Thyroid Stimulating Antibodies Are Highly Prevalent in Hashimoto s Thyroiditis and Associated Orbitopathy

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1 ORIGINAL ARTICLE Thyroid Stimulating Antibodies Are Highly Prevalent in Hashimoto s Thyroiditis and Associated Orbitopathy George J. Kahaly,* Tanja Diana,* Jennifer Glang, Michael Kanitz, Susanne Pitz, and Jochem König Departments of Medicine I (G.J.K., T.D., J.G., M.K.), Ophthalmology (S.P.), and Institute of Medical Biostatistics, Epidemiology and Informatics (J.K.), Johannes Gutenberg University Medical Center, Mainz, Germany Context: Thyroid-associated orbitopathy (TAO) rarely occurs in patients with Hashimoto s thyroiditis (HT). Objective: There is evidence that TSH receptor stimulating antibodies (TSAb) play a role in the pathogenesis of TAO. In this report, the prevalence of TSAb in HT patients with and without TAO was studied. Design: This is a longitudinal observational study. Setting: The study took place in an academic joint thyroid-eye clinic. Subjects: A total of 1055 subjects were included. Methods: TSAb was measured with a Food and Drug Administration cleared bioassay that uses Chinese hamster ovary cells expressing a chimeric TSH receptor and a camp response elementdependent luciferase. Results of TSAb activity were reported as percentage of specimen-to-reference ratio (SRR%, cutoff 140%). Main Outcome Measure: We measured the association of TSAb with the risk of TAO in patients with HT. Results: Of 700 consecutive and unselected patients with HT, 44 (6%) had overt TAO. Patients with HT TAO were older (P.001), heavier smokers (P.032), and clustered less with autoimmune diseases (P.005). All healthy controls were TSAb negative. In contrast, serum was TSAb positive in 30/44 (68.2%) and 36/656 (5.5%, P.001) patients with HT TAO and HT, respectively. Compared to patients with HT only, serum TSAb levels were higher in HT TAO (median SRR%, 25th and 75th percentiles): 45, vs 192.5, , P.001. Highest TSAb values were noted in patients with active and severe TAO vs those with mild and inactive TAO: 486, vs 142, ; P.001. The odds ratio of TSAb positivity for the risk of TAO adjusted for gender and age was 55.9 (95% confidence interval [CI], , P.0001), whereas the odds ratio per 10-fold change in TSAb SRR% (quantitative TSAb) was 133 (95% CI, , P.0001). The area under the receiver operating characteristic curve for qualitative and quantitative TSAb was 87.2% (95% CI, ) and 89.4% (95% CI, ), respectively. Conclusions: TSAb is strongly associated with TAO in HT and TSAb may contribute to the pathophysiology of TAO. (J Clin Endocrinol Metab 101: , 2016) ISSN Print X ISSN Online Printed in USA Copyright 2016 by the Endocrine Society Received January 27, Accepted March 4, First Published Online March 10, 2016 * G.J.K. and T.D. equally contributed equally as first authors to the manuscript. Abbreviations: CHO, Chinese hamster ovary; CI, confidence interval; GD, Graves disease; HT, Hashimoto s thyroiditis; OR, odds ratio; RLU, relative light units; ROC, receiver operating characteristic; SRR%, percentage of specimen-to-reference ratio; TAO, thyroid-associated orbitopathy; TBII, TSH binding inhibitory immunoglobulins; TBAb, thyroid blocking autoantibodies; Tg, thyroglobulin; TPO, thyroid peroxidase; TSAb, thyroid stimulating autoantibodies; TSHR, TSH receptor 1998 press.endocrine.org/journal/jcem J Clin Endocrinol Metab, May 2016, 101(5): doi: /jc

2 doi: /jc press.endocrine.org/journal/jcem 1999 Hashimoto s thyroiditis (HT) or chronic autoimmune thyroiditis is the most common cause of hypothyroidism in both adults and children in areas of iodine sufficiency (1, 2). Thyroid failure is seen in up to 10% of the population and its prevalence increases with age (3). It is characterized clinically by gradual thyroid failure, goiter formation, or both, resulting from autoimmune-mediated destruction of the thyroid gland involving apoptosis of thyroid epithelial cells. Patients have high serum concentrations of antibodies against one or more thyroid antigens; diffuse lymphocytic infiltration of the thyroid, which includes predominantly thyroid-specific B and T cells; and follicular destruction. The cause of HT is thought to be a combination of genetic susceptibility and environmental factors. The familial association with Graves disease (GD) and the fact that GD may evolve into HT (and vice versa) indicate that the two disorders are closely related pathophysiologically despite their different clinical presentations (1, 4, 5). TSH receptor (TSHR) antibodies of the stimulating variety were first identified by their prolonged thyroid-stimulating activity when serum from patients with GD was injected into animals (6). Subsequently, IgG fractions of serum from these patients were found to