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

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dc.contributor.advisorHeckmann, Jeannineen_ZA
dc.contributor.advisorPrince, Sharonen_ZA
dc.contributor.advisorAbrahams, Amaalen_ZA
dc.contributor.authorAuret, Jennifer Maryen_ZA
dc.date.accessioned2014-07-30T17:31:42Z
dc.date.available2014-07-30T17:31:42Z
dc.date.issued2013en_ZA
dc.descriptionIncludes abstract.
dc.descriptionIncludes bibliographical references.
dc.description.abstractMyasthenia 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.en_ZA
dc.identifier.apacitationAuret, J. M. (2013). <i>Identifying the molecular basis for treatment resistance in a subset of myasthenia gravis patients of African ancestory</i>. (Thesis). University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology. Retrieved from http://hdl.handle.net/11427/4237en_ZA
dc.identifier.chicagocitationAuret, Jennifer Mary. <i>"Identifying the molecular basis for treatment resistance in a subset of myasthenia gravis patients of African ancestory."</i> Thesis., University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 2013. http://hdl.handle.net/11427/4237en_ZA
dc.identifier.citationAuret, J. 2013. Identifying the molecular basis for treatment resistance in a subset of myasthenia gravis patients of African ancestory. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Auret, Jennifer Mary AB - 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&gt;G SNP (C&gt;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&gt;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&gt;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&gt;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&gt;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&gt;G DAF lymphoblast protein expression and therefore holds promise as a potentially effective treatment for MG patients with the C&gt;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&gt;G DAF lymphoblasts to cell lysis (82% C&gt;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&gt;G SNP may have an increased susceptibility to complement-mediated damage when treated with prednisone, which further represses C&gt;G DAF expression. DA - 2013 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2013 T1 - Identifying the molecular basis for treatment resistance in a subset of myasthenia gravis patients of African ancestory TI - Identifying the molecular basis for treatment resistance in a subset of myasthenia gravis patients of African ancestory UR - http://hdl.handle.net/11427/4237 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/4237
dc.identifier.vancouvercitationAuret JM. Identifying the molecular basis for treatment resistance in a subset of myasthenia gravis patients of African ancestory. [Thesis]. University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 2013 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/4237en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDepartment of Molecular and Cell Biologyen_ZA
dc.publisher.facultyFaculty of Scienceen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherCell Biologyen_ZA
dc.titleIdentifying the molecular basis for treatment resistance in a subset of myasthenia gravis patients of African ancestoryen_ZA
dc.typeMaster Thesis
dc.type.qualificationlevelMasters
dc.type.qualificationnameM Meden_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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