Variation of HLA-DPB1 Gene in Hepatitis B Infection

Background: Hepatitis B virus (HBV) affects approximately 68 million people in China. 10-15% of adult infected with HBV will develop to chronic HBV, liver cirrhosis (LC), liver failure and hepatocellular carcinoma (HCC). The human leukocyte antigen HLA-DPB1 gene polymorphism and expression were identied to be associated with HBV susceptible and spontaneous clearance. We evaluated the role of HLA-DPB1 gene polymorphism in HBV infection. Methods: In this study, HLA-DPB1, rs9277535 polymorphisms were investigated in 259 HBV infection patients (CHB) and 442 healthy controls (HC) by using a sequence based typing. The mRNA of HLA-DPB1 were measured by real-time polymerase chain reaction (RT-PCR). Results: The results shown that DPB1 gene, rs9277535 were all associated with HBV infection in the Sichuan Han population. Rs9277535A, DPB1*04:02 play a protected role in HBV infection, Rs9277535G, DPB1*05:01 prone to susceptible to HBV infection. rs9277535GG have signicantly higher HLA-DPB1 mRNA expression in HBV group compared that in HC group. DPB1*05:01 and DPB1*21:01 have a signicantly lower HLA-DPB1 mRNA expression in HBV infection group compared than HC group. The meta-analysis revealed that HLA-DPB1*02:01, DPB1*02:02, DPB1*04:01 and DPB1*04:02 protected against HBV infection, while DPB1*05:01, DPB1*09:01, and DPB1*13:01 were risk factors for HBV infection susceptibility. DPB1*02:01 and DPB1*04:01 were prone to HBV spontaneous clearance, while DPB1*05:01 and DPB1*13:01 were associated with chronic


Samples
The samples involved in this study were from physical examination center of Deyang people hospital, Sichuan province, China. A total of 499 participants, including 259 chronic HBV carriers (CHB) and 441 healthy controls (HC).
The HBV subjects were determined based on the serological results of Hepatitis B surface antigen (HBsAg), antibody against Hepatitis B surface antibody (HBsAb), Hepatitis Be antigen (HBeAg), antibody against Hepatitis Be (HBeAb) and total antibody against HBV core antigen (HBcAb) tests. Volunteers and different ethnic genetic populations, they revealed that HLA-DPB1 and rs9277535A/G not only related with susceptible HBV infection but also affect the results of HBV infection, to be spontaneous clearances or to be chronic HBV infections.
Recent years, researchers [12] found that risk alleles of HLA-DP decrease liver mRNA expression of HLA-DP in HBV patients, suggesting that expression of HLA-DP genes is important in control of HBV in non-Hispanic European. Thomas and his colleagues found [13] that the rs9277534A/G variant distinguishes the most protective HLA-DPB1 allele (DPB1*04:01) from the most susceptible (DPB1*01:01) after HBV infection, they also confers rs9277534GG have signi cantly higher levels of HLA-DPB1 surface protein and transcript level expression in healthy donors compared than rs9277534AA genotype, rs9277534A/G can be an HLA-DPB1 expression marker [14] , Decreased expression of DPB1 mRNA are associated with HBV reactivation in patients treated with immunomodulatory agents [15] , and increased HCV-related liver disease and correlated with HCV related disease progression [16] . These results suggesting that differences HLA-DPB1 alleles and DPB1 expression may in uence the risk of persistent HBV infection.
The latest research found [17] that subset of HLA-DP molecules, such as HLA-DP401, which interact with NKp44 trigger functional NK cell responses. This interaction between DP alleles and NKp44 implicates HLA class II as a component of innate immune response, much like HLA-C and KIR molecular. It may provide a potential mechanism [18] for the relationship between HLA-DP alleles and disease outcomes, including HBV infection. It's speculates that during acute HBV infection, NKp44 interacts with HLA-DP401 allele expressed on the surface of infected hepatocytes, IFN-γwas secreted both by Th1 and NK cells which contributing to lysis of HBV infected cells. In HBV infected individuals carrying HLA-DP301, NKp44 is unable to bind to HLA-DP301 molecules, resulting in ine cient lysis of infected hepatocytes by NK cells and lead to higher risk of chronic HBV infection.
