Background: The pathogenesis of Graves’ ophthalmopathy (GO) and the mechanism for its link to thyroid autoimmunity is poorly understood. Our present research focuses on the role of the skeletal muscle calcium binding protein calsequestrin (CASQ1). Earlier studies showed that the CASQ1 gene was up-regulated in thyroid tissue from patients with GO compared to those with Graves’ hyperthyroidism (GH) without eye signs, however, the protein levels remained the same in both groups, raising the possibility that the orbital autoimmune reaction begins in the thyroid gland. Here, we measured the concentration of the CASQ1 protein in normal and Graves’ thyroid tissue, correlating levels with parameters of the eye signs, CASQ1 antibody levels and the CASQ1 gene polymorphism rs3838216, shown previously to be a risk factor for ophthalmopathy. Methods: The CASQ1 protein was measured by quantitative Western blotting. Following electrophoresis, samples were transblotted to polyvinyl difluoride (PVDF) membranes incubated with a 1:1000 dilution of a rabbit anti-CASQ1 antibody and incubated with an horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody, or anti-mouse antibodies for Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The protein concentrations were determined from density quantification using the Quantity One 4.4.0 ChemiDoc program and expressed as pmol/mg total protein by reference to CASQ1 standards. Results: Western blot analysis showed the presence of two forms of CASQ1 in the thyroid, of 50 and 60 kDa molecular weight respectively. In thyroid tissues, the mean (± SD) (GO 54.9±86.8 pmol/mg) concentration of the CASQ1 protein (GH 37.1±51.8 pmol/mg) was significantly reduced in patients with Graves’ disease , with or without ophthalmopathy, compared to normal thyroid tissues from control subjects with multi-nodular goitre or thyroid cancer (144.3±162.5 pmol/mg). The difference between GO and GH was not significant. The decreased CASQ1 protein levels in Graves’ thyroid tissues correlated with the homozygous genotype of the rs3838216 CASQ1 polymorphism. A two-fold increase in Levels of CASQ1 protein in toxic nodules compared to Graves’ hyperthyroidism was markedly significant. Conclusions: Decreased CASQ1 in the thyroid tissues of patients with Graves’ disease compared to normal thyroid tissues from control subjects may reflect consumption of the protein in the course of an autoimmune reaction against CASQ1 in the thyroid. Comparing CASQ1 protein levels in thyroid tissue from five patients with toxic nodular goitre (74.5±52.8) to controls showed no significance. The relative two fold increase in CASQ1 levels in toxic nodules compared to Graves’ disease suggests that this is due to the autoimmune reaction rather than the hyperthyroidism.
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- Graves’ disease
- thyroid gland
- Western blotting