A Functional Variant In The OAS1 Gene Is Associated With Sj?gren's Syndrome Complicated With Hbv Infection

Recently,Sj?gren's syndrome(SS),as a common autoimmune disease,has drawn researchers' attention by the complex pathogenesis.Even though genome-wide association study(GWAS)has identified multiple SS-associated susceptibility genes,there are several genetic association studies have also elucidated different single nucleotide ploymorphisms(SNPs)and people discovered that genes involved in interferon(IFN)signaling pathway regulating SS development at genetical and molecular immunological level.However,the particular pathogenesis of SS remains unclear,which would further limit the development of clinical therapy and new drug design for SS.Summarily,investigate the pathogenesis of SS is critical and meaningful.Our group collected Chinese Han Sj?gren's syndrome patient samples for a long time.Base on a cohort of 1455 healthy controls and 588 SS patients,we extracted genomic DNA from anticoagulant blood and isolated peripheral blood mononuclear cells(PBMC),which were further separated for PBMC m RNA and immortalized B lymphoblast cell lines(LCLs),according to experimental needs.Firstly,we identified the genetic association of a functional variant rs1074671 in the oligoadenylate synthetase 1(OAS1)gene in Chinese Han Sj?gren's syndrome by Taqman PCR(P = 0.0398,odds ratio/OR = 0.85).When combining the clinical anti-SSA antibody information,statistical analysis indicated that rs10774671 is associated with anti-SSApositive SS(P = 8.0×10-5,OR = 0.85),and is not associated with anti-SSA-negative SS(P = 0.1725).If anti-HBc antibody information was merged,this variant would be genetically associated with anti-SSA-positive/anti-HBc-positive SS(P = 0.0059,OR = 0.36)and fail to associate with anti-SSA-positive/anti-HBc-negative SS(P = 0.3746,OR = 1.24).OAS1 plays an essential role in the IFN signaling pathway,with functional genetic locus rs10774671 on intron 5 who is genetically associated with multiple autoimmune diseases,including Multiple sclerosis and(MS)and Type I diabetes(T1D).Our study identified a genetic association of the OAS1 locus rs10774671 with Sj?gren's syndrome in Chinese Han population,and the genetic association is consistent with the genetic association of this locus in the European Sj?gren's syndrome population.Meanwhile,rs10774671 is associated with anti-SSA-positive SS.As an indicator of SS,antigen SSA/Ro60 is believed to involve in disease pathogenesis,which suggests there may be a potentially synergistic pathogenic relationship between OAS1 and SSA/Ro60.Another correlation analysis data indicates that rs10774671 is associated with antiSSA-positive/anti-HBc-positive SS.This result demonstrates the functional variant in the OAS1 gene is associated with Sj?gren's syndrome complicated with HBV infection for the first.HBV infection shows different correlations among different population of SS patients.Given the high incidence of HBV infection in China and China's special epidemiological characteristics,identifying the role and molecular mechanism of the genetic association between HBV infection and SS in Chinese population have important clinical significance.Upon the genetic association between rs10774671 and disease,we further identified the association of this SNP and OAS1 gene expression in SS samples.Firstly,we examined the total OAS1 gene expression in the control and disease samples with the “G/G”,“G/A”,and “A/A” genotypes in rs10774671,and found that in both patients with SS and normal healthy people the rs10774671 locus was not associated with total OAS1 gene expression.However,the total OAS1 gene expression was significantly increased in anti-SSA-positive patients,compared with anti-SSA-negative patients.Since OAS1 is interferon-inducible gene,we also examined the relationship of rs10774671 and OAS1 gene expression in interferon-stimulated SS LCLs.And there's no correlation.We also analyzed the bioinformatics database and found that the SNPs in linkage disequilibrium(r2>0.95)with rs10774671 is partially distributed in the promoter region of OAS3 genomic gene.In order to clarify the role of OAS3 playing in SS pathogenesis,we also examined the association of rs10774671 locus with total OAS3 gene expression in control,SS,and interferon-stimulated SS.It showed rs10774671 had no association with OAS3 gene expression.Based on the previous data,rs10774671 has a significant genetic association with SS.However,gene expression data indicates that this variant is not associated with total OAS1 m RNA expression.To further elucidate the pathogenic mechanism of rs10774671 risk allele susceptibility in SS development,we detected the association of OAS1 transcript variant(TV)gene expression with the variant genotype in disease samples.It is known allele "G" to "A" mutation at the rs10774671 locus results in that OAS1 m RNA cleavage precursor recognizes mistake sequence to form three new transcript variants(OAS1 TV2,TV3,TV4,corresponding to the wild type OAS1 TV1).According to the sequence characteristics,we designed the specific OAS1 isoform primers and identified four isoforms gene expression in the disease samples with different genotypes.The results suggested that OAS1 TV1 gene expression was enhanced in wild homozygous samples,while the expression of OAS1 TV2,TV3,TV4 was enhanced in people carrying allele “A”.It reveals that OAS1 isoforms gene expression is dependent on the variant locus allele,as the new isoforms fail to respond to immunity,we clarify the mechanism of rs10774671 risk allele is more susceptible to SS development at molecular biology level.It is reported that OAS1 inhibit the infection and replication activity of HBV virus through Jak-STAT signaling pathway.To explore the molecular mechanism of rs10774671 locus associating with HBV infection in this study,we constructed four expression plasmids of OAS1 isoform,respectively.They were co-transfected into Hep G2 cell with HBV virus expression construct,and the expression of HBV in cells and supernatant was detected by Western Blot,ELISA and real-time PCR.The antiviral ability of OAS1 TV1 was significantly stronger than the three others.Therefore,when the rs10774671 variant is mutated,with OAS1 TV1 expression decreasing and the expression of the OAS1 TV2,TV3,and TV4 enhancing,the antiviral ability of the new isoforms is decreased.This conclusion reveals people carrying allele "A" is more susceptible to HBV infection.This study identified the genetic association of the functional SNP rs10774671 in the OAS1 gene with Chinese Han Sj?gren's syndrome complicated with HBV infection.We also verify the molecular mechanism of rs10774671 regulating Sj?gren's syndrome and HBV infection,which will help to strengthen the recognization of the susceptibility genes and pathogenesis of Sj?gren's syndrome and would build the foundation for the advancement of disease treatment.