have thyroidstimulating actions qualitatively similar to those of TSH in many bioassays and to block binding of radiolabeled TSH to thyroid membranes; the IgG fraction contained TSHR antibodies that acted as TSH agonists (7). Later, with the use of TSH binding competition assays, TSHR antibodies were detected in the serum of some patients with HT (8, 9). However, these antibodies, which are usually polyclonal (5), block the action of TSH more than activating thyroid tissue. Stimulating and blocking antibodies bind to the leucine rich repeat region of the extracellular domain of the TSHR (10). Patients who had HT and GD at different times have also been described; at the appropriate times, their serum contained a predominance of TSHR-blocking or TSHR-stimulating (TSAb) antibodies (5, 11 13). TSHR mrna has been detected in several tissues, and TSH can modulate bone cell and adipocyte function (14, 15). The orbital expression of the TSHR has been implicated in thyroid-associated orbitopathy (TAO), which rarely occurs in patients with HT. Because TSAb plays a role in the pathogenesis of TAO (16 18), in a longitudinal observational study, we looked for the presence of TSAb in HT patients, with and without associated TAO, followed at a specialized referral center for autoimmune thyroid diseases. The main outcome measure of this study was the association of TSAb with the risk of HT TAO. Secondary outcome measures were the prevalence and serum levels of TSAb in patients with combined HT TAO and the correlation of TSAb levels with the clinical phenotype of TAO. Materials and Methods Patients The medical records or files of 700 consecutive and unselected patients with HT were analyzed and supplemented by serial measurements taken in 228 patients within this longitudinal study. All patients were followed at the endocrine outpatient clinic and at the joint thyroid-eye clinic of the Johannes Gutenberg University Medical Center, Mainz, Germany. HT was defined as the presence of at least 5-fold increased serum level of antibodies to thyroid peroxidase (TPO) with or without positive thyroglobulin autoantibodies, and euthyroidism or hypothyroidism, and a heterogeneous hypoechoic pattern in thyroid ultrasound imaging, and, when available, a low uptake in the thyroid radionuclide scintigraphy. All patients were screened for signs of orbital involvement. TAO was classified as clinically active/inactive and as mild/moderate to severe, and sight-threatening as recommended by the European Group on Graves orbitopathy (19). A group of 53 patients with GD was also included in this study. GD was defined as overt hyperthyroidism with positive TSHR antibodies, a diffuse and markedly increased radionuclide uptake in the thyroid scan, and an enlarged gland with typical thyroid storm pattern in the Doppler ultrasound imaging of the neck. Further, a large group of 302 healthy persons devoid of thyroid, endocrine, and autoimmune disorders served as controls. The study protocol was consistent with the principles of the Declaration of Helsinki. Written informed consent to be included in the database and to draw blood was obtained from all participants and approval by the local ethics committee was obtained. This study was observational and did not include any interventions aside from those commonly falling within the daily routine and no decoded patient-related data were passed to third parties. Thyroid function and autoantibody tests Serum levels of TSH, free T3, free T4, anti-tpo and antithyroglobulin (Tg) antibodies (CLIA, Abbott), thyroid binding inhibiting immunoglobulins (TBII, automated binding assay, Kryptor), and Tg (ECLIA, Cobas e411, Roche Diagnostics) were measured in all patients with commercially available kits according to the manufacturers instructions. TSAb bioassay Serum TSAb activity was measured with the Thyretain bioassay (Quidel) according to the manufacturer s instructions (20). Briefly, a frozen vial of Chinese hamster ovary (CHO)-MC4 cells were seeded and grown to a confluent cell monolayer in 96-well plates for hours. Samples, positive, reference, and normal controls were diluted 1:11 in reaction buffer, added to the cell monolayers, and each plate was incubated for 3 hours at 37 C, 5% CO 2. Subsequently, the CHO-MC4 cells were lysed and the relative light units (RLU) were quantified in a luminometer (Tecan Infinite M200). The results were reported as percentage of specimen-to-reference ratio (SRR%) and converted into miu/l. SRR% values were calculated according to the following formula: SRR% Average TSAb specimen RLUs/average reference standard RLUs 100. The percentage coefficient variation was calculated according to the formula: percentage coefficient variation (SD RLU specimen/mean RLU specimen) 100. The assay cutoff is at the SRR% of 140.

