Abstract
The dystrophin complex stabilizes the plasma membrane of striated muscle cells. Loss of function mutations in the genes encoding dystrophin, or the associated proteins, triggers instability of the plasma membrane and myofiber loss. Mutations in dystrophin have been extensively cataloged providing remarkable structure-function correlation between predicted protein structure and clinical outcomes. These data have highlighted dystrophin regions necessary for in vivo function and fueled the design of viral vectors and now, exon skipping approaches for use in dystrophin restoration therapies. However, dystrophin restoration is likely more complex, owing to the role of the dystrophin complex as a broad cytoskeletal integrator. This review will focus on dystrophin restoration, with emphasis on the regions of dystrophin essential for interacting with its associated proteins and discuss the structural implications of these approaches.
Keywords: muscular dystrophy, dystrophin, spectrin repeat, exon skipping, sarcoglycan, sarcolemma
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INTRODUCTION
Muscular dystrophy is a collection of inherited diseases characterized by skeletal muscle weakness and degeneration. Muscular dystrophies are progressive disorders because over time healthy muscle fibers are lost and replaced by fibrosis and fat, making muscle tissues less able to generate force for everyday activity. As muscle wasting ensues, patients experience weakness, although muscle groups may be targeted differently in specific forms of muscular dystrophy. Respiratory failure, resulting from the weakening of breathing muscles, may limit lifespan in muscular dystrophy unless mechanical support is instituted. In some forms of muscular dystrophy, the heart is also affected resulting in cardiac complications including heart failure and irregular heart rhythms.
Duchenne muscular dystrophy (DMD) is one of the most common forms of muscular dystrophy. DMD is caused by recessive mutations in the dystrophin gene on X chromosome, affecting 1 in 3,500 to 5,000 newborn males worldwide (82). Boys with DMD show signs of muscle weakness early in childhood, typically between 2 and 7 years of age, and often lose ambulation around the time of puberty. DMD boys may have delayed development of motor skills such as sitting, walking and talking. Becker muscular dystrophy (BMD) is also caused by mutations in the DMD gene that encodes dystrophin. Individuals with BMD share similar signs and symptoms with DMD boys but with later onset and more varied time course. Like DMD, the heart can be affected in BMD.
The dystrophin gene is the largest known human gene, containing 79 exons and spanning > 2,200 kb, roughly 0.1% of the whole genome (96). The most common mutation responsible for DMD and BMD is a deletion spanning one or multiple exons. Such deletions account for 60–70% of all DMD cases and 80~85% BMD cases (58, 147). Point mutations are responsible for around 26% of DMD cases and 13% BMD cases. Exonic duplications account for 10 to 15% of all DMD cases and 5% to 10% BMD cases. Subexonic insertions, deletions, splice mutations and missense mutations account for the rest of the cases. DMD is associated with mutations that disrupt the protein’s reading frame causing premature stop codons. These mutated transcripts are susceptible to nonsense mediate decay, and the carboxy-terminal truncated protein products are also unstable and subject to degradation, leaving little or no protein produced in cells. In contrast, BMD patients usually have in-frame deletions that maintain the correct reading frame. Furthermore, nonsense mutations have been associated with both BMD and DMD. However, nonsense mutations associated with BMD are more prone to induce exon skipping than those found in DMD (59). The resulting protein products in BMD are internally truncated and expressed at lower levels than normal muscle. However, these internally truncated proteins are expressed at higher levels than in DMD and remain partially functional. Within one BMD affected family, three males carrying the same nonsense mutation in exon 29 displayed phenotypes from severe, mild to asymptomatic. This nonsense mutation is located in an exon recognition sequence in exon 29 and induces partial skipping of exon 29, producing an internally truncated dystrophin. A considerable amount s