A Translational Exploration of Graves’ Disease: Clinical, Laboratory and Animal Studies

Abstract

Graves’ disease (GD) is an autoimmune disorder, mediated by the thyroid stimulating hormone (TSH) receptor antibody (TRAb). This bioactive antibody causes thyrotoxicosis and it may contribute to extra-thyroidal manifestations, particularly Graves’ ophthalmopathy and pre-tibial myxoedema. Despite its pathogenic role, TRAb is not universally measured to confirm the diagnosis of GD in a hyperthyroid patient with a non-pathognomonic presentation. The modern TRAb assays only quantitate the serum TRAb level without determining its function. Furthermore, the mainstream animal models of GD focus upon induction of Graves’ disease and related immune responses while there are very few models that have been developed to explore the translational role of human TRAb. This PhD study was designed to investigate the role of TRAb in a clinical setting; in laboratory testing; and in an animal model. To address these issues, this thesis is divided into three complementary components: (1) establishing roles of TSH receptor antibody in clinical management of Graves’ disease [Chapters 2 & 3]; (2) comparing and contrasting TSH receptor antibody diagnostic assays [Chapters 4 & 5]; and (3) undertaking an in-vivo pilot pre-clinical model of Graves’ disease to investigate the effect of TSH receptor antibody in mice [Chapter 6]. In the clinical studies, based on the review of our in-house GD database, TRAb measurement was found to be comparable to thyroid scintigraphy in confirming the diagnosis in most of the patients who presented with GD. In patients where GD recurred, the initial TRAb level was not a significant risk factor. However, younger age of disease onset was strongly associated with recurrence. TRAb was shown to be an independent risk factor associated with liver enzyme abnormality at diagnosis. However, the relationship was attenuated by multivariate analysis including both FT3 and FT4, showing liver enzyme abnormality was associated more strongly with the thyroid hormone status than the TRAb level. GGT was the most commonly elevated liver enzyme in GD, followed by ALT, ALP and AST, most of which normalised with hyperthyroidism treatment. There also appeared to be an ethnic difference in the pattern of liver enzyme abnormality. The laboratory study compared the current FDA-approved Thyroid Stimulating Immunoglobulin (TSI) assays and found ThyretainTM was the only bioassay able to distinguish between the different functional status of TRAb. At the same time, an in-house TSI assay using cAMP measurement was established using a commercial Chinese Hamster Ovary cell line overexpressing human TSH receptor. The cell line was also used to explore the TSH receptor intra-cellular signalling pathway associated with TRAb binding. Data demonstrated that while TSH stimulates both cAMP cascade and Phosphatidylinositol 4,5-bisphosphate (PIP2) pathway, TRAb exclusively triggers the cAMP cascade. In the third main section of studies in GD, a pilot translational murine model of GD was commenced by injecting the purified serum of a patient, who had pathognomonic GD and very high TRAb level, into mice. Thyroid overactivity, both biochemically and histologically, was induced. However, it was not a sustained response and therefore further studies are needed to explore the options for augmenting an established response and to examine the subsequent physiological changes induced. Overall, this PhD thesis has refined Graves’ disease clinical linkages with TRAb in liver enzymes and risk recurrence. It has also provided clarity in TRAb and related assays. Furthermore, it has aided in the exploration of an animal model of GD. In time, it is envisaged that the outcomes of this work will serve as a foundation in the establishment of a GD Centre of Excellence with TRAb and related laboratory measures as a focal point.