The distribution of HLA-DPB1 molecules in Chinese populations were similar to those in Asian populations, HLA-DPB1 expression in HBV infection in Asian population haven't reported yet. In this research, we typed HLA-DPB1 and associated rs9277535A/G in a population-based case control study in Chinese Han population, including 259 HBV cases and 441 controls from Sichuan province, also identi ed the different HLA-DPB1 expression in different alleles of these two groups. patients with seropositive for HBsAg combine with seropositive for HBeAg and HBcAb or HBeAb and HBcAb and positive of HBsAg at least lasting 6 months were considered to be CHB carriers, the HC who were seronegative for both HBsAg and HBeAg. All blood samples were negative for Hepatitis C virus (HCV) and Human immunode ciency virus (HIV).
Subjects who were seropositive for HBsAg, had normal serumalanine aminotransferase (ALT) levels (lower than 35 U/L), had no obvious clinical symptoms were considered to be asymptomatic carriers and had not use any antiretroviral drugs. Each subject gave his/her written informed consent before enrolment.
rs9277535 and HLA-DPB1 genotyping The peripheral blood of all samples and DNA extraction were performed as consistent with previous reported [19,20] . Genotyping was performed using sequence based typing (PCR-SBT). Ampli cation primers were following: rs9277535: Forward was 5'-TAACTGTGTGTGTTGCTGC, reward was 5'-CTGCGTGGTGAGAAAACAGG, for amplify HLA-DPB1 exon 2, forward primer was 5'-CTGCGTGGTGAGAAAACAGG, 5'-CCTGACAAGCTCCAGATGGG, reward primer was 5'-TTCCTTTATGGTGTTGCTCCT. For each PCR mix (total volume, 10uL) contained 1ul DNA and 5uM primers and 5uL GoTaq Green Master Mix (Promega, Madison, USA). The DPB1 ambiguities were resolved by group-speci c sequencing primers (GSSPs) sequencing, each primer was referenced by EBI-IMGT database. primers were shown in Table 1. Thermal cycling conditions were as followed: 96°C for 3 min, 30 cycles at 95°C for 20s, 62°C for 15s, 72°C for 1min, and 72°C for 5 min. The PCR products were analyzed by an ABI 3730 DNA Sequencer (Applied Biosystem, Foster City, CA). Group-speci c sequencing primers used to resolve alternative genotypes, the primers based on ambiguous genotypes obtained HLA-DPB1 mRNA level measurement RNA were prepared from cell suspensions of each sample freshly isolated peripheral blood mononuclear cell (PBMC) using a TRIzol method (Invitrogen), the RNA extraction and cDNA preparation were also consistent with previous reported [19,20] .
The expression of HLA-DPB1 were quanti ed by SYBR green quantitative PCR (qPCR) using the threshold cycle (CT) method in CFX96 Touch PCR machine (Bio-Rad) with primers Forward: 5'-GTGCATTGCAGAAGGTCAGA-3' Reward: 5'-CTGGTGATAGGCCATCAGGT-3'. Each PCR were triplicate including 12.5ul of FastStart Universal SYBR Green Master (Roche), 200nM primers, and 2.5ul of cDNA in a total volume of 25ul, qPCR protocol was using the recommended by Roche speci cation. The speci city of the DPB1 primers were con rmed by melt curve analysis using the dissociation step with single-peaked. All reactions were standardized to the reference gene GAPDH expression.

Linkage of HLA-DPB1 and SNPs
Genotype frequency was tested for Hardy-Weinberg equilibrium using Arlequin v 3.5.1.2. D' was calculated as described by Paximzadis et al [21] . The maximum-likelihood (ML) method was used to estimate the LD, an Expectation-Maximization (EM) algorithm for multi-locus genotypic data were used by the observed data with an unknown gametic phase. ELB algorithm was used to estimate the gametic phase of the genotype data generated at all the polymorphic positions within the gene region when the recessive alleles are present. The statistical signi cance of the linkage disequilibrium between each of the allele pairs was evaluated by the approximate X 2 reported by previously [19] . All statistical signi cance was de ned as P<0.05.

