DMD pseudoexon mutations: splicing efficiency, phenotype, and potential therapy.
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.


CONCLUSIONS
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.
Gurvich
Olga L
OL
Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT, USA.
Tuohy
Therese M
TM
Howard
Michael T
MT
Finkel
Richard S
RS
Medne
Livija
L
Anderson
Christine B
CB
Weiss
Robert B
RB
Wilton
Steve D
SD
Flanigan
Kevin M
KM
D000293:Adolescent; D000328:Adult; D017398:Alternative Splicing / Q000235:genetics*; D002478:Cells, Cultured; D002648:Child; D004252:DNA Mutational Analysis; D016189:Dystrophin / Q000235:genetics*; D005091:Exons / Q000235:genetics; D020022:Genetic Predisposition to Disease / Q000235:genetics*; D005820:Genetic Testing; D015316:Genetic Therapy / Q000379:methods; D005838:Genotype; D006801:Humans; D008297:Male; D018485:Muscle Fibers, Skeletal / Q000187:drug effects; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics* / Q000503:physiopathology / Q000628:therapy; D016254:Mutagenesis, Insertional / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology / Q000627:therapeutic use; D010641:Phenotype; D012322:RNA Precursors / Q000235:genetics; D022821:RNA Splice Sites / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics
D016189:Dystrophin; D016376:Oligonucleotides, Antisense; D012322:RNA Precursors; D022821:RNA Splice Sites