| Objective:This study aimed to investigate the association between single nucleotide polymorphisms?SNPs?of IL2RA/miR182/FoxO1 genes and susceptibility to Graves'disease?GD?in Southwest Chinese Han population,and to explore the preliminary molecular mechanisms possibly involved in the effect of significantly associated SNP on the occurrence of GD.Methods:Based on the previous association study between genetic polymorphisms and autoimmune diseases,we selected eight SNPs in three genes related to T helper cell 17?Th17?and regulatory T cell?Treg?,including IL2RA/?rs3118470,rs2104286,rs7093069?,miR182/rs76481776 and FoxO1/?rs2297626,rs17592236,rs9549241 and rs12585277?.A three-stage case-control study was performed in 650 GD patients and1300 normal controls using polymerase chain reaction-restriction fragment length polymorphism?PCR-RFLP?assay.Quantitative polymerase chain reaction?qPCR?was used to detect the gene mRNA expression in peripheral blood mononuclear cell?PBMC?obtained from healthy controls.Cytokine secretion levels were quantified by enzyme linked immunosorbent assay?ELISA?.Statistics analysis were performed by SPSS software version 17.0.Results:?1?In the first stage,300 GD patients and 600 normal controls were enrolled to investigate the association of eight candidate SNPs with GD susceptibility.Our results showed that the frequency of IL2RA/rs3118470 TT genotype increased significantly in GD patients than normal controls?Pc=0.024,OR=1.688?.A significantly increased frequency of IL2RA/rs2104286 AA genotype was also identified in GD patients as compared to healthy controls?Pc=0.024,OR=1.658?,while the frequency of AG genotype in GD patients was significantly lower than normal controls(Pc=4.104×10-3,OR=0.530).No statistically difference was found for the frequencies distribution of allele and genotype in remaining six SNPs?rs7093069,rs76481776,rs2297626,rs17592236,rs9549241 and rs12585277?between GD patients and normal controls?Pc>0.05?.?2?In the second stage,a total of 350 patients with GD and 700 normal controls were included to verify the association between the two SNPs?rs3118470,rs2104286?and susceptibility to GD.The results showed that the frequency distribution of IL2RA/rs2104286 AA/AG genotype was significantly different between GD patients and normal controls(AA genotype:Pc=0.006,OR=1.618;AG genotype:Pc=3.233×10-5,OR=0.494,respectively),while the allele and genotype frequencies of IL2RA/rs3118470 showed no significant difference between GD patients and normal controls?Pc>0.05?.The combined data of IL2RA/rs2104286 locus showed that the frequencies of rs2104286 A allele and AA genotype were significantly higher in GD patients than normal controls(A allele:Pc=0.004,OR=1.322;AA genotype:Pc=8.772×10-5,OR=1.636,respectively),while the frequencies of G allele and AG genotype were significantly lower in patients with GD(G allele:Pc=0.004,OR=0.757;AG genotype:Pc=1.282×10-8,OR=0.510,respectively).Stratification analysis revealed that IL2RA/rs2104286 was associated with Graves'ophthalmopathy?GO?susceptibility(AA genotype:Pc=6.150×10-3,OR=1.851,AG genotype:Pc=4.710×10-6,OR=0.348,respectively).?3?In the third stage,the function research results showed that IL2RA/rs2104286 AA genotype individuals had significantly lower IL2RA mRNA?P=0.021?expression and IL-10 cytokine production?P=0.015?as well as significantly higher IL-17 cytokine production(P=1.467×10-4)than AG genotype individuals in anti-CD3/CD28 antibody stimulated PBMC obtained from healthy controls.No significant difference was found in IL-6 and TGF-?1 secretion levels between rs2104286 AA and AG genotype carriers?P>0.05?.Conclusion:Our study identified that IL2RA/rs2104286 was significantly associated with GD and GO susceptibility in Southwest Chinese Han population,which may be involved in the occurrence of GD and GO by affecting the mRNA expression of IL2RA gene and the cytokine production. |
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