3 2000 Kahaly et al TSAb in Hashimoto s Orbitopathy J Clin Endocrinol Metab, May 2016, 101(5): Table 1. Demographic Data of 700 Consecutive Patients With Autoimmune HT or Without or With TAO HT HT TAO P Value N (%) 656 (94) 44 (6) Age (y) 35.2 ( ) 49.3 ( ).001 Duration of HT (y) 0.9 (0 5.6) 2.4 (0.1 9).028 Female 543 (83) 37 (84).823 Children ( 18 y) 171 (26.1) 4 (9.1).012 Heavy smoker (20 cigarettes/d/y) 84 (16.6) 12 (30).032 L-T4 therapy, N (%) 376 (58.4) 28 (65.1).385 Monoglandular disease 345 (52.6) 33 (75).004 Polyglandular disease 147 (22.4) 2 (4.5).005 HT type 1 diabetes 107 (16.3) 1 (2.3).013 HT Addison s disease 23 (3.5) 1 (3.5).663 HT autoimmune gastritis 68 (10.4) 3 (6.8).450 HT pernicious anemia 25 (3.8) 2 (4.5).802 HT celiac disease 36 (5.5) Values are represented in median and quartiles (25% and 75% percentiles). Thyroid-blocking antibody bioassay CHO cells expressing a chimeric TSHR and a camp response element dependent luciferase were used to measure blocking activity as previously described (21, 22). Blocking activity was defined as percent inhibition of luciferase expression relative to induction with bovine TSH alone (100 (1-sample bovine TSH/bovine TSH alone). The assay cutoff for the chimeric CHO cells is at 40% inhibition. Serum thyroid-blocking antibody (TBAb) activity was measured in all TSAb-positive samples of patients with HT, in all patients with HT TAO, in all control patients with GD, as well as in all healthy controls. Statistical analysis For statistical data analysis, we used the statistics software program SPSS version 23 (SPSS), and the R package (R Core Team, 2015). All P values are two-sided and are, descriptively, referred to as statistically significant if they do not exceed.05. We statistically compared any two groups of patients with respect to serum levels (of TSAb and TBII at first measurement) by Mann Whitney U test. The effect of serum levels on the probability of having TAO was quantified by fitting a logistic model to first measurements. We transformed TSAb levels by taking the logarithm to the base of ten and reported results as odds ratio (OR) per 10-fold change in TSAb value. We fitted another logistic model using TSAb positivity defined via the cutoff 140 SRR%, which we refer to as qualitative TSAb finding. We adjusted both models for age and gender. We assessed the diagnostic accuracy of TSAb for indicating TAO by construction of receiver operating characteristic (ROC) curves for both quantitative and qualitative TSAb finding and reported the area under ROC curve with 95% confidence intervals (CI). We fitted an additive model for correlated data to obtain smooth estimates of the time course of TSAb. We defined time on study as the time since first TSAb measurement. We fitted the model separately to patients with and without TAO, included a random intercept, and allowed for a smooth fixed effect of time on study (R package mgcv). Results Demographic and clinical data The demographic and clinical data of the 700 consecutively seen and unselected HT patients (aged 4 weeks to Table 2. Serological Data of 700 Consecutive Patients With HT Without or With TAO and of 53 Control Patients With GD Without or With TAO HT HT TAO P Value GD GD TAO P Value N (%) 656 (94) 44 (6) TSAb-positive patients, N (%) 36 (5.5) 30 (68.2) (100) 27 (100) TSAb-positive samples, N (%) 67/1128 (8) 103/141 (73).001 TSAb SRR% (patients) 45 (35 65) 193 ( ) ( ) 654 ( ).001 TSAb SRR% (samples) 46 (35 63) 304 ( ).001 TSAb SRR% in smokers 43 (34 61) 176 (85 291) ( ) 645 ( ).019 TSAb SRR% (hypothyroid patients) 47 (35 67) 189 ( ).001 TBII positive, N (%) 35 (5) 22 (51) (100) 27 (100) TBII ( 1.8 IU/liter) 0.5 ( ) 1.8 ( ) (3.6 16) 12 (6 32).027 TSH ( mu/liter) 1.8 ( ) 0.9 ( ) ( ) 0.01 ( ).146 FT3 ( pg/ml) 3 ( ) 2.9 ( ) ( ) 11 ( ).182 FT4 ( ng/dl) 1.2 (1 1.4) 1.1 (1 1.3) ( ) 2.2 ( ).218 Tg ( 55 ng/ml) 3.3 ( ) 2.9 ( ) (41 224) 249 (97 815).043 Tg-Ab ( 4.1 IU/ml) 59 ( ) 74 ( ) (2.4 40) 5.4 ( ).593 TPO-Ab ( 6 IU/ml) 182 (38 526) 158 (73 903) (37 956) 299 (4 786).693 Values are represented in median and quartiles (25% and 75% percentile).

