THYROID Volume 16, Number 10, 2006 © Mary Ann Liebert, Inc. Study of Serum Antibodies Against Three Eye Muscle Antigens and the Connective Tissue Antigen Collagen XIII in Patients with Graves’ Disease With and Without Ophthalmopathy: Correlation With Clinical Features Bamini Gopinath, 1 Reilly Musselman, 1 Cherie-Lee Adams, 1 Junichi Tani, 1 Nicole Beard, 2 and Jack R. Wall 1 Objective: The extraocular muscles are one of the primary tissues implicated in the autoimmune-mediated in- flammation of thyroid-associated ophthalmopathy (TAO). Our aim was to determine the prevalence and level of antibodies against three candidate eye muscle antigens and the orbital fibroblast membrane antigen colla- gen XIII, in well-characterized patient groups. Study design and patients: The study cohort consisted of patients with Graves’ hyperthyroidism with and without ophthalmopathy, controls patients with other thyroid or other autoimmune disorders and healthy subjects. The presence of eye muscle antibodies was determined using an optimized and standardized enzyme-linked immunosorbent assay. We measured antibodies against (i) the 67- kDa flavoprotein (Fp) subunit of the mitochondrial enzyme succinate dehydrogenase; (ii) G2s, a 141 amino acid fragment of the winged-helix transcription factor FOXP1; (iii) calsequestrin, a 63-kDa calcium-binding protein; and (iv) collagen XIII, a connective tissue protein that is closely linked to the congestive ophthalmopathy sub- type of TAO. Eye muscle antibody levels were correlated with clinical diagnosis and presence or not of oph- thalmopathy. Results: Prevalences of positive antibody tests to calsequestrin (75.0%) and collagen XIII (43.8%) were significantly greater in Graves’ disease (GD) patients with ophthalmopathy than in healthy subjects, whereas modest significance was demonstrated with antibodies against Fp, but not G2s. Significantly greater serum levels of antibodies against calsequestrin, G2s, and collagen XIII, but not Fp, were found in GD patients with ophthalmopathy compared to control patients without eye disease and healthy subjects. Conclusions: Calse- questrin and collagen XIII antibodies are the most specific to TAO, whereas antibodies against G2s, and to a lesser extent Fp, are also markers of ophthalmopathy, but less reliable. These results are unique in that it is the first time the significance of a panel of three candidate eye muscle antibodies and a connective tissue antibody have been evaluated in the same patients with ophthalmopathy. 967 Introduction O PHTHALMOPATHY IS A COMMON MANIFESTATION of autoim- mune thyroid disease occurring in 25% to 50% of pa- tients with Graves’ hyperthyroidism (1–3). Because ophthal- mopathy is also present in a small proportion of patients with Hashimoto’s thyroiditis, the combination of Graves’ hy- perthyroidism or Hashimoto’s thyroiditis and ophthal- mopathy is best called “thyroid-associated ophthalmopathy (TAO)” (4). TAO comprises two main subtypes, namely, con- gestive ophthalmopathy and ocular myopathy (5). Conges- tive ophthalmopathy is characterized by swelling of the eyelid and peri-orbital connective tissues, whereas ocular myopathy reflects an autoimmune attack against the ex- traocular muscle tissue manifest as impaired extraocular muscle function and diplopia. The pathogenesis of TAO is best explained by an autoim- mune reaction directed against a thyroid and orbital tissue shared antigen(s) (6,7). It has been proposed previously that the initial inflammatory reaction occurs in the orbital and adipose connective tissues leading to orbital fibroblast stim- ulation and congestive eye signs, with secondary damage to the eye muscle fibres (8,9). A good candidate for the con- nective tissue antigen is the thyrotropin receptor (TSH-r), which has been localized, in both orbital fat and eye muscle tissue (10). However, because diplopia and eye muscle dys- function are sometimes found in the absence of periorbital congestion (11,12) an alternative hypothesis is that the ex- traocular muscles are also primary targets in TAO. Several antibody markers of immune-mediated damage to eye muscle have been identified, and the great majority of 1 Department of Medicine, The University of Sydney, Nepean Hospital, Penrith, New South Wales 2751, Australia. 2 Division of Molecular Bioscience, The John Curtin School of Medical Research, Canberra City, ACT 2601, Australia.