Genomic and functional genomic investigations of neuromuscular disorders

Kristen Jean Nowak

This thesis is divided into two parts. In part I genomics techniques were used to identify the genes and mutations causing a selected group of neuromuscular disorders. In Part II functional genomic techniques were used to investigate some of the mutant proteins identified. Part I i. Functional cloning was utilised to identify a novel missense mutation in the gamma-sarcoglycan gene and a large deletion encompassing this gene, in two sisters with limb girdle muscular dystrophy. ii. Postional candidate cloning was used to identify a novel missense mutation in the myelin protein zero gene in a four generation family diagnosed with intermediate hereditary and motor sensory neuropathy. iii. A candidate gene cloning approach identified the first mutations within the skeletal muscle alpha-actin gene causing the disease actin myopathy. iv. The skeletal muscle alpha-actin gene was also investigated as a candidate gene for nemaline myopathy. This has resulted in the identification of 44 different mutations within this gene producing nemaline and other congenital myopathies in patients world-wide. v. The human muscle cofilin gene, also a candidate gene for nemaline myopathy, was studied to determine its genomic sequence and structure, then screened for mutations in 50 nemaline myopathy patients, with none being discovered. Part II To obtain more information about the disease process in actin and nemaline myopathies, functional genomics was used to explore the skeletal muscle alpha-actin mutations further. i. The Baculovirus Expression Vector System was used to produce functional recombinant skeletal muscle alpha-actin. This method of protein production will allow interaction studies in the future. ii. ii. Wild-type and two of the mutant skeletal muscle alpha-actin proteins (each tagged with Enhanced Green Fluorescent Protein) were expressed in mouse muscle cells in culture. The 2 mutant proteins, which caused early death in humans, were lethal for H2K muscle cells. The genomic and functional genomic analyses prepare for future work aimed at therapies for the sarcomeric protein diseases. There have been six publications from the thesis work to date and two entries into Genbank.

Genomic and functional genomic investigations of neuromuscular disorders

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