Meta-analysis study
In order to improve the in uence of the HLA-DPB1 on the susceptible and spontaneous clearance of HBV infection, we performed a meta-analysis of them. Relevant studies were searched and identi ed by a computerized literature search of electronic databases, including Pubmed, EBSCO, Elsevier, and Web of Science, with English only as the language restriction.
The following index terms were used: 'Hepatitis B' and 'HLA-DP'. The inclusion criteria were: (a) genotype frequencies of HLA-DPB1 can be obtained in healthy controls, HBV carriers, chronic HBV infections and HBV spontaneous clearance; (b) the study included speci c criteria for enrolling the samples; (c) the study had a case-control design; and d) the numbers of cases and controls, and the alleles frequencies were clear stated. Studies that did not meet these criteria were excluded.

Statistics analysis
The Hardy-Weinberg equilibrium (HWE) of the genotype distributions and the linkage disequilibrium (LD) of the SNPs were examined using Arliquin 3.5 software, the differences for categorical variables and continuous variables were compared using the χ2 test and Student's t test, respectively. We used a logistic regression model to calculate the age-and gender-adjusted odds ratios (ORs) and 95% con dence intervals (CIs) between the HLA-DPB1 variants and the risk of HBV infection. The metaanalysis was performed by Review Manager 5.2 software, the odd ratios (OR) with 95% con dence intervals (CI) were used to assess the strength of HLA-DPB1 polymorphisms in HBV susceptibility. Values of P lower than 0.05 were considered statistically signi cant.

Results
Relationship between HLA-DPB1 and rs9277535A/G in HBV infection  HLA-DPB1 shows highly polymorphic, the most prevalence allele in our populations were HLA-DPB1*05:01, which (1.41, 95%Cl: 1.14-1.76) was signi cantly associated with HBV susceptibility in Sichuan Han population. Both HLA-DPB1*04:02 (0.31, 95%Cl: 0.16-0.59) were signi cantly associated with HBV protection in our study population (Table 3).       [22] . After the introduction of the HBV vaccine into the immunization program in China in 1992, the government administered free HBV vaccine to newborns to reduce the spread of HBV. At the same time, China has adopted a variety of methods to prevent and control HBV, strengthen the standardized management of HBV screening and medical treatment for blood donors, and effectively control iatrogenic HBV infection. All the above measures have signi cantly reduced the infection rate of new HBV infections, and the total HBV prevalence rate in China has dropped to 5.49% by 2015 [1] . Therefore, it is of great signi cance to study the infection mechanism of hepatitis B for the prevention and treatment of hepatitis B.
HLA-II genes play a key role in viral antigen presentation to mediate cellular and humoral immune responses. Since Kamatani and his colleagues [4] did GWAS in large HBV infection cohort shown that HLA-DPB1 and related single nucleotide polymorphism rs9277535A/G are a strongly risk factors for persistent infection with hepatitis B virus. Thereafter, many researches focused on and successfully repeated the association between HLA-DPB1 and HBV infection and infection outcomes. they found [11] that HLA-DPB1 *09:01 increased HBV susceptibility, DPB1*02:01 was not susceptible to HBV, and DPB1*02:01 was also associated with reduced risk of HBV progression to chronic HBV. Studies [13] in American populations also found that the allele with the greatest risk of infection for HBV was DPB1*01:01, and the allele with the greatest protection against HBV infection was DPB1*04:01.
