Association Analysis Between Single Nucleotide Polymorphism Of TSHR,CTLA-4 And FCRL3 And Graves' Disease In Han Population In Northern Anhui

BackgroundGraves' disease(GD)is an organ-specific autoimmune disease caused by genetic,immune and environmental factors.The main manifestation of GD is increased excitability and hypermetabolism of nervous,circulatory,digestive systems and so on.GD is the main cause of hyperthyroidism,however,the pathogenesis of GD remains unknown so far.Patients with GD have abnormal immune function and produce antibodies against thyrotropin receptor(TSHR),that is,thyrotropin receptor antibody(TRAb),TRAb is a group of polyclonal antibodies,including thyroid stimulating antibodies(TSAb)and thyroid blocking antibodies(TBAb),TSAb are the main autoantibodies to GD and exist in almost all cases of GD.In addition,the severity of GD is related to the level of TSAb.TSAb competitively bind TSHR with similar biological function of TSH,which initiate intracellular cascade reaction,stimulate and exite thyroid gland,and cause thyroid tissue to proliferate.Moreover,this stimulating effect is not regulated by pituitary TSH,while uncontrolled continuous stimulation leads to the pathological synthesis and secretion of excessive thyroid hormones in the thyroid gland.As a specific gene of autoimmune thyroid diseases(AITD),TSHR is an unique GD susceptible gene and the first GD tested related non-major histocompatibility complex(MHC)gene,reflecting the importance of TSHR in the pathogenesis of GD.TSHR encoded protein is not only related to the clinical manifestations of GD,but also the direct target of GD autoimmune response.Therefore,TSHR gene is always the research focus.TSHR gene is located on chromosome 14q31,consists of 10 exons and encodes a G protein-coupled receptor,and plays a central role in regulating thyroid growth,development and function.Over the last dozen years,with the development of measure technology,the ability to measure the genetic variation of hundreds of markers in a large number of individuals come true,which has greatly contributed to the association between common genetic variation and complex diseases.The application of genome-wide association studies(GWAS)in large-scale case-control studies has made it possible to identify disease-related single nucleotide variants and to discover disease susceptibility genes.The development of GWAS further establishes a strong association between susceptible genes and GD.In addition to TSHR and thyroglobulin as specific thyroid genes,GD susceptible genes also include a class of immunomodulatory genes,such as forkhead winged helix transcription factor p3(Foxp3),CD25,CD40,cytotoxic T lymphocyte antigen 4(CTLA 4),Fc receptor-like 3(FCRL3),human leukocyte antigen(HLA),etc.FOXP3 and CD25 play important roles in the establishment of peripheral tolerance,while CD40,CTLA-4 and HLA genes play key roles in T lymphocyte activation and antigen expression.FCRL3 encodes a member of the immunoglobulin receptor superfamily,and is expressed in T cells,B cells,NK cells and other immune cells.In addition,FCRL3 is involved in the regulation of immunity and plays a role in the pathogenesis of autoimmune diseases.Generally,the polymorphism of these immunomodulatory genes has an important effect on the GD susceptibility.The single nucleotide polymorphism(SNP)in immunomodulatory genes may functionally hinder the normal development of central and peripheral tolerance and change the interaction between T cells and antigen presenting cells(APCs)in immune synapses.Therefore,the investigation of gene polymorphism is of great significance to clarify the occurrence and development of GD,the difference of clinical phenotype,the sensitivity and prognosis of drug therapy and the guidance of individualized treatment.This study is the first time to simultaneously detect three GD susceptibility gene loci of TSHR?CTLA-4?FCRL3 discovered by GWAS in the larger Han population in northern Anhui in recent years.We analyzed the association between intron 1 of susceptible gene TSHR,CTLA-4 susceptible loci,FCRL3 susceptible loci and GD.These susceptible loci may play an important role in the pathogenesis of the disease.The objective of this study was to explore the relationship between SNPs of TSHR,CTLA-4 and FCRL3 and GD,so as to provide a theoretical basis for revealing the pathogenesis of GD and further functional analysis of susceptible genes in the futureResearch objectives1.To explore the association between six SNPs loci(rs 179247,rs2284722,rs12101261,rs4903964,rs2300525 and rs17111394)in intron 1 of TSHR and GD susceptibility.2.To explore the association between four SNPs loci(rs231804,rs1024161,rs231726 and rs10197319)of CTLA4 and GD susceptibility3.To explore the association between rs3761959 SNP of FCRL3 gene and GD susceptibility4.To analyze the false positive reporting probability of each susceptible gene locus and the interaction among the three genesResearch methodsHan population in northern Anhui was taken as the research object,a case-control study was conducted,including 597 cases in the GD case group and 620 cases in the normal control group.The relevant clinical data were collected in the outpatient department.All the subjects signed the informed consent form,the peripheral venous whole blood of them were collected to extract DNA.Six SNPs loci in intron 1 of TSHR,four SNPs loci of CTLA4 gene and rs3761959 locus of FCRL3 gene were genotyped by Taqman probe on Fluidigm EP1 platform,and the distribution of genotypes and alleles in the two groups were analyzed.In addition,the association analysis between SNPs and GD susceptibility,and the association between linkage disequilibrium,gene haplotype and GD were analyzed.The false positive reporting rate was calculated for the related susceptible loci,and the association between genotypes of every locus and clinical manifestations of the case group was also analyzedStatistical analysisQuantitative data were described by x±s,while qualitative data were described by percentage or constituent ratio.The differences of