Identification And Functional Analyses Of Regulatory SNPs Of Interleukin-10 And Estrogen Receptor α Gene In HBV-related Liver Diseases

Hepatitis B virus (HBV) infection is one of the major infectious diseases with more than 350 million chronic carriers worldwide, which causes a broad spectrum of liver diseases ranging from asymptomatic carrier (AsC), acute hepatitis (AHB), chronic hepatitis (CHB), liver cirrhosis (LC), liver failure (LF) and hepatocellular carcinoma (HCC). Persistent HBV infection and HBV-related liver diseases have been considered as a multifactorial and polygenic disorder with viral, environmental and host genetic components. Host factors, especially host gene, are played an important role in the pathology of HBV-related diseases. Segregation analysis and twin studies strongly support the role of host genetic components in determining the chronicity of HBV infection. With the completion of the human genome project, attention is now rapidly shifting towards the study of individual genetic variation. The most abundant source of genetic variation in the human genome is represented by single nucleotide polymorphisms (SNPs), which can account for heritable inter-individual differences in complex phenotypes. Identification of SNPs that contribute to susceptibility to complexs diseases, such as HBV-related diseases, will provide highly accurate diagnostic information that will facilitate early diagnosis, prevention, and treatment of human diseases.For genetic susceptibility studies of common complex diseases, the population-based linkage disequilibrium / association study has much higher power than classic family-base linkage analysis. SNP is a powerful tool for identifying genetic susceptibilities to common complex diseases in recent years. Our study group have systematically screened the SNPs in the promoter and exon region of 15 HBV infection susceptibility candidate genes by sequencing and have confirmed that the polymorphisms of estrogen receptorα(ESR1) and interleukin-10 (IL-10) gene are related with susceptibility to chronic HBV infection and disease process of HBV-related liver diseases by the case-control association study, transmission disequilibrium test. Several studies from the other groups also have shown the important between their genetic polymorphisms of ESR1 and IL-10 and chronic HBV infection and HBV-related liver diseases. However, the majority of previous researches have some limits. First, since different studies have different sample size and different ethnic backgrounds, the results of different studies are not the same. Second, and the most important, the previous researches are lack of functional studies to validate the biological significance of positive associated SNP loci. The positive SNP loci associated with diseases can be divided into two different kinds. One kind is indirect association which is simply an associated mark to the disease and has no biological consequence. The number of this indirect association SNP is the majority of positive SNP loci associated with diseases. Anther kind is direct association which plays an important role in the occurrence and development of diseases. For direct associated SNPs in coding regions, it is often possible to predict consequences for protein structure and function, for direct associated SNPs in non-coding DNA, it may affect gene regulation. So it is important and necessary to carry out functional studies to identify SNPs that affect transcriptional regulation.The association of genetic polymorphisms of IL-10 gene with the susceptibility to HBV infection and HBV-related diseases has been recently emphasized. However, the association between functional SNPs in the promoter region of IL-10 and HBV-related acute liver failure (ALF) has not been established yet. Some reports indicated that high levels of IL-10 contribute to the monocytes paralysation and poor clinical outcome in ALF. Although some data concerning the influence of polymorphisms in the IL-10 gene on IL-10 production are still contradictory, most studies have prompted that polymorphisms in the promoter region of IL-10 may affect IL-10 production and confer susceptibility to inflammatory diseases, which show evidence of balancing selection. It is necessary to conducted further functional analyses to verify the biological significances of the IL-10 associated genetic variations.Several studies have demonstrated that ESR1 participates in the pathogenesis of