CD40 polymorphisms were associated with HCV infection susceptibility among Chinese population

Background CD40, encoded by TNFRSF5, participates in the survival of B cells, process of antigen presentation and generation of CD8+ T cell memory. It also has an important effect on HCV antiviral immune response. This study aims to investigate whether TNFRSF5 gene polymorphisms are associated with HCV infection outcomes among Chinese population. Methods Three single nucleotide polymorphism (SNPs) (rs1535045, rs1883832, rs4810485) on TNFRSF5 were genotyped by TaqMan assay among Chinese population, including 1513 uninfected subjects, 496 spontaneous viral clearance subjects and 768 persistent HCV-infected subjects. Logistic analysis was used to compare these SNPs among different groups in this cross-sectional study. Functional annotations of the identified SNPs were further evaluated by bioinformatics analysis. Results After adjusted by age, gender and routes of infection, the results of logistic analysis indicated that individuals carrying rs1535045 T allele had a higher risk to infect HCV compared with C allele (in recessive model, adjusted OR = 1.368, 95%CI = 1.070-1.749, P = 0.012). Subjects carried rs1535045 TT genotype were more likely to infect HCV than wild CC genotype (adjusted OR = 1.397, 95%CI = 1.078-1.809, P = 0.011). For rs1883832, T allele was significantly associated with an increased risk of HCV infection (in recessive model, adjusted OR = 1.337, 95%CI = 1.069-1.673, P = 0.011). Subjects with TT genotype had more possibility to infect HCV (adjusted OR = 1.351, 95%CI = 1.060-1.702, P = 0.015). In the stratified analysis, rs1535045 and rs1883832 were remained in various subgroups and the heterogeneity test showed no pronounced heterogeneity in any pairwise comparison (all P > 0.05). In addition, the results of the cumulative effects showed a tendency of that the more risk alleles (rs1535045 T and rs1883832 T) subjects carried, the more possibility of HCV infection exhibited (P<0.001). In haplotype analyses, compared with the CC haplotype, CT, TC and TT was correlated with an increased risk to infect HCV (P = 0.029, P = 0.047 and P<0.001, respectively). Conclusions In conclusion, CD40 polymorphisms were significantly associated with the susceptibility to HCV among Chinese populations.


Background
It is estimated that more than 184 million people around the world are infected with hepatitis C virus (HCV). HCV infection has brought about large burden on many countries resulting from the highly correlated hepatic and extrahepatic disorders such as cirrhosis, hepatocellular carcinoma and hepatic failure [1]. Furthermore, current vaccines are not available for effective prevention of HCV infection [2].
As is known, host genetic factors play an important role in infection outcome of HCV and the response to antiviral therapies [3]. IL-28B rs12979860 and HLA class II rs4273729 are independently associated with spontaneous resolution of HCV infection [4]. Genome-wide association studies (GWASs) of patients with chronic hepatitis C have identified the association between IL28B variations, especially rs12979860 and rs8099917, and response to treatment with peg-interferon alpha and ribavirin [5,6]. Therefore, it is still essential to figure out the atlas of disease related genetic factors.
CD40, also known as tumor necrosis factor receptor superfamily 5 (TNFRSF5), which is principally expressed on the B cells, participates in the survival of B cells, process of antigen presentation and generation of CD8+ T cell memory [7]. It also has an important effect on antiviral immune response. In HCV-associated chronic liver diseases, CD40 is highly expressed on liver tissues and has a key role in hepatocyte survival [8]. To be more specific, CD40 can inhibit HCV replication and mediate viral clearance, and disruptions by activated innate immune mechanism [9]. CD40 gene polymorphisms have been found to be associated with many immune related disorders, for instance, Kawasaki Disease, systemic lupus erythematosus and lung cancer [10][11][12]. According to previous study, rs1535045 T allele was higher than C allele in Coronary Artery Disease (CAD) patients [12]. It also has been found that rs1883832 T allele was associated with higher risk of sepsis [13]. One metaanalysis showed a significant association between the CD40 rs4810485 T allele and rheumatoid arthritis (RA) [14] However, so far, no relevant research has addressed the association between CD40 genetic variants and HCV infection susceptibility or outcomes. Hence, this study aimed to explore the relationships between CD40 genetic variants and HCV infection outcomes among a Chinese high-risk population, including rs1535045, rs1883832 and rs4810485.

