Identifying the molecular basis for treatment resistance in a subset of myasthenia gravis patients of African ancestory

Myasthenia gravis (MG) is an autoimmune disease in which pathogenic antibodies block, target or destroy the acetylcholine receptors of the muscle endplate resulting in failure of neuromuscular transmission and fatigable weakness. We have previously shown that a subpopulation of South African MG patients of African genetic ancestry develop a severe extraocular muscle (EOM) phenotype whilst receiving standard immunosuppressive drug therapies. This phenotype associates with a functional c.-198C>G SNP (C>G SNP) in the regulatory region of decay accelerating factor (DAF), a complement regulatory protein, which is essential for protection against complementmediated damage. MG patients are treated with prednisone as the first-line immunosuppressant and frequently, an additional steroid-sparing agent, such as azathioprine, cyclosporin A or methotrexate. We hypothesised that MG patients with the C>G SNP when treated with immunosuppressant drugs, may have lower DAF protein levels contributing to increased susceptibility to autologous complement-mediated damage at their EOMs. This study tests this by comparing the effect of prednisone, azathioprine, cyclosporine and methotrexate individually and prednisone in combination with each of these steroid-sparing agents on wild-type and C>G DAF protein (western blotting) and mRNA (quantitative real time PCR) levels and promoter activity (luciferase reporter assays). These experiments were performed in EBV transformed lymphoblastoid cell lines from control individuals with wild-type DAF and MG patients carrying the C>G SNP. As a more representative model for this study the experiments were repeated in mouse skeletal muscle cells because there was no available EOM cell line. In support of the hypothesis of this study, prednisone, cyclosporin A and azathioprine individually was shown to repress C>G DAF protein levels while having either no effect on wild-type DAF or slightly activating it. Importantly, methotrexate was the only drug that was able to increase C>G DAF lymphoblast protein expression and therefore holds promise as a potentially effective treatment for MG patients with the C>G SNP. Moreover, using a calcein assay, clinically relevant doses of prednisone in combination with MG patient sera was shown to significantly increase the susceptibility of C>G DAF lymphoblasts to cell lysis (82% C>G DAF lymphoblasts vs. 9% wild-type DAF lymphoblasts). These results suggest that MG patient sera contain factor(s) that together with prednisone may also be responsible for the susceptibility of the EOMs in these patients to injury. The results show that the levels of DAF protein and mRNA did not always match which suggests that the drugs tested may regulate the DAF protein at a posttranscriptional level. In a mouse skeletal muscle model, Daf (equivalent to human wild-type DAF) protein expression was consistently repressed by prednisone treatment but activated by cyclosporine A, azathioprine and methotrexate. Furthermore, co-treatment of prednisone with either of the steroid-sparing drugs showed that azathioprine, and low doses of cyclosporin A and methotrexate were able to overcome the repressive effect of prednisone-only treatment on Daf expression. These observations indicate that the regulation of Daf may be species and/or cell-type specific. Together the results from this thesis suggest that in EOMs, where DAF is already downregulated, compared to other muscles, our MG patients with the C>G SNP may have an increased susceptibility to complement-mediated damage when treated with prednisone, which further represses C>G DAF expression.

Grantor dc:publisher.institution

Department of Molecular and Cell Biology

Year dc:date.issued

2013

Author dc:creator

• Auret, Jennifer Mary

Advisors dc:contributor.advisor

• Heckmann, Jeannine
• Prince, Sharon
• Abrahams, Amaal

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/11427/4237

OAI identifier oai:identifier

oai:open.uct.ac.za:11427/4237