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Characterisation of the ovine model of McArdle's disease: development of therapeutic strategies

Walker, Kendall Rae (2006) Characterisation of the ovine model of McArdle's disease: development of therapeutic strategies. PhD thesis, Murdoch University.

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McArdle's disease (OMIM 232600) is one of the most common glycogen storage diseases affecting skeletal muscle. It is inherited in an autosomal recessive manner and is caused by a defect in the muscle glycogen phosphorylase gene (PYGM) (Ch 11q13), a key metabolic enzyme. As a result, patients are unable to mobilise muscle glucose stores to provide the energy for muscle contraction; and suffer from exercise intolerance (myalgia, tachycardia, breathlessness and early fatigue) as well as cramps / contractures and occasionally rhabdomyolysis and myoglobinuria after bouts of strenuous exercise. Histopathologically, McArdle's disease is characterised by an increase in subsarcolemmal storage of glycogen and reduced or absent glycogen phosphorylase activity. McArdle's disease displays significant genetic heterogeneity, with 47 different disease-causing mutations in PYGM having been identified in human sufferers. In keeping with this genetic heterogeneity, significant molecular heterogeneity is observed in patients with regard to the effect of the disease-causing mutation on the PYGM mRNA transcript. In contrast, little biochemical heterogeneity is associated with the disease, with approximately 90% of human patients having no residual pygm protein and therefore no enzyme activity.

A naturally occurring ovine model of McArdle's disease was identified a number of years ago in a flock of merino sheep in Western Australia. The disease causing mutation responsible for ovine McArdle's disease was identified and published in 1997 by Tan et al., (1997) [1]. The mutation occurs in the 3' acceptor splice-site of intron 19 of the ovine PYGM gene, resulting in the activation of a cryptic splice-site site in exon 20 and the premature termination of the transcript. Hypothetically this mutant pygm protein should be 31 amino acids smaller than the wild-type. When I began my PhD, it was known that the ovine sufferers displayed exercise intolerance and histological examination of affected muscle revealed an excess subsarcolemmal storage of glycogen and absent phosphorylase, as occurs in their human counterparts. Due to their similarities in muscle mass to humans and the relative ease and low cost of maintenance, the ovine model of McArdle's disease is an important and highly relevant test-bed for therapeutic strategies.

This thesis can essentially be divided into two parts:
1) The characterisation of the ovine model of McArdle's disease-
In particular, determining the effect of the disease-causing mutation on both the PYGM mRNA and protein, characterising the sequences of the ovine glycogen phosphorylase isoforms and their developmental and tissue specific expression patterns, as well as determining the identity and the activity of the glycogen phosphorylase isoforms expressed in certain muscle groups (cardiac, extraocular and smooth muscles) which appear to be protected from the disease.

Based upon this data and making use of the ovine model;
2) Development of therapeutic strategies-
A number of potential therapeutic strategies were investigated including:
* The upregulation of a functionally related gene, through the re-expression of non-muscle isoforms of glycogen phosphorylase via notexin induced muscle regeneration.
* As well as the replacement of the PYGM gene using modified adenovirus 5 (AdV5) and adeno-associated virus 2 (AAV-2) vectors.

Based upon the ability of the non-muscle isoforms of glycogen phosphorylase to function in-vivo in McArdle's muscle in the absence of the muscle isoform, the ability of tributyrin (a butyrate prodrug and potent histone deacetylase inhibitor) to re-express brain glycogen phosphorylase in mature mouse muscle was also investigated.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Veterinary and Biomedical Sciences
Supervisor(s): Laing, Nigel
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