| Objective:This study aimed to explore the association of vitamin D receptor?VDR?gene polymorphisms with Graves'disease?GD?and Graves'ophthalmopathy?GO?in Southwest Chinese Han population.Methods:Four single nucleotide polymorphism?SNP?sites of VDR genes identified to be associated with autoimmune diseases previously,including Apa??rs7975232?,Bsm??rs1544410?,Fok??rs2228570?and Taq??rs731236?,were selected in this study.A total of 650 GD patients?180 male,470 female;Average age:38.8±14.8 years old?and 1,209 healthy controls?372 male,837 female;Average age:39.3±12.9 years old?in Southwest China were included in this three-stages case-control study.Among 650 GD patients,148 patients were diagnosed with GO?46 male,102 female;Average age:37.5±14.7 years old?.The polymerase chain reaction-restriction fragment length polymorphism?PCR-RFLP?assay was applied for genotyping,and direct sequencing of PCR products was conducted to verify the accuracy of PCR-RFLP genotyping result.The Hardy-Weinberg equilibrium was tested in control groups.The allele and genotype frequencies of candidate SNPs were compared between GD/GO patients and healthy controls by the?2 test using the SPSS version 17.0,which was also used to calculate the P value and odds ratio?OR?.For multiple comparisons,Bonferroni correction was applied,and the corrected P value was recorded as the Pc value.If the Pc value is less than 0.05,it is considered to be statistically significant.In addition,real-time PCR and ELISA were performed to quantified the mRNA expression of VDR gene and the production of cytokines?IL-17,TGF-?1?in peripheral blood mononuclear cells?PBMCs?with different genotypes of candidate SNP.The statistical analysis was performed by SPSS version 17.0 in this study.Results:?1?In the first stage,among the four SNPs of the VDR gene,the frequency of VDR/Apa?AA genotype was significantly higher in GD patients than that in the healthy control(Pc=1.67×10-2,OR=1.98).No significant differences in the genotype and allele frequencies of other three investigated SNPs?Bsm?,Fok?and Taq??were detected between GD patients and healthy controls.?2?In the second stage,a larger sample size of case and control was enrolled to verify the significant association identified in the first stage.Our results confirmed that the AA genotype frequency of Apa?was significantly higher in GD patients than that in the healthy control(Pc=4.29×10-2,OR=1.82)in the second stage.The combined data from the two stages showed that the frequencies of Apa?A allele and AA genotype in the GD patients was significantly higher than that in the healthy control(Pc=2.62×10-2,OR=1.20;Pc=3.45×10-4,OR=1.87,respectively).Apa?A allele and AA genotype may be the risk factor in the occurrence of GD.Stratification analysis revealed that the frequency of VDR/Apa?AA genotype was significantly higher in GO patients than that in the healthy control(Pc=8.69×10-5,OR=2.84),which suggested that the AA genotype may be the risk factor involved in the occurrence of GO.?3?The functional research results in the third stage showed that the significantly decreased VDR gene mRNA expression(P=1.41×10-2;P=6.50×10-3,respectively),significantly increased IL-17 production(P=6.55×10-3;P=3.80×10-3,respectively)and significantly decreased TGF-?1 production(P=3.41×10-2;P=1.17×10-4,respectively)were observed in Apa?AA genotype carriers than those in Apa?Aa or aa genotype carriers when PBMCs were stimulated with anti-CD3+/CD28+antibodies.However,there was no significant differences in VDR gene mRNA expression and TGF-?1 production among the individuals with each Apa?genotype when PBMCs were stimulated with LPS.Conclusion:The VDR/Apa?is significantly associated with the genetic susceptibility to GD and GO in Southwest Chinese Han population,which may be involved in the occurrence of GD and GO by affecting the mRNA expression of VDR gene and the cytokines production. |
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