Treatment of the Graves’ hyperthyroidism with antithyroid medications or radioactive iodine was initiated according to the discretion of the referring endocrinologist. Medical records were reviewed for relevant clinical findings including symptoms of thyroid disease, the presence or absence of a goiter, and the presence or absence of eye findings. Demographic data for each patient were extracted and relevant past medical history and family history of autoimmune or thyroid disease recorded. The presence of any other, nonthyroid, medical conditions was recorded, as was each patient’s medications. In the case of positive eye findings, the type of finding was recorded. All available data were then entered into the study’s database. Serum T4 and TSH serum levels were recorded for each patient. In most cases, T4 and TSH measurements and EMAb testing were performed on the same serum sample. Otherwise, the serum for the T4 and TSH values was drawn 1–3 days before or after the EMAb test serum.

Enzyme-Linked Immunosorbent Assay (ELISA)

This method has been described in previous publications from this laboratory (11,12). Concentrations of antigens used in these studies were Fp 1.25 mg=ml and G2s 0.25 mg=ml. G2s fusion protein was purified using a pFLAG ATS Escherichia coli expression system. Human recombinant Fp was kindly supplied by Dr. B.A.C. Ackrell (University of California, San Francisco). Serum dilution was 1:50. Second antibody was a rabbit antihuman IgG at a dilution of 1:2000. Results were expressed as optical density (OD). For the purposes of this study, a positive test was taken as an OD > mean (þ2SD) for a panel of normal sera, for each antibody.

RESULTS

EMAb Studies from a Single Center

EMAb test results were compared with the thyroid diagnosis for each patient and analyzed by w2 testing. A statistically significant association, at 95% confidence, was found between both positive anti-Fp and positive anti-G2s results with the

Table 3 Correlation of Eye Muscle Antibodies with Thyroid Diagnoses from One Center

diagnosis of Graves’ disease. No statistically significant association was found between the EMAb test results and other thyroid diagnoses (Table 3). The clinical findings of goiter or positive family history were also compared to EMAb test results. No statistically significant associations were found (w2 tests, p ¼ NS). EMAb test results were compared to each patient’s clinical eye findings at the time of the test. No statistically significant associations were found between anti-Fp antibody results and eye findings (Table 4). A statistically significant association, at 95% confidence, was found between a positive anti-G2s antibody results and the presence of any eye findings. A positive anti-G2s antibody result was significantly associated with the ocular myopathy subtype of eye findings, but not with the congestive ophthalmopathy subtype (Table 4).

Table 4 Correlation of Eye Muscle Antibodies with Eye Findings from One Center

Pearson w2 p values.

ap < 0.05, NS ¼ not significant. bEOM ¼ extraocular muscle.

Table 5 Mean T4 and TSH Values for Each Group of Test Results from One Centre

NS ¼ not significant.

aDifference between EMAB positive and EMAB negative groups, assessed using two-tailed Student’s t-test.

Mean T4 level was slightly greater in EMAb-positive patients than in EMAb-negative patients, for both anti-Fp and anti-G2s antibodies (Table 5). Mean serum TSH was slightly lower in EMAb-positive patients compared to EMAb-negative patients. The differences in these means were not statistically significant (two-tailed Student’s t-tests, p ¼ NS).

Prospective Study of Graves’ Disease Patients

Data on the first 15 patients with Graves’ hyperthyroidism followed for 1 year or more are available. These comprise 12 females and 3 males, aged 18–56 (mean age 38), of whom 5 were smokers and 7 had a positive family history of thyroid or other autoimmune disease. Two of these patients were excluded from analysis due to insufficient follow-up. Of the remaining 13, 4 were being treated with antithyroid drugs and 9 received radioactive iodine. There were no differences in congestive or ocular muscle subtype between smokers and nonsmokers, or between patients with and without positive family history. None of the four medically treated patients developed EMAb or extraocular muscle involvement. On the other hand, anti-Fp or anti-G2s antibodies were detected in serum from eight of nine radio iodine-treated patients, in five cases following therapy. Six patients had eye muscle involvement as defined by increased intraocular pressure on upgaze, diplopia, or restricted motility, all of

whom received radioiodine and had positive EMAb tests. None of these results were statistically significant, possibly due to the small numbers involved.

DISCUSSION

The objective of this study was to assess the association of EMAb test results with the clinical presentation of TAO in a single center. Firstly, we showed that both EMAb tests were associated with Graves’ disease, with or without ophthalmopathy, but not with other thyroid diagnoses, in patients from a single center. A statistically significant association between positive anti-G2s results and the presence of clinical eye findings was demonstrated. Although a higher proportion of positive anti-Fp results was found in patients with TAO as compared to the controls, this difference did not achieve statistical significance.

While the pathogenesis of TAO is still debated, it does appear that autoimmune attack of extraocular muscle antigens plays an important role (2,5). In the current study, the association between EMAb test results and the clinical findings of TAO supports this hypothesis. In particular, the association between anti-G2s results and features of eye muscle disease suggests that these antibodies may play a role in the ocular myopathy subtype of TAO. However, a number of limitations in this study prohibit drawing any definitive conclusions. These include: a relatively small sample population, potential discrepancies in the description of eye findings reported, and little information available on other risk factors for TAO. Despite these limitations, this study did demonstrate some significant associations between EMAb testing and clinical findings. While not addressed in this study, positive EMAb test results in individuals without any signs of TAO may represent a risk factor for the development of future eye problems. If this were the case, many of the ‘‘false positives’’ found in this study may eventually develop into true positives, thereby improving the association found between the EMAb test results and the eye findings.