RhoA with Associated TRAb or FT3 in the Diagnosis and Prediction of Graves' Ophthalmopathy

Abstract

During Graves' disease (GD) treatment, Graves' ophthalmopathy (GO) is often ignored because only mild ocular symptoms are present in early GD. Therefore, we performed isobaric tags for relative and absolute quantification (iTRAQ) analysis and measured relevant endocrine hormones to identify predisposing factors of GO. Serum samples from 3 patients with mild GD and GO and 3 patients with GD but without GO were analyzed by iTRAQ. Based on their clinical data, 60 patients with GD were divided into the GO-free and GO groups. All patients were followed up for 7 months. Their eye conditions and changes in related biochemical indexes were recorded. The iTRAQ results showed that RhoA expression was upregulated and correlated significantly with the tight junction pathway and immunity. The changes in FT3 and RhoA from baseline to 7 months, the FT3 and RhoA baseline levels, and the TRAb titer levels in patients with GD significantly differed between the groups. ELISA and western blotting for RhoA, TRAb, and FT3 in the serum samples from GO patients showed significant upregulation, as well as elevated serum RhoA and TRAb levels in the mild stage of GO. At 7 months, the serum RhoA and FT3 levels were elevated. RhoA is a potential biomarker for mild GO. In GD patients, if an elevated serum RhoA level is accompanied by an elevated TRAb or FT3 level, GO is highly likely to occur, even when obvious ocular symptoms are absent.

Figure 1

iTRAQ results. (a) Protein quantitative results were analyzed by principal component analysis. The control group and the experimental group aggregated significantly into different clusters. Blue: control group; red: experiment group. Heatmap (b) and volcano plots (c) protein expression between the control and experimental groups (fold change > 1.5 and adjusted P value < 0.05). Red: upregulated; blue: downregulated. (d) GO enrichment analysis was performed for upregulated proteins. The results showed that the upregulated proteins are mainly involved in actin filament polymerization, focal adhesion, and actin binding. (e) GO enrichment analysis was performed for downregulated proteins. The results showed that the downregulated proteins are mainly involved in the protein activation cascade, blood microparticles, and antigen binding. (f) KEGG pathway analysis was performed for upregulated proteins. The results showed that the upregulated proteins are mainly involved in the regulation of actin cytoskeleton, platelet activation, pathogenic Escherichia coli infection, and tight junction. (g) KEGG pathway analysis was performed for downregulated proteins. The results showed that the downregulated proteins are mainly involved in complement and coagulation cascades, Staphylococcus aureus infection, prion disease, and systemic lupus erythematosus.

Figure 2

Results of bioinformatics analysis and results of ELISA and western blotting. (a) GSEA analyses of gene sets for tight junctions. (b) Ridge plot reflecting protein expression of the experimental group and the control group. Blue: experimental group; red: control group. (c) Perform protein-protein interaction analysis on differential proteins related to tight junctions, and RhoA is at the center of the interaction. (d) Pearson correlation analysis was performed on proteins involved in the tight junction pathway of GSEA. Serum samples from 3 patients with newly diagnosed GO complicated with GD (experimental group) and 3 with GD without GO (control group). These serum samples were detected by WB and ELISA. (e) Western blotting for RhoA. (f) Results of FT3 ELISA and results of TRAb ELISA. (g) The grayscale value of the RhoA band (P < 0.0001).

Figure 3

RhoA, FT3, FT4, and TSH levels in patients in the GO-free and GO groups over a 7-month follow-up period. (a–c) After eliminating the interaction between hormones and time by the Greenhouse-Geisser correction method, monthly RhoA, FT4, and FT3 levels in the GO group were higher than those in the GO-free group. (d) After eliminating the interaction of hormones and time by the Greenhouse-Geisser correction method, no significant differences in TSH levels were observed between the GO-free and GO groups.

Figure 4

Differences in thyroid antibody titer at first diagnosis of GD. (a) The difference in TRAb titer levels was statistically significant (P = 0.004). (b, c) TPOAb (P = 0.972) and TgAb (P = 0.929) titer levels were not statistically significant. Median (line), interquartile range (box), and total range (whiskers) for TRAb, TPOAb, and TgAb titers between the GO-free group and the GO group when the two groups of patients were diagnosed with GD.

Figure 5

Receiver operating characteristic (ROC) curves were generated to evaluate the diagnostic significance of RhoA, FT3, FT4, TSH, TPOAb, and TgAb for onset of GO. (a) The area under the ROC curve (AUC) refers to the area formed by the ROC curve and the x-axis, (1, 0) -(1, 1). The basal FT3 level and difference between the basal FT3 level and endpoint value had diagnostic value. (b) TRAb titer also had diagnostic value and was superior to FT3. FT4, TSH, TPOAb, and TgAb titers had no diagnostic value. (c) The basal RhoA level and difference between the basal RhoA level and endpoint value had diagnostic value.