The skeletal and cardiac calsequestrin proteins share 68.4% amino acid homology. Previous work has shown that RNA levels of skeletal muscle calsequestrin are 4.7 times higher in extraocular muscle (EOM) than in masticatory skeletal muscle (jaw), and cardiac calsequestrin is expressed 2.7 times more in EOM. We postulate that up-regulation of casq2 gene in the thyroid of patients with GH may lead to the production of autoantibodies and sensitized T-lymphocytes, which cross-react with calsequestrin in the EOM of patients who develop ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO) is an orbital autoimmune disorder of the extraocular and eyelid muscles and surrounding connective and adipose tissue. Although mononuclear cell infiltration of orbital tissue is a characteristic feature of TAO the likely role of T lymphocyte reactivity against eye muscle antigens in the initiation of eye muscle damage in TAO has not been explored in detail. Therefore, we tested for T lymphocyte sensitisation to three eye muscle antigens namely, calsequestrin, G2s and flavoprotein (Fp), in patients with Graves' ophthalmopathy (GO), Graves' hyperthyroidism (GH) without ophthalmopathy and age and sex matched normal subjects. T lymphocyte reactivity was determined in a proliferation assay, results being expressed as stimulation index (SI). Mean ( +/- SE) SI for patients with GO, but not GH without ophthalmopathy, were significantly greater than that for normal subjects for calsequestrin and Fp, but not G2s. Mean ( +/- SE) SI was also significantly increased in patients with active ophthalmopathy, but not chronic ophthalmopathy, compared to normal subjects, for calsequestrin and Fp, but not G2s. Overall, positive lymphocyte proliferation to calsequestrin was demonstrated in 59% of patients with GO and 33% of patients with GH, which was significantly greater than in normals for both groups. In conclusion, we have demonstrated significant T lymphocyte reactivity to calsequestrin and, to a lesser extent, Fp in patients with GO. Because calsequestrin is located in the cell membranes of the eye muscle cell during the myotubular stage of the cell cycle, its targeting might be the primary reaction which leads to extraocular muscle inflammation in patients with GH.

Extra-ocular and upper eyelid (levator) muscle damage in thyroid orbitopathy may be due to autoimmunity against eye muscle auto antigens. The main antigen appears to be the calcium binding protein calsequestrin. In this study we have tested for T lymphocyte sensitization to calsequestrin in patients with Graves' disease, with and without orbitopathy, in standard proliferation assay. We have also tested total RNA prepared from thyroid tissue of patients with Graves' disease with and without orbitopathy for expression across 20,589 genes using micro array analysis technology. We were looking for differences in gene expression between the two groups which might provide information about the early thyroid events that lead to the development of eye muscle autoimmunity. Positive lymphocyte reactivity to calsequestrin was demonstrated in 59% of Graves' patients with orbitopathy, 33% without evident ophthalmopathy and in 43% of patients with Hashimoto's thyroiditis and upper eyelid retraction (UER). Two hundred and ninety six genes were identified to be differentially expressed between in patients with Graves' disease with and without orbitopathy. Of these, the cardiac calsequestrin gene CASQ2 was the most highly up regulated, 2.2-fold. The closely related skeletal muscle calsequestrin gene CASQ1 was also up-regulated, 4.1 fold, but this was not significant, while genes encoding the thyroid antigens thyroglobulin, thyroid peroxidase and the TSH-receptor were not differentially expressed. These findings provide further evidence for a prominent role of autoimmunity against calsequestrin in the pathogenesis of the eye muscle components of thyroid orbitopathy.