Objective: The degenerative muscle diseases Duchenne (DMD) and Becker muscular dystrophy result from mutations in the DMD gene, which encodes the dystrophin protein. Recent improvements in mutational analysis techniques have resulted in the increasing identification of deep intronic point mutations, which alter splicing such that intronic sequences are included in the messenger RNA as "pseudoexons." We sought to test the hypothesis that the clinical phenotype correlates with splicing efficiency of these mutations, and to test the feasibility of antisense oligonucleotide (AON)-mediated pseudoexon skipping.
Methods: We identified three pseudoexon insertion mutations in dystrophinopathy patients, two of whom had tissue available for further analysis. For these two out-of-frame pseudoexon mutations (one associated with Becker muscular dystrophy and one with DMD), mutation-induced splicing was tested by quantitative reverse transcription polymerase chain reaction; pseudoexon skipping was tested using AONs composed of 2'-O-methyl-modified bases on a phosphorothioate backbone to treat cultured primary myoblasts.
Results: Variable amounts of pseudoexon inclusion correlates with the severity of the dystrophinopathy phenotype in these two patients. AON treatment directed at the pseudoexon results in the expression of full-length dystrophin in a DMD myoblast line.
Interpretation: Both DMD and Becker muscular dystrophy can result from out-of-frame pseudoexons, with the difference in phenotype being due to variable efficiency of the newly generated splicing signal. AON-mediated pseudoexon skipping therapy is a viable approach to these patients and would be predicted to result in increased expression of wild-type dystrophin protein.