The Association Between Gene Polymorphismss Of ER Gene And Graves Ophthalmopathy In Women

Objective1. To investigate the relationship between polymorphisms of the estrogen receptor-a (ER-a) and the estrogen receptor-P (ER-β) with the female patients with Graves ophthalmopathy.2. To study the proportion of CD4+CD25+FoxP3+T cells, the concentration of E2, IL-10, TGF-β1, TNF-a and thyroid function under different genotypes of the Xba I estrogen receptor a in GD(GO).3. To examine the association between Xba I estrogen receptor a polymorphisms and the response to treatment of female patients with active GO with prednisone.Methods1.Whole blood samples were obtained from162female patients with GD(the group of GD:67patients without eye symptoms after the diagnosis of GD at least18months; the group of GO-S:56patients underwent eye symp-toms after the time diagnosis of GD in18months and the group of GO-L:40patients with eye symptoms after the diagnosis of GD over18months)and126healthy female with controls. Genomic DNA was extracted from these samples followed by the analysis of polymorphism at Pvu II and Xba I sites of estrogen receptor alpha with PCR-RFLP. Genomic DNA was extracted from these samples followed by the analysis of polymorphism at Rsa I and Alu I sites of estrogen receptor beta with PCR-RFLP. The frequencies of alleles and genotypes were compared between the controls and patients with GD in women.2. E2、TSH、FT3、FT4、TPOAb、TGAb、CD4+CD25+FoxP3+T cells、IL-10、 TGF-β1、TNF-α were determined in72untreated female patients with GD(incIuding22patients with GO) and50female control cases. The blood from all females was collected in the first phase (between days three and seven) of menstrual cycle. E2、 TSH FT3、FT4、TPOAb、TGAb were examined with electrochemiluminescence system. TRAb were examined with automated chemiluminecence system. IL-10、 TGF-β1、TNF-a were examined using ELSIA. CD4TCD25+ToxP3+T cells were examined by Flow Cytometry (FCM). 3. There were84female patients with active GO selected in the study. Before and after treatment with prednisone, the clinical activity scores (CAS) were recorded. Isolating the fresh PBMCs from the patients and and extracting the total RNA followly. Then the mRNA expression of ER-a and FoxP3were measured semi quantitatively by reverse transcriptase-Polymerase chain reaction(RT-PCR). PCR product bands were scanned and analyzed by gel electrophoresis system. E2were examined with electroc hemiluminescence system.Results1.The frequency of AA genotype in the group of GO-S was80.4%and in controls, GD group、GO-L group are46.8%,59.1%and57.5%, respectively. A statistically significant difference was observed between the patients and controls in the frequency of the Xba I polymorphism (P=0.005). Furthermore, the frequency of AA genotype in GD-S has statistically significant difference with that of the control, GD group and GO-L group(P=0.000,0.04and0.042, respectively).The frequency of AA genotype in the total163patients was66%and the controls was46.8%.There was a statistically significant difference between them (P=0.004). The rate of A allele in the group of GO-S, GD and GO-L are88.4%,75.4%and59.4%, respectively. The odds ratio (OR) for A allele was2.11times (OR=2.1195%CI:1.203-3.699) between GO-S group and GD group. The odds ratio (OR) for A allele was2.45times (OR=2.4595%CI:1.312-4.575) between GO-S group and GO-S group. The gene polymorphism of Puv II didn’t have the statistically significant difference in the controls and the group of GO-S. GD and GO-L (P=0.698). In the rs4986938gene polymorphism the frequency of AA genotype in the group of controls、GD、GO-S and GO-L has a statistically significant difference (P=0.032).The differences of the rate of A allele in the four groups were statistically significant(P=0.016).Furthermore, The frequency of AA genotype in the group of GD, GO-S and GO-L had no statistically significant difference (P=0.910).The frequency of AA genotype in the total163patients and the controls had a statistically significant difference (P=0.001). The odds ratio (OR) for A allele was1.71times (OR=1.7195%CI:1.272-2.299) between the total163patients and the controls.The gene polymorphism of Alu I didn’t have the statistically significant difference in the four groups (P=0.836). 