persistent HBV infection. Our study group has reported that the genetic variation at the ESR1 locus influences susceptibility to persistent HBV infection and HBV-related HCC in a Chinese population. We observed that the subjects bearing ESR1 29T/T genotype had an increased susceptibility to persistent HBV infection and HBV-related HCC compared to those bearing at least one 29C allele. In accordance, the relative messenger RNA levels of the risk C allele of T29C were consistently higher than those of the T allele in heterozygous cells. The associated T29C in exon 1, which is a synonymous polymorphism (Ser/Ser), may not have functional consequences. Linkage disequilibrium (LD) mapping analysis indicated that the T29C polymorphism contained within a LD block located from promoter region to intron 3 of ESR1, suggesting that the strong association detected with T29C in ESR1 originated from ESR1 itself. To our knowledge, there is no nonsynonymous SNP (which results in amino acid change) with frequency above 5% in the ESR1 gene. It is very likely that there exist(s) as-yet-unidentied causative regulatory polymorphism(s) that is (are) in LD with the observed associated T29C in the 140-kb region. Indeed, there is a PvuII RFLP(IVS1 T-401C) in intron 1, about 400 bp upstream of exon 2. Bioinformatics and recently a report showed that the PvuII polymorphism is located within a potential c-Myb binding site, which is able to regulate transcription efficacy of a reporter gene. So, further studies are needed to determine whether the PvuII polymorphism is a regulatory polymorphism and clarify its functional molecular mechanisms for this association with chronic HBV infection and HBV-related diseases.Therefore, we select IL-10 and ESR1 gene as two candidate genes and examine the relationship between their polymorphisms (especially the polymorphisms in the non-coding region, such as promoter and intron regions) and the susceptibility to HBV-related liver disease (ALF and/or LC) in large sample of case-control cohort. In addition, we also validate the biological functions of positive associated SNP loci positive and investigate the significance of the relationship between the gene polymorphism and susceptibility to HBV-related liver diseases from the perspective of population genetics.In this study, The clinical date and blood samples of near 6,000 in-patients or out-patients with HBV infection were collected and genomic DNAs were extracted from peripheral blood leukocytes by using standard phenol/chloroform protocols. All the samples were classified different HBV-related disease phenotypes according to the recognized diagnostic criteria. In a hospital-based case-control cohort composed of 451 unrelated healthy blood donors (BDs), 469 asymptomatic HBV carriers(AsC),468 HBV-related liver cirrhosis patients(HBV-LC) and 359 HBV-related ALF patients (HBV-ALF), three polymorphisms in the IL-10 gene promoter and three polymorphisms in the ESR1 gene were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay, tetra-primer amplification refractory mutation system-PCR (ARMS-PCR) and / or PCR-based direct sequencing. Haplotypes were constructed by PHASE program. Pair-wise linkage disequilibrium (LD) between SNPs was analyzed by LDA.χ2 tests and unconditional logistic regression models were used for calculating odds ratios (95% confidential intervals) and corresponding P values controlling age and sex. To further validate the biological functions of positive associated SNP loci, electrophoresis mobility shift assay (EMSA) , allele specific chromatin immunoprecipitation (haploChIP), dual-luciferase plasmid reporter-gene expression assay (DLRA), allele specific small RNA interference(SiRNA) and gene expression assay were used to examine the functional significant of IL-10 A-592C and ESR1 IVS1 T-401C.The main results of our study were listed as follow:1. The clinical date and blood samples of more than 1,000 new in-patient or out-patient with chronic HBV infection were collected and the classification of disease phenotype of all the samples previous collected (more than 5,000 cases) was completed.2. The IL-10 gene promoter polymorphisms (A-1082G, T-819C and A-592C) were successfully genotyped in 414 unrelated healthy blood donors, 367 asymptomatic HBV carriers and 345 HBV-related ALF patients. The allele frequencies of IL-10 -592C and -819C were significantly higher in HBV-related ALF patients than in blood donors and asymptomatic HBV