4 doi: /jc press.endocrine.org/journal/jcem years) with and without TAO are shown in Table 1. Patients with HT and TAO were significantly older and included more heavy smokers ( 20 cigarettes per day per year). This group encompassed fewer children and had significantly fewer patients with associated autoimmune endocrine and nonendocrine disorders. No differences were noted in gender distribution, prevalence of hypothyroidism, or rate of levothyroxine replacement. Of the 53 GD patients (42 female median, [25% 75% percentile]; range) age 47 years [34 55]; years), 26 and 27 patients had no (11/26 smokers, 42%) or overt TAO (14/27 smokers, 52%), respectively. Thyroid ultrasound imaging showed a significantly larger thyroid volume in GD patients compared to HT subjects. All 302 healthy control donors(155 female, aged 25, 24 29; years) had a negative personal and family history of thyroid, endocrine, autoimmune, and tumor diseases. All were negative for thyroid-related autoantibodies and had baseline levels of serum TSHFT3/FT4 within the normal range. Thyroid-related parameters and autoantibodies Baseline serum TSH was significantly higher in patients with HT without eye disease vs those with TAO (Table 2). All patients with HT were TPO antibody positive. No relevant differences were noted between the two HT Figure 1. Receiver operating characteristic (ROC) curve for discriminating between patients with and without TAO by quantitative and qualitative TSAb measurements (using the cut-off 140 SRR %). groups in levels of serum free T4 and free T3, serum Tg, and in titers of anti-tg and anti-tpo autoantibodies. In contrast, in all patients with GD, baseline serum TSH was suppressed and serum free T3 was increased. Free T4 and serum thyroglobulin were also increased in 49/53 (92.5%) GD patients, whereas 13 (24.5%) and 40 (75.5%) patients with GD were TPO antibody negative and positive, respectively. TSAb in HT vs HT TAO All healthy controls were TSAb negative (median SRR%, 25th and 75th percentile: 52, ). In contrast, TSAb were present in all 53 patients with GD (Table 2) with highest peak values in patients with associated TAO. In comparison, TSAb was present in only 9% of patients with HT. Interestingly, TSAb was present in only 5.5% of HT patients without TAO, but in more than twothirds in HT patients with TAO. Furthermore, approximately three-quarters of 141 serum samples from HT TAO patients were TSAb positive. The ROC curve for both quantitative and qualitative TSAb measurements during the first visit of the HT patients is shown in Figure 1. Furthermore, in comparison to TSAb, TBII positivity was less prevalent in the HT TAO group. Compared to patients with HT only, serum TSAb levels were markedly higher in HT TAO. Also, in smokers, serum TSAb levels were strongly higher in HT TAO vs HT only. In comparison, serum TSAb were more likely to be positive in HT patients with recent onset and TSAb levels negatively correlated with the duration of the disease in patients with thyroidal involvement only (r 0.169, P.001). However, this was not true in those with associated TAO (r 0.131, P.396), where TSAb positivity was sustained. In samples with combined HT TAO, serum TSAb titers correlated (r 0.628, P.001) with serum TBII, whereas in the HT group, TSAb correlated positively with TPO antibodies (r 0.243,P.001),Tgantibodies(r 0.171, P.001), serum TSH (r 0.191, P.001), and negatively with free T4 (r 0.180, P.001) and free T3 (r 0.113, P.01). Serum TSHR blocking activity (TBAb) was measured in all TSAbpositive samples of patients with HT, in all patients with TAO, in all GD patients as well as in all healthy controls. All 170 TSAb-positive HT samples, all

5 2002 Kahaly et al TSAb in Hashimoto s Orbitopathy J Clin Endocrinol Metab, May 2016, 101(5): Table 3. Clinical and Serological Data in 44 Patients With HT and TAO According to the Clinical Activity and Severity of TAO Mild/Inactive Severe/Active P Value Patients, n 44 (%) 29 (66) 15 (34) Samples, n Age (y) 46 (31 55) 51 (44 57).185 Female 25 (86.2) 12 (80).594 Smoker 9 (35) 3 (20).385 Hypothyroid 20 (69) 10 (67).877 Euthyroid 7 (24) 3 (20).756 Hyperthyroid 2 (7) 2 (13).481 (subclinical) a TSAb positivity 13 (44.8) 15 (100).001 (patients) TSAb positivity 35 (48.6) 68 (99).001 (samples) TSAb SRR% levels 142 (73 193) 486 ( ).001 (patients) TSAb SRR% levels 133 (68 195) 512 ( ).001 (samples) TBII positivity 10 (34) 12 (80).006 (patients) TBII levels ( 1.8 IU/liter) 