quantitative data between groups were analyzed by t test,and the differences of qualitative data between groups were described by ?2 test.The distribution of genotypic frequency and allele in case group and control group and the relationship between SNPs variation and GD were analyzed by binary univariate and multivariate logistic regression model.Then OR value and 95%CI were calculated to evaluate the association between each genotype and the incidence of GD,the rank data were tested by rank sum test,?2 test was used to analyze the relationship between genotypes and GD clinical manifestations,and the above statistical analysis was completed by SPSS 19.0 software.The distribution of SNPs loci in the control population was detected by Haploview4.2 software,and the association between haplotypes and GD was analyzed.The association between gene-gene interaction and the pathogenesis of GD was analyzed by generalized multifactor dimensionality reduction(GMDR).All the tests were double test with test level ?=0.05,and P<0.05 indicated that the difference was statistically significant.Results1.In the control group,the genotypic distribution of 6 loci in intron 1 of TSHR was in accordance with Hardy-Weinberg genetic equilibrium(P>0 05).For rs179247,rs2284722 rs12101261,rs4903964 and rs17111394 loci,there were significant differences in the distribution of corresponding alleles or genotypes between the two groups(P<0 05).Association analysis between six SNPs loci and GD showed that rs179247 allele G,rs12101261 allele C and rs4903964 allele G were negatively correlated with the incidence of GD in the whole population,male population and female population.The risk of GD in rs179247G allele carriers was 0.57,0.56 and 0.57 times higher than that in A allele carriers,respectively,and the risk of GD in rs12101261C allele carriers was 0.58,0.58 and 0.58 times higher than that in T allele carriers,respectively,and the risk of GD in rs4903964G allele carriers was 0.58,0.60 and 0.58 times higher than that in A allele carriers,respectively.Rs2284722 allele A and rs17111394 allele C were positively correlated with the incidence of GD in the whole population and female population,and the risk of GD in rs2284722A allele carriers was 1.34 and 1.47 times higher than that in G allele carriers,respectively;and the risk of GD in rs17111394C allele carriers was 1.29 and 1.37 times higher than that in T allele carriers,respectively.Rs2300525 allele C was only positively correlated with the incidence of GD in female population,and the risk of GD in C allele carriers was 1.25 times higher than that in T allele carriers.There was a positive correlation between haplotype AGTA,AATA in haplotypes composed of rs 179247,rs2284722,rs12101261 and rs4903964 and the risk of GD(OR=1.27,95%CI=1.07-1.50,P=0.005;OR=1.45,95%CI=1.21-1.75,P<0.001).Haplotype GGCG and GACG were negatively correlated with the risk of GD(OR=0.56,95%CI=0.46-0.67,P<0.001;OR=0.40,95%CI=0.18-0.92,P<0.031).Haplotype CC composed of rs2300525 and rs17111394 was positively correlated with the risk of GD(OR=1.32,95%CI=1.08-1.60,P=0.006).There was no significant correlation between TSHR genotype and GD goiter,exophthalmos and TRAb(P>0.05).2.In the control group,the genotypic distribution of the four SNPs loci of CTLA-4 gene was in accordance with the law of Hardy-Weinberg genetic equilibrium(P>0.05).There was no difference in the distribution of alleles or genotypes of rs231804,rs1024161 and rs10197319 loci between the two groups(P>0.05),and there was no difference in the distribution of genotype of rs231726 loci between the two groups(P>0.05).There was significantly difference in the distribution of allele of rs231726 loci between the two groups(?2=4.47,P=0.035).There was a negative correlation between rs231726 allele G and the risk of GD in the whole population.The risk of GD in rs231726 allele G carriers was 0.83 times higher than that in A allele carriers,while no correlation was found between rs231804,rs1024161 loci and the risk of GD.Among the four haplotypes composed of rs231804,rs1024161 and rs231726,there was a positive correlation between AAA and the risk of GD(OR=1.21,95%CI=1.02-1.43,P?0.029),but no association was found between other haplotypes and GD.There was no significant correlation between CTLA-4 genotype and GD goiter,exophthalmos and TRAb(P>0.05).3.There was no significant difference in the distribution of genotypes of FCRL3 rs3761959 locus between the control group and the case group(?2=0.278,P=0.598),and there was no significant difference in alleles between the two groups(?2=0.187,P=0.666).Association analysis between SNP of FCRL3 rs3761959 locus and GD showed that there was no correlation between rs3761959 locus and GD risk in dominant model,recessive model,heterozygous model and homozygous model in the whole population,male population and female population(P>0.05).4.The false positive report probability of susceptible genes rs179247,rs12101261,rs4903964 were all less than the preset critical value 0.2.The interaction between the three genes was analyzed by GDMR software,and the best cross-validation consistency was only the rs179247 model in the single factor model.The training equilibrium accuracy was 0.59,the test set equilibrium accuracy was 0.59?and the consistency rate of cross verification was 10/10(P>0.05)Conclusions1.The SNP locus(rsl79247,rs2284722,rs12101261,rs4903964,and rs17111394)in intron 1 of TSHR were associated with GD susceptibility.Rs179247G,rs12101261C,rs4903964G alleles and haplotypes of GGCG and GACG were protective factors of GD,while Rs2284722A,rs17111394C alleles and haplotypes of AGTA and AATA were the susceptible factors of GD.2.The rs231726 SNP of CTLA4 gene was related to GD susceptibility,and allele A was a risk factor for GD.Among four haplogypes composed of rs231804,rs 1024161 and rs231726,AAA was a risk factor for GD.3.The SNP of FCRL3 rs3761959 locus was not associated with GD susceptibility.4.SNPs of rs179247 rs12101261 rs4903964 may be truly correlated with GD.There was no interaction among TSHR gene,CTLA-4 gene and FCRL3 gene.