg-negative CHB patients include two possibilities: one is with normal ALT and low or undetectable serum HBV DNA, the other is with elevated ALT and detectable HBV DNA[33]. Our results showed that HBeAg-positive and HBeAg-negative CHB patients had different rates of genotype frequency and allele frequency. HBeAg-positive patients showed a larger difference from SLHBV than HBeAg-negative patients, especially on rs3077 and the INDEL sequence of ATAAATGTTGA. Moreover, HBeAg-negative patients, with or without virus in serum, showed different immune states that may depend on the host genetic factors.

In humans, major histocompatibility complex (MHC) class II molecules are encoded by three different loci, HLA-DR, HLA-DQ and HLA-DP, and polymorphism is a notable feature of MHC class II genes[34]. HLA class II proteins are cell-surface molecules that present antigens to T cells, and their expressional regulation is crucial to the immune reaction. Sequence variation at the regulatory region can directly affect the gene expression level[35,36]. Variations in different HLA-DP loci maybe associate with different immune states, in CHB patients including HBeAg-positive and HBeAg-negative. Total CHB and HBeAg-positivity were associated with HLA-DPB1-rs9277535, HLA-DPA1-rs3077 and the INDEL sequence of ATAAATGTTGA. However, in HBeAg-negative patients, the association showed contradiction. Total HBeAg-negative and having no virus in serum were associated with HLA-DPB1-rs9277535. The INDEL sequence of ATAAATGTTGA and rs3077 showed no correlation among HBeAg-negative patients. HLA-DPB1-rs9277535 was a stronger tag than HLA-DPA1-rs3077, and the INDEL sequence of ATAAATGTTGA was associated with CHB. Patients with the combination of rs9277535 and rs3077 (AA+TT, AA+CT and AG+CC) were more likely to get rid of virus during HBV infection.

IL-28B rs12979860 variation was found to be significantly associated with the response to poly-ethylene glycol (PEG)-IFN-α and ribavirin for patients with chronic genotype 1 hepatitis C virus (HCV) infection in three groups studied: Hispanics, individuals of European ancestry, and African Americans[37]. Moreover, our data showed that the rs12979860 genotypes could be considered a predictive marker for better response in the Han Chinese patients with HCV infection and the high rs12979860 CC genotype rate might explain why its sustained virological response (SVR) rate was higher than the average rate of the global population (Antiviral Therapy, in press). Genetic variation in IL-28B is the strongest and most significant genetic factor associated with spontaneous clearance of HCV[38]. It seems likely that the rs12979860 variation is involved in the innate immunity of HCV infection. However, our data showed that rs12979860 variance had no correlation with outcomes of HBV infection.

In conclusion, this study demonstrated that genetic variants in the HLA-DP loci, but not IL-28B, were strongly associated with outcomes of HBV infection in the Han Chinese population, of which mechanism needs to be further investigated.

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