CD40 polymorphisms were associated with HCV infection susceptibility among Chinese population

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 [10‐12]. 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 meta-analysis 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.

Up to now, the pathogenesis of HCV has not been illuminated clearly enough because a number of immune-related 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 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 intron1 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 RNA-seq 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 genotype had a lower CD40 mRNA levels, it was consistent with the results found by Rau SJ et al. that CD40 could inhibit HCV replication and mediate viral clearance, and disruptions [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.