Study subjects
This study incorporates three high risk populations up to 2777 subjects. We recruited 720 hemodialysis (HD) subjects from nine hospital hemodialysis centers from October 2008 to May 2015, 459 intravenous drug users recruited from Nanjing compulsory detoxification center from December 2008 to November 2012 and 1598 paid blood donors from six villages in Zhenjiang City from October 2008 to September 2016. Exclusion criteria are as follows: 1. concurrently infected with other virus (human immunodeficiency virus or hepatitis B virus); 2. suffered from any other liver diseases (e.g. alcoholic, autoimmune or metabolic liver diseases); 3. accepted any antiviral therapies during this whole study. All participants were categorized into three groups. Group A were health subjects who were tested both anti-HCV and HCV-RNA negative. Group B were the spontaneous clearance group who were tested anti-HCV positive and HCV-RNA negative. Group C were called persistent infection group whose anti-HCV and HCV RNA were both seropositive. It is worth noting that all serological results were verified by three independent experiments within six consecutive months.
Demographic data, dangerous behavior exposure and medical histories of HCV infection were collected through structured questionnaires designed by professionals. Only achieved the rigorous professional trainings could the investigators carry out the interview for each participant. Quality control was throughout the entire process of investigation in order to make sure that the collected data was true and reliable.

Viral testing
After the interview, an approximately 10-mL morning fasting venous blood was collected from each participant. The serum and white blood cells were isolated at the speed of 4000 rpm for 10 min immediately and refrigerate at − 80°C before using. Taking appropriate approaches for detection of anti-HCV antibodies, HCV-RNA and diverse HCV genotypes followed by the standard operating protocols. The reagents used for each step were the third-generation enzyme-linked immunosorbent assay (for anti-HCV antibody), Trizol LS Reagent (for HCV-RNA) and Murex HCV Serotyping 1-6 Assay ELISA kit (for HCV genotype).

SNPs selection and genotyping
SNP searching strategies: 1) Candidate TNFRSF5 tagSNPs were download on the 1000 Genomes Project SNP database (www.internationalgenome.org) and selected through Haploview 4.2 software, 2) Potential functional SNPs predicted by following databases (UCSC, HaploReg v4.1, GTEx Portal, SNP Function Prediction, and microRNA-related SNP), 3) Relevant SNPs discovered by others scholars associated with viral hepatitis or other liver and immune-related disorders, 4) According to the criteria, the minor allele frequency (MAF) of selected SNPs must be more than 5% among Chinese Han population. Finally, three SNPs (rs1535045, rs1883832 and rs4810485) were chosen into this study.
Genomic DNA of each subject was extracted from peripheral blood leukocytes by proteinase K and phenolchloroform respectively, and further purified by ethanol precipitation. We used TaqMan allelic discrimination assay to genotype three SNPs on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). For quality control, two blank controls were set in each 384-well plate, and a 100% concordance was achieved in 10% random samples. The success rates of genotyping for selected SNPs were all above 95%. The samples failed for genotyping were excluded from the statistical analyses. The genotyping results were read by SDS 2.3 software (Applied Biosystems, Foster City, CA, USA). The primers and probes sequences for those SNPs were listed in Additional file 1: Table S1.

Statistical analysis
For further susceptibility analysis, Group B and Group C were combined as HCV infected group, then compared with Group A which are health subjects. For HCV chronicity analysis, Group C was contrasted with Group B. Differences of the distribution in demographic and clinical characteristics were compared between subjects among three groups. Wherever appropriate, these differences were evaluated using Student's t test, χ 2 -test, Kruskal-Wallis test or one-way analysis of variance. A goodness of fit χ 2 test was used to test Hardy-Weinberg equilibrium (HWE) for each SNP among healthy subjects. Linkage disequilibrium (LD) parameters (r 2 and D ′) of selected SNPs in CHB population were estimated by Haploview software (version 4.2). The odds ratios and 95% confidence intervals were used to estimate the association between SNPs and the HCV infection outcomes by adjusting age, gender and routes of infection using both univariate and multivariate logistic regression models in this cross-sectional study. P<0.05 in a two-sided test was considered statistically significant. Bonferroni correction was used to conduct multiple comparisons among genotypes and the P value was adjusted to 0.0167 (0.05/3). All the analyses were carried out by Stata/SE (V.12.0 for Windows; StataCorp LP, College Station, TX, USA).