0.7 ( ) 6.3 (2.1 16).001 Values are represented in median and quartiles (25% and 75% percentiles) are shown. a Subclinical hyperthyroidism with serum TSH below the normal range and normal serum levels of free T4 and free T3. TAO samples, all GD sera, and all samples from the control donors tested negative for the presence of serum TBAb. TSAb and clinical activity/clinical severity of TAO Two-thirds of the patients with HT TAO had a mild and inactive thyroid eye disease (Table 3). TSAb was positive in all 15 patients with clinically moderate-to-severe and active HT TAO. In contrast, only 80% of sera from these patients were positive when measured for TBII. Also, serum TSAb levels were markedly higher in patients with moderate-to-severe/active eye disease vs those with mild and inactive TAO independent of the thyroid function and of the smoking status. Duration of thyroid eye disease was 6 years (range, years) in all 44 TAO patients, whereas it was shorter in patients with active/moderateto-severe vs those with inactive/mild disease (4 years, years vs 7 years, years; P.132). No significant differences were noted between the two groups pertaining to gender, age, thyroid function, and smoking. Logistic regression analysis and time course The OR of TSAb positivity for the risk of TAO adjusted for gender and age (Table 4) was 55.9 (95% CI, , P.0001). For quantitative TSAb, the OR per 10-fold change in TSAb SRR% adjusted for gender and age was Table 4. Association of the First TSAb Measurement With TAO Assessed by Means of Logistic Regression Predictor Adjustments OR (95% CI) P Value TSAb positive None 36.9 ( ).0001 (SRR 140%) Age, sex 55.9 ( ).0001 TSAb (per 10- fold change) 133 (95% CI, , P.0001). The area under the ROC curve was 87.2% (95% CI, ) for qualitative TSAb and 89.4% (95% CI, ) for quantitative TSAb (cutoff SRR% 140). Analysis of longitudinal TSAb measurements (203 without and 25 patients with TAO) did not reveal any long-term trends (Figure 2). Discussion None 102 (38 273).0001 Age, sex 133 (45 390).0001 To the best of our knowledge, this is the first study that demonstrates a strong association between functional TSAb and thyroid eye disease in a large group of consecutive, unselected, well-characterized, and well-defined patients with autoimmune HT. All HT patients had high titers of anti-tpo autoantibodies, a typical hypoechoic and strongly heterogeneous ultrasound imaging pattern of a small thyroid gland, and the vast majority was hypothyroid or euthyroid on levothyroxine replacement. The very few HT patients who were mildly or subclinically hyperthyroid had a HT of very recent onset and a normal or slightly enlarged thyroid gland. In HT patients, TSAb was highly prevalent in those with clinically overt and associated TAO compared to HT patients without eye disease. Also, logistic regression analysis and the ROC curve clearly demonstrated the strength of association and the high discriminative power of the first qualitative and quantitative serum TSAb measurement to discriminate between patients with and without TAO. All patients with HT TAO were TBAb negative. The presence of TSAb was neither related to gender nor to thyroid function. On the other hand, patients with isolated thyroidal involvement had a very low percentage of TSAb positivity that was primarily present in those with recent onset of the disease. This phenomenon maybe related to the hypertrophy of the thyroid gland which can be observed at the early stage of the disease. This is in line with the registered negative correlation of the TSAb positivity with the duration of the disease as well as with the fact that all patients with small thyroid volume and/or long-term chronic disease were TSAb negative. There are very few articles in the literature describing the association of HT with orbitopathy; most of them are case reports encompassing only one or two patients with

