Characterising the molecular consequences of LMNA mutations in congenital muscular dystrophy

Storey, Emily Carolyn

Abstract

LMNA-related congenital muscular dystrophy (L-CMD) is a rare disorder predominantly causing muscle weakness and wasting, over time, leading to development of dysphagia and life-threating respiratory insufficiency, and sometimes cardiac arrhythmias. There are no pharmacological therapies for L-CMD, and treatment focuses on managing symptoms of the condition. L-CMD is caused by mutations in LMNA, a gene encoding nuclear lamina component lamin A/C. Many mechanisms downstream of LMNA mutations in L-CMD remain elusive, making the identification of non-genetic therapeutic targets difficult. Here, research focused on identifying conserved cellular and molecular defects across three myoblast cell lines derived from L-CMD patients, each harbouring different mutations in LMNA (R249W, del.K32, L380S). Throughout this thesis, a combination of molecular biology and biochemistry techniques and systematic reviewing were used. In the first results chapter, coimmunoprecipitation and mass spectrometry revealed new putative lamin A binding partners that were lost or gained across the three L-CMD myoblast cell lines, and new putative lamin A interactors that were present across all myoblast lines, as well as lamin A interactors that have been previously identified in other cells/tissues. In the second results chapter, a targeted approach uncovered nuclear defects, including abnormal nuclear morphology, in L-CMD myoblasts and myotubes compared to controls. Lamin A/C, emerin and SUN2 were all significantly decreased in expression in L-CMD myotubes compared to controls. In the third results chapter, quantitative proteomics revealed hundreds of differentially expressed proteins in L-CMD myoblasts and myotubes compared to healthy controls. Ingenuity pathway analysis identified downregulation of the necroptosis signalling pathway, and upregulation of the synaptogenesis signalling pathway in L-CMD myoblasts, and upregulation of the insulin secretion signalling pathway and Huntington’s disease signalling in L-CMD myotubes. In the final results chapter, genotype-phenotype correlations were determined in the genes encoding nuclear envelope (NE) proteins using a systematic review. Clusters of LMNA mutations causing striated muscle diseases were found to be located on exon 6 of LMNA, whilst metabolic-disease associated LMNA mutations were common in the region encoding the tail domain of lamin A/C. LMNA was the only NE protein to cause many distinct diseases. Identification and comparison of lamin A interactors in L-CMD and control myoblasts offers insight into the debated theory of whether LMNA mutations result in loss- or gain-of function. Cells derived from other laminopathy patients have previously exhibited nuclear defects, whilst reduced lamin A/C expression has been found in myoblasts and myotubes from the LmnaΔK32/ΔK32 mouse model of L-CMD. Confirmation of decreased lamin A/C expression in L-CMD myotubes suggests this may be a key feature of the disease that should be targeted for upregulation in future therapy development. Dysregulated pathways identified in LCMD myoblasts and myotubes including the necroptosis pathway have previously been implicated in other neuromuscular disorders, so may contribute to L-CMD pathophysiology and should be considered for further study. Taken together, these results have identified numerous defects that are common to each of the three L-CMD myoblast cell lines harbouring different LMNA mutations. This thesis has offered insight into mechanisms which may underlie the pathophysiology of L-CMD, which may be targeted in future studies for the development of therapies, or alternatively for biomarker purposes, to track disease or treatment progression.

Citation

Storey, E. C. (2023). Characterising the molecular consequences of LMNA mutations in congenital muscular dystrophy. (Thesis). Keele University. Retrieved from https://keele-repository.worktribe.com/output/674333

• Thesis Type: Thesis
• Deposit Date: Dec 21, 2023
• Publicly Available Date: Oct 23, 2024
• Public URL: https://keele-repository.worktribe.com/output/674333
• Additional Information: Embargo on access until 3 October 2024 - The thesis is due for publication, or the author is actively seeking to publish this material.
• Award Date: 2023-12