carriers. Logistic regression analysis and stratification analysis with adjustment for age and sex indicated that the polymorphisms of A-592C and T-819C were associated with susceptibility to HBV-related ALF (P=6.9×10-7), and the -1082A-819C-592C haplotype in the IL-10 gene promoter was associated with an increased susceptibility to ALF in HBV carriers ( dominant model, P = 0.0002, odds ratio = 1.60, 95% CI 1.25-2.07).3. The ESR1 gene polymorphisms (IVS1 T-401C, T29C and A252966G) were genotyped in 469 asymptomatic HBV carriers, 468 HBV-related LC patients and 359 HBV-related ALF patients. The allele frequencies of ESR1 IVS1 -401C and 29C were significantly higher in HBV-related ALF and HBV-related LC patients than in asymptomatic HBV carriers. Logistic regression analysis and stratification analysis with adjustment for age and sex indicated that the polymorphisms of ESR1 IVS1 -401C and 29C were associated with susceptibility to HBV-related ALF(P=0.0246, P=0.0071, separately) and HBV-related LC (P=3.0×10-4, P=5.4×10-6, separately), and the 29C -401C haplotype in the ESR1 gene was associated with an increased susceptibility to HBV-related ALF (dominant model, P =0.0116, odds ratio=1.46, 95% CI 1.08-1.96) and to HBV-related LC (dominant model, P = 0.0000, odds ratio=1.83, 95% CI 1.38-2.44).4. EMSA showed that the IL-10 A-592C polymorphism is a nuclear proteins binding site and the ability of nuclear protein binding between two alleles of A-592C was different in vitro. ChIP showed that IL-10 -592C allele has much more ability to binding RNA polymeraseⅡthan IL-10 -592A allele in vivo, which suggested disease susceptible -592C allele had a higher transcription regulation activity compared with -592A allele. In respond to LPS stimulation, the IL-10 mRNA expressions of PBMC were significantly higher in -592CC homozygous genotype compared with individuals heterozygous and homozygous with other genotypes (P<0.01). In the serum of ALF patients, the IL-10 level was associated with ALT level. IL-10 -592C allele contributes to the risk of occurrence of HBV-related liver failure by increasing expression of IL-10.5. From a serials of functional experiments, such as, EMSA, haploChIP, DLRA and SiRNA, we proved that the ESR1 IVS1 T -401C polymorphism is a c-Myb binding site and the ability of c-Myb binding between two alleles of T-401C was different in vitro. ESR1 IVS1 -401C allele has much more ability to binding c-Myb than IVS1 -401T allele in vivo and in vitro, which suggested disease susceptible -401C allele had a higher transcription regulation activity compared with -401T allele. ESR1 IVS1 T-401C polymorphism is a cis-regultory variant and IVS1 -401C allele contributes to the risk of occurrence of HBV-related liver failure and cirrhosis by binding c-Myb and altering ESR1 expression.In conclusion, based on population genetic association study and genetic functional analysis of the cadidate genes and loci, our study emphasizes the importance of IL-10 and ESR1 in the pathophysiology of HBV-related liver disease (ALF and LC) on the population level. According to our large sample of case-control study in groups,we find the IL-10 gene promoter polymorphisms (T-819C and A-592C) and the ESR1 gene polymorphisms (T29C and IVS1 T-401C) were associated with the susceptibility to HBV-related liver disease (ALF and LC) in a Chinese population. Through series of functional studies, we confirmed the positive causative loci (IL-10 A-592C and ESR1 IVS1 T-401C) seem to be two functional regulatory SNPs, which is not simply a mark of the indirect link, but is a direct link to susceptibility to HBV-related liver disease. IL-10 -592C and ESR1 IVS1 -401C alleles of the two functional regulatory SNPs alter the nuclear protein biding abilitity and influence the gene expression. These findings suggest that IL-10 -592C and ESR1 IVS1 -401C alleles contribute to the risk of occurrence of HBV-related liver failure and cirrhosis by increasing expression of IL-10 and ESR1. Our study suggest that estrogen signaling pathway and Th2 cytokine signaling pathway play an important role in the pathogenesis of chronic HBV infection and HBV-related disease from a new view. It not only provides researchers new clues to the further basic research of pathogenesis of chronic HBV infection and HBV-related liver diseases, but also provides the clinicians new ideas to personalized prevention and treatment strategies for patients of HBV infection and HBV-related liver disease.