6 doi: /jc press.endocrine.org/journal/jcem 2003 Figure 2. Time course of TSAb. Predicted values and 95% confidence bands based on fitting an additive model for correlated data to longitudinal TSAb measurements from 25 patients with and 203 patients without TAO with more than one measurement. For patients with TAO, measurements did not start before the year of reported onset of TAO. The model describes the average outcome, which is allowed to depend smoothly on time of study. HT-associated orbitopathy (23 25). These clinical demonstrations report on the phenotype, clinical course, and management of the disease; however, no published reference has systematically dealt with this association. On the other hand, a French group has shown that thyroid status can modulate thyroid autoimmunity expression, such as TSHR antibodies and TPO antibodies, in patients with euthyroid or hypothyroid goitrous HT (26). The same authors demonstrated in vivo, that, in contrast to TSH, TSHR antibodies are related to both thyroid hormonal production and volume in euthyroid or hypothyroid patients with HT (27). To date, however, there are no scientific papers which have evaluated patients with HT for functional anti- TSHR antibodies measured using cell-based bioassays and anti-tshr binding antibodies measured in binding assays with commercially available semiautomated devices. Anti-TSHR antibodies measured in binding assays do not differentiate between neutral, blocking, and stimulating antibodies and do not allow an accurate assessment of a possible switch and/or variation of ratios between these antibodies. We have previously noted a higher analytical performance of the TSHR bioassay when compared to an automated binding assay which enabled us to detect anti- TSHR antibodies activity at significantly lower concentrations (28). This may explain why, in the current paper, the prevalence of TSAb was higher than TBII in our patients with HT and TAO. Taken together, the observations described in this study suggest that stimulating TSHR antibodies may be relevant to the pathophysiology of orbital involvement in HT. Although no causality has been proven, that more than two-thirds of the HT patients with associated orbitopathy are TSAb positive points to the possibility that circulating TSAb binding to TSHR-bearing target cells in orbital connective tissue contribute to the clinical phenotype of thyroid eye disease. Furthermore, because the prevalence of TSAb in HT TAO patients is higher than the prevalence of TBII, it can be further speculated that the functionality of TSAb may be directly relevant to the pathophysiology of the orbitopathy and that TSAb may stimulate TSHR-dependent signaling pathways in eye tissue that is involved in promoting the orbital pathology. Whether these signaling pathways are similar to what occurs in thyroid tissue that involves camp-mediated transduction or whether other signaling pathways are involved will be an interesting area of future investigation. Demographically and as previously noted when looking at a large collective of 1400 consecutive patients with thyroid autoimmunity at our institution (29), patients with HT and TAO had significantly less associated endocrine and nonendocrine autoimmune diseases (Table 1). Most specifically, the prevalence of the autoimmune polyglandular syndrome was 10-fold higher in patients with HT alone in contrast to those with extrathyroidal orbital involvement. Thus, HT clusters significantly with other autoimmune disorders, but to a much lower degree in HT patients with TAO, suggesting a protective effect of orbitopathy on the development of nonthyroidal autoimmune disease. Alternatively, instead of the suggested protective effect of TAO on nonthyroid autoimmunity, these findings could also point toward the presence/coexistence of GD in these patients. GD is less associated with nonthyroid autoimmunity than HT, and the coexistence of GD may be the protective factor. Furthermore, as recently shown in a

7 2004 Kahaly et al TSAb in Hashimoto s Orbitopathy J Clin Endocrinol Metab, May 2016, 101(5): large series of consecutive patients with TAO (30), patients with euthyroid or hypothyroid HT and TAO had a milder ophthalmic phenotype compared to patients with GD and associated eye disease. This is in line with the noted findings in this paper, where less than one-third of HT patients with TAO showed an active and severe orbitopathy. In conclusion, serum TSAb is strongly associated with TAO in patients with HT and, as with GD (16 18), TSAb may be relevant to the pathophysiology of the associated orbital involvement. Acknowledgments We thank Paul D. Olivo, MD, PhD, Washington University, St Louis, Missouri, for the thorough and critical evaluation of the manuscript. This paper includes parts of the doctorate thesis of J. Glang. Address all correspondence and requests for reprints to: G.J. Kahaly, Johannes Gutenberg University Medical Center, Mainz , Germany. kahaly@ukmainz.de. 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