Splicing behaviour and exotic mutations in the DMD gene

DMD is the largest gene in the human genome, spanning over 2.2Mb of the X chromosome, and more than 99% of the gene content is intronic sequence. DMD encodes dystrophin, a crucial protein for protecting muscle fibres from mechanical damage. Mutations to DMD can cause any one of a family of diseases, known as the dystrophinopathies. The most severe of these is Duchenne muscular dystrophy, a progressive and global muscle wasting disease that is fatal to affected males in early life. Therapies for dystrophinopathies have progressed substantially in recent years, but at present there is no cure. The projects comprising my MPhil research aim to improve our understanding of splicing and mutation in DMD transcripts. DMD splicing is necessarily complex due to the size of the gene and its multiple spliceoforms. As such, the full effects of a given mutation within the gene can be unpredictable. Because the first step of describing a mutation should be elucidation of the sequence, a new method (Fractal PCR) has been devised for efficiently determining the intronic breakpoints of whole-exon deletions in DMD. Existing research into DMD and NF1 pseudoexons was used to inform PCR designs, and this strategy successfully discovered rare alternative transcripts of these two genes in normal human RNA. For the bioinformatics component of this project, results were compiled for hundreds of putative exon-skipping antisense oligomers (AOs) and a search was conducted for patterns in the splice factor (SF) motifs targeted or avoided by the most effective of these molecules. The intent of this work was to generate a predictive model for optimal AO design. While the power of this model was equivocal in some regards, the characteristics of the SF motifs identified as targets unexpectedly point to a clear link between DMD transcript splicing and myotonic dystrophy