General demographic information
Demographical and clinical characteristics of all subjects were listed in Table 1. The distribution of age and gender were balanced among Group A, Group B and Group C (all P > 0.05), while the ALT, AST and routes of infection were significantly different among three groups (all P < 0.001). HCV genotypes of partial HCV patients were statistically different between Group B and Group C (P< 0.05). The three SNPs were all in accordance with Hardy-Weinberg equilibrium in the Group A (P = 0.553 for rs1535045, P = 0.130 for rs1883832 and P = 0.723 for rs4810485).

Association between polymorphisms in CD40 gene and susceptibility to HCV infection
The distributions of rs1535045, rs1883832, and rs4810485 genotypes among Group A, Group B, and Group C were shown in Table 2. To explore the association between CD40 polymorphisms and hepatitis C susceptibility, logistic regression methods were used by constructing three genetic models (additive, dominant and recessive). After adjusted by age, gender and routes of infection, the logistic regression analysis discovered that rs1535045 T allele was remarkably bound up with higher risk to infect HCV compared with C allele (in recessive model, adjusted OR = 1.368, 95%CI = 1.070-1.749, P = 0.012). Subjects carried rs1535045 TT genotype were more likely to infect HCV than wild CC genotype (adjusted OR = 1.397, 95%CI = 1.078-1.809, P = 0.011). For rs1883832, T allele was significantly associated with an increased risk of HCV infection (in recessive model, adjusted OR = 1.337, 95%CI = 1.069-1.673, P = 0.011). Subjects with TT genotype had higher possibility to infect HCV (adjusted OR = 1.351, 95%CI = 1.060-1.702, P = 0.015).

Association of CD40 polymorphisms with the spontaneous clearance of HCV infection
When exploring the association of TNRSF5 polymorphisms with the spontaneous clearance of HCV infection, we proceeded association study by comparing the distribution of genotypes of selected SNPs in the persistent infection group and spontaneous clearance group after the adjustment by gender, age and routes of infection. No significant association was discovered in any of the three SNPs (rs1535045, rs1883832 and rs4810485) with the virus spontaneous clearance both in the additive, dominant, recessive models (all P>0.05, Table 2).

Cumulative effects of rs1535045 and rs1883832 on the susceptibility of HCV infection
The analysis of combined risk alleles (rs1535045 T allele and rs1883832 T allele) came to conclusions that subjects carried three risk alleles or four risk alleles were more likely to be infected when compared with subjects carried no risk alleles (adjusted OR = 1.947, 95%CI = 1.741-2.179, P<0.001, Table 4). Nevertheless, those carried one and two risk alleles showed no higher possibility to infect HCV than subjects who carried risk-free alleles. These two SNPs had trend influence on more possibility of being infected by HCV after the Cochran-Armitage trend test (P<0.001).

