Context: Autoimmune polyendocrine syndrome type 1 (APS1) is characterised by multiple autoimmune endocrinopathies and results from mutations in the AIRE (autoimmune regulator) gene. Approximately 80% of patients present with hypoparathyroidism which is suggested to result from autoimmune responses against the parathyroid glands. The calcium-sensing receptor (CaSR), which plays a pivotal role in maintaining calcium homeostasis by sensing blood calcium levels and regulating release of parathyroid hormone, has been identified as a parathyroid autoantibody target in APS1. Aims: The main aims of this study were to characterise APS1 patient anti-CaSR autoantibodies in relation to their prevalence, disease associations, epitopes, specificity, IgG subclass and effects upon CaSR function, and to develop an ELISA to detect CaSR autoantibodies in patient sera. Methodology: Immunoprecipitation; radioligand binding assays; phage-display; ELISA; bioassays; protein expression. Results: Autoantibodies against the CaSR were detected in 16 out of 44 (36%) APS1 patients and in none of 38 healthy control subjects (P = < 0.0001). No statistically significant associations were found between the presence of CaSR autoantibodies and the disease manifestations of APS1 including hypoparathyroidism. The detection of CaSR autoantibodies had a specificity of 83%, and a sensitivity of 39% for the diagnosis of hypoparathyroidism. There were no significant associations between the presence of CaSR antibodies and either sex, age or disease presentation age. However, a significant association between a shorter APS1 duration (< 10 years) and positivity for CaSR autoantibodies was noted (P = 0.019). CaSR autoantibody epitopes were identified between amino acids 41-69, 114-126, 171-195 and 260-340 in the extracellular domain of the receptor. Autoantibodies against CaSR epitopes 41-69, 171-195 and 260-340 were exclusively of the IgG1 subclass. Autoantibody responses against CaSR epitope 114-126 were predominantly of the IgG1 with a minority of the IgG3 subclass. CaSR autoantibodies were analysed for their ability to increase Ca2+-dependent inositol phosphate accumulation in HEK293 cells expressing the CaSR. The results indicated that 4/16 (25%) APS1 patients had anti-CaSR-activating autoantibodies, suggesting that although the majority of APS1 patients do not have CaSR-stimulating autoantibodies, there may be a small minority of patients in whom the hypoparathyroid state is the result of functional suppression of the parathyroid glands. As part of this study, an ELISA was developed using the extracellular domain of the CaSR expressed in Escherichia coli as the antibody-capture antigen. Although this assay was able to detect the presence of autoantibodies in APS1 patient sera, the experimental method still requires further optimisations in order to attain a validated and robust ELISA, as this was not achievable during the present study. Conclusion: The study provides a detailed analysis of the characteristics of CaSR autoantibodies in APS1 patients, although further investigations are required to determine the exact role played by the autoimmune response against the CaSR in the pathogenesis of APS1.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:694497 |
| Date | January 2016 |
| Creators | Habibullah, Mahmoud |
| Contributors | Kemp, Elizabeth H. ; Weetman, Tony |
| Publisher | University of Sheffield |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://etheses.whiterose.ac.uk/14411/ |
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