Abstract:
The thrust of this thesis concerns the analysis of the dystrophin gene and, in part, dystrophin’s paralogue utrophin. The approach that was taken could be considered a bridge between diagnostics and disease modelling. In terms of the former, the analysis of the dystrophin gene is pivotal in confirming a clinical diagnosis of Duchenne/Becker Muscular Dystrophy (D/BMD), but this gene comprises multiple exons, which creates a significant challenge in the context of identifying point mutations. In order to address this challenge, an automated primer design programme was written to allow subsequent high throughput sequencing of dystrophin gene exons. As a by-product, this programme allowed primer designs and in-silico evaluations to be made against all coding exons of all genes in the human genome. A streamlined sequencing approach was designed to screen for all point mutations in the dystrophin gene coding exons using a Sanger-type sequencing method. This approach was validated using a number of D/BMD patient DNA samples; however, in order to improve the throughput, a pilot study was undertaken to evaluate a Next Generation Sequencing (NGS) approach. This study identified the key practical consideration that would allow NGS to be applied generally to molecular diagnostics. The second part of the work presented here concerns the long term objective of modelling the effects of mutations in the dystrophin gene using the small freshwater vertebrate species, Danio rerio (Zebrafish). While the intention was to identify dystrophin gene mutations in D/BMD patients then determine the effects of these mutations in zebrafish (and ultimately test therapeutic interventions in the mutant zebrafish), the thrust of the research was largely fundamental in determining the expression profile of the zebrafish dystrophin gene during embryonic development. An analysis of the zebrafish utrophin gene was also undertaken in that reduced expression of dystrophin (brought about by mutations) has been reported to lead to up-regulation of utrophin. Taken together, the twin approaches described in this thesis has led to improvements in screening the dystrophin gene for mutation events, and provided a foundation for subsequent studies in the modelling of a fatal human disease in zebrafish.