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

Abstract

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 2Ј-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.

Mutation spectrum of the dystrophin gene in 442 Duchenne/Becker muscular dystrophy cases from one Japanese referral center

“…Because the completely normal 79 exons of the dystrophin gene were maintained in these cases, induction of pseudoexon skipping would be expected to result in completely normal expression of dystrophin, the ultimate goal of dystrophinopathy treatment. 41 Our cases can now be tested for any potential to be treated by exon skipping. In contrast to the deletions and duplications, which were localized within two hotspots, nonsense mutations were detected throughout the gene, with 30.4% (21) Japan, reports from outside Japan have described lower nonsense mutation rates up to 13.2%.…”

Progress in therapeutic antisense applications for neuromuscular disorders

“…20 For both mutations in in-frame and out-offrame exons, exon skipping led to dystrophin re-expression in cultured myogenic cells 16,17,21 and two dystrophic mouse models (mdx and mdx 4cv ), which carry point mutations in mouse exons 23 and 53, respectively. 22,23 Furthermore, a point mutation in a splice site causing exon 7 skipping in a dog model of the disease has been corrected by exon 6 and 8 skipping in cultured cells and in vivo 24,25 AONs have been used to restore normal splicing in very rare small intronic mutations that induce the inclusion of cryptic exons 26 and to restore the reading frame for a single patient with an inversion of exons 49 and 50. 27 For duplications (present in 7% of DMD patients 28 ) exon skipping is more challenging, as the original and the duplicated exons are indistinguishable for the AONs.…”

A Targeted Deleterious Allele of the Splicing Factor SCNM1 in the Mouse

“…Splice-site mutations compose $15% of all diseasecausing mutations, and a similar number occur in splice enhancer and suppressor sequences (Nissim-Rafinia and Kerem 2005). Splice-site mutations are often associated with clinical heterogeneity within families, as described for Duchenne and Becker muscular dystrophy, cardiac sodium channelopathy (SCN5A), familial adenomatous polyposis (APC), severe combined immunodeficiency ( JAK3), and neurofibromatosis type 2 (NF2) (Kluwe et al 1998;Frucht et al 2001;Mohamed et al 2003;Rossenbacker et al 2005;Gurvich et al 2008). There is evidence that the clinical severity of these disorders is influenced by splicing efficiency.…”