Subsequently, the genotypes of DPB1 * 05:01, 09:01, and DPB1 * 02:01, 04:01, and 04:02 were again found to be susceptible to HBV in Japanese [9] and Chinese [10] populations. The studies also found that some DPB1 alleles and rs9277535G related to weak HBV vaccines response [23] , low sensitive of HBV drug therapy [15] , incidence of occult HBV infection [24] , and also prone to hepatocellular carcinoma development [25] . In our study, we found the strongly association between rs9277535 and HLA-DPB1 alleles and HBV infection susceptibility. We found the rs9277535A, HLA-DPB1*04:02 were protective factors for chronic HBV infection in this study population, the results were accordingly to previously.
rs9277535G and HLA-DPB1*05:01 were signi cantly associated with HBV susceptibility in Sichuan Han population. Evidence reported here in suggests that HLA-DP have key role in HBV infection progression.
The in uence of the HLA-DPB1 region on HBV recovery is due to levels of HLA-DPB1 expression and less likely to differences in the peptides presented by different HLA-DPB1 alleles. The HLA-DPB1 expression were signi cantly different rs9277535AA and rs9277535GG, the protect genotype 9277535AA has signi cantly lower HLA-DPB1 mRNA expression level compared than the risk genotype rs9277535GG in healthy controls, Thomas [13] and his colleagues also demonstrated that rs9277534GG genotype, which confers susceptibility to HBV persistence with signi cantly higher levels of HLA-DP surface protein and transcript level expression in healthy donors, rs9277534GG have lower levels of HLA-DPB1 mRNA in HBV group, O'Brien [12] had reported that rs9277535AA with higher HLA-DPB1 mRNA expression in liver associated with lower HBV odds ratios with chronic HBV, our study also get a similar result with HLA-DPB1 expression in PBMC. The HLA-DPB1*05:01 and DPB1*21:01 in HBV group showed signi cantly lower HLA-DPB1 mRNA expression levels compared to the healthy control groups. Previous research [7] observed that decreased expression levels of DPA1 and DPB1 mRNA were correlated with the HBV activation after 2 years of treatment with HBV drugs, the high expression of DPA1 mRNA was associated with lower HBV viral load [21] . These results suggesting that the persistence HBV infection may in uenced by differences HLA-DPB1 expression, lower HLA-DPB1 expression may lower the function of antigen peptide present, at last, decrease the immune response to HBV viral, resulting HBV persistence infection risks.
Studies also found [10] that amino acids at position 84-87 of the second exon antigen presentation sequence of DPB1 had a complete linkage with the DPB1 alleles, and there were two major amino acids at position 84-87 with GGPA and DENA, through bioinformatics analysis found that the four amino acids is located at the groove contacting peptide residues pocket-1, can be caused different antigen presenting functions, at last, caused different immunity function to HBV infection. Class II HLA molecules expression on the surface of antigen-presenting cells, to combine with antigenic peptide to CD4 + T helper cells. The T cell paly a crucial role in HBV response in host immune response.
Usually, HLA class II molecules present exogenous antigen peptide, However, HLA-DP molecules with beta-chains encoding DPGly84 [26] does not bind invariant chain (Ii) via the class II-associated invariant chain peptide (CLIP) region to constitutively present endogenous peptides. And processed by the proteasome and transported to the ER by the transporter associated with antigen processing (TAP).
Therefore, DP84Gly can uniquely uses both class I and II antigen processing pathways to present peptides derived from intracellular and extracellular sources, DP84Asp has not such endogenous antigen presentation function. Therefore [27] , this polymorphism has different functions in autoimmune, antiviral and tumor mechanisms through the different function of antigen presentation .
The DP84Gly genotype not only plays a unique antiviral function in adaptive immunity, but also as a ligand of NKp44 [17] to activate NKT cells to play an antiviral role in natural immune function. Compared with healthy controls, NK cells in CHB patients show inhibitory phenotypes, in which the expression of activated receptors NKp44 and NKp46 are down-regulated [28] . Whether the DP84Gly genotype can promote the spontaneous clearance of HBV by binding NKp44 to activated NK after HBV infection has not been studied.
HLA-DPB1 expression associated with HBV infection was not veri ed in Chinese population, to the best of our knowledge, this is the rst research to investigate the HLA-DPB1 mRNA expression in different HLA-DPB1 alleles and SNPs alleles, and the association between mRNA expression and HBV infection.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.