2. The level of plasma TSH、FT3、FT4、TPOAb、TGAb、TRAb、E2、IL-10. TGF-β1and TNF-α in the controls of different genotypes had no statistically significant difference(P>0.05). The portion of CD4+CD25+FoxP3+T cell in the PBMCs in the controls of different genotypes had no statistically significant difference, too.(P>0.05). The level of plasma TSH、FT3、FT4、TPOAb、TGAb、TRAb. E2and TGF-(31in the patients with GD of different genotypes had no statistically significant difference(P>0.05). The patients with GD had higher level of plasma E2and TNF-a than that of controls(P=0.000and P=0.000). The portion of CD4+CD25+FoxP3+T cell in the PBMCs in the controls was lower than that of controls, too.(P=0.000).GD patients bearing GG genotypes showed the highest level of CD4+CD25+FoxP3+Tregs as well as IL-10and the lowest TNF-α serum level in comparison to patients carrying GA or AA genotypes. GD patients bearing AA genotypes showed the lowest level of CD4+CD25+FoxP3+Tregs as well as IL-10and the highest TNF-α serum level(P=0.015).3. Before treatment, the values of CAS and the level of plasma E2had no difference in the patients carrying GG,GA and AA genotypes (P=0.684and P=0.714).After treatment, the values of CAS and the level of plasma E2descended in all patients. But the values of CAS of the patients with GG genotypes descended more and the patients with GG genotypes descended less(P=0.028).Before treatment, there was higher expression of ER-a mRNA in the PBMCs of the patients than that of the controls. The patients with GG genotypes has the higher expression of ER-α mRNA than that of patients with GA genotypes and AA genotypes(P=0.029and0.002).After treatments, the expression of ER-α mRNA in the PBMCs of the patients descended significantly (P<0.05). The expression of ER-α mRNA was higher in the patients with GG genotypes than that of patients with GA genotypes and AA genotypes. Before treatment, there was lower expression of FoxP3mRNA in the PBMCs of the patients than that of the controls. The patients with GG genotypes has the higher expression of FoxP3mRNA than that of patients with GA genotypes and AA genotypes(P=0.039).After treatment, the expression of FoxP3mRNA in the PBMCs of the patients increased significantly (P<0.05).The expression of FoxP3mRNA was higher in the patients with GG genotypes than that of patients with GA genotypes and AA genotypes (P=0.002)Conclusion1.The frequency of Xba I polymorphism has the statistically significant difference between the patients and the controls, and AA genotype allele is associated with susceptibility to GD. Furthermore, the AA genotype allele is associated with the time the eye symptoms onset and is risk factor for GD to develop GO early. The frequency of Alu I polymorphism has the statistically significant difference between the patients and the controls, and AA genotype is associated with susceptibility to GD. Furthermore, the AA genotype is not associated with the time the eye symptoms onset.The frequency of PvuⅡ polymorphism and Rsa I polymorphism are not associated with susceptibility to GO.2. Because the less level of IL-10and high level of TNF-α,the GD patients bearing AA genotypes showed the lowest level of CD4+CD25+FoxP3+Tregs. So, the GD patients bearing AA genotypes have less ability to suppress the actived T cell and inflammation. It may be reasons for the patients bearing AA genotype developing GO earlier.3. Because the high level of ER-a mRNA and high level of Foxp3mRNA,the GD patients bearing GG genotypes showed a better response to the treatment. So, because of the Xba I estrogen receptor a polymorphism,the patients bearing GG genotypes have more quantity of ER and the ER can enhance the E2to strengthen the functions of CD4+CD25+FoxP3+Tregs. The higher level of Foxp3mRNA can demonstrate the phenomenon.