Discussion
Up to now, the pathogenesis of HCV has not been illuminated clearly enough because a number of immunerelated pathways and a series of genes involve in the process of HCV infection. Growing researches on HCV are paid attention to find more genetic variations in order to identify relevant biological markers of the disease. We explored the possible associations between three CD40 SNPs (rs1535045, rs1883832 and rs4810485) and HCV infection outcomes. In this study, we found that genetic variants of CD40 (rs1535045 T allele and rs1883832 T allele) were significantly associated with an increased risk of HCV infection among Chinese population at the fist time.
TNFRSF5 encodes a costimulatory protein called CD40, which have been found on antigen presenting cells. The CD40 has been found to play an essential role in mediating a broad variety of immune and inflammatory responses including T cell-dependent immunoglobulin class switching, memory B cell development, and germinal center formation [7,15]. Various cytokines can control the expression level of CD40, particularly the IFN-γ, IL-1β, and TNF-α enhancing the expression on monocytes [16], endothelial cells [17,18], thymic epithelium [19] and in six HCC cell lines [20]. In HCV-associated chronic liver diseases, CD40 might have important effects on hepatocyte survival and death in HCV-associated chronic liver diseases [8]. Studies demonstrated that CD40 signal can activate NF-κB in a dose-dependent manner, then NF-κB inhibits Fas or Tumor Necrosis Factor Receptor (TNFR) mediated apoptosis by blocking the activation of caspase-8 [21,22]. Specifically, CD40 is up-regulated on hepatocytes during infection with The P value, OR and 95% CIs of Group C versus Group B were calculated on the basis of the logistic regression model, adjusted by gender, age and routes of infection Bonferroni correction was applied and the P value was adjusted to 0.0167 (0.05/3) Bold type indicates statistically significant results hepatitis C virus, can inhibit HCV replication and mediate viral clearance, and disruptions by activated innate immune mechanism [9].
In our study, we found rs1535045 (located in in-tron1 of TNFRSF5) and rs1883832 (located in 5'UTR of TNFRSF5) have an association with susceptibility to HCV infection. However, no significant association was found among the three candidate SNPs and the spontaneous clearance of HCV, which may be related to the smaller sample size included in the analysis. Therefore, this result should be further verified in large sample studies. These SNPs had been broadly researched in different genetic backgrounds. It turned out that rs1535045 had connection to many diseases, such as coronary artery calcification [23] and small cell lung cancer [11], and rs1883832 was related to systemic lupus erythematosus [24] and acute coronary syndrome [25].
According to the bioinformatics information from UCSC, SNP rs1535045 was situated in highest peak of the H3k4me1, H3k4me3 and H3k27AC histone marker in 7 cell lines which were potential regulatory elements region (Fig. 1). We found rs1883832 was located at peak of transcription level assayed by RNAseq on 9 cell lines from ENCODE and H3k27AC histone marker in 7 cell lines (Fig. 1). Recent studies have found that probable pathogenic SNPs are enriched in the enhancer region and regulatory elements region, particularly in the H3K27Ac region, indicating that these mutations may have possible functions on gene transcription and expression process and then effect the disease susceptibility and outcomes [26,27]. Besides, we identified whether these SNP could cause any differential expression of mRNA in different tissues by eQTL analysis. It turned out that different genotypes of rs1883832 could cause differential mRNA level of CD40 in whole blood (P = 2.7 × 10 − 9 ) (Fig. 2). Subjects carried the risk TT    [9].
In the stratified analysis, considering that the genetic pattern of positive SNPs is recessive, we conducted the analysis in recessive model. We found that rs1535045 and rs1883832 significantly increased the risk of HCV infection in some subgroups. Although different gender and route of infection can bring complex infection environment and immune response, rs1535045 T allele and rs1883832 T allele remained meaningful in various subgroups. In addition, the results of the cumulative effects and haplotype analyses showed a tendency of that the more risk alleles (rs1535045 T and rs1883832 T) subjects carried, the more possibility of HCV infection exhibited.
This research did have some limitations. First of all, the samples included in this study were poorly representative and lacked important information that might affect the outcome, such as HCV genotype and IL28B genotyping results. Second, this is a cross-sectional study and the association should be better verified in cohort study. Third, the evidence based on bioinformatics analysis was obtained on the computer simulation, further functional experiment should be carried out. In addition, this study only included high-risk populations of HCV infection, which may overestimate the association between candidate SNPs and the susceptibility of HCV infection. Therefore, our results must be verified in larger cohorts in the future.

Conclusion
CD40 polymorphisms was significantly associated with the susceptibility to HCV among Chinese populations. Further functional studies are needed to confirm the biological significance of these positive SNPs. This study provided the genetic evidence of association between CD40 polymorphisms and the outcome of Fig. 1 The functional annotations of rs1883832, rs4810485 and rs1535045 in UCSC genome browser were listed in this figure. SNPs marked with blue vertical lines (rs1883832, rs4810485, rs1535045 arranged from left to right) are located within the putative promoter transcription regulatory region characterized by Transcription Levels, histone protein modification (H3k4me1, H3k4me3 and H3k27AC) and DNase Hypersensitivity clusters HCV infection, and they may lead other scientists and clinician to identify new targets for therapy and personalized medical-based strategy by combined with other molecular genetics information.
Additional file 1: Table S1. Probes and primers of investigated TNFRSF5 SNPs for TaqMan assay.  showed that minor T allele of rs1883832 were pronouncedly associated with decreased expression levels of CD40. The P values were derived from linear regression model. This data was obtained from Genotype-Tissue Expression project (GTEx V6p) Portal