Frameshift deletions of exons 3-7 and revertant fibers in Duchenne muscular dystrophy: mechanisms of dystrophin production.

Duchenne muscular dystrophy (DMD) patients with mutations that disrupt the translational reading frame produce little or no dystrophin. Two exceptions are the deletion of exons 3-7 and the occurrence of rare dystrophin-positive fibers (revertant fibers) in muscle of DMD patients. Antibodies directed against the amino-terminus and the 5' end of exon 8 did not detect dystrophin in muscle from patients who have a deletion of exons 3-7. However, in all cases, dystrophin was detected with an antibody directed against the 3' end of exon 8. The most likely method of dystrophin production in these cases is initiation at a new start codon in exon 8. We also studied two patients who have revertant fibers: one had an inherited duplication of exons 5-7, which, on immunostaining, showed two types of revertant fibers; and the second patient had a 2-bp nonsense mutation in exon 51, which creates a cryptic splice site. An in-frame mRNA that uses this splice site in exon 51 was detected. Immunostaining demonstrated the presence of the 3' end of exon 51, which is in agreement with the use of this mRNA in revertant fibers. The most likely method of dystrophin production in these fibers is a second mutation that restores the reading frame.     

Targeted skipping of human dystrophin exons in transgenic mouse model systemically for antisense drug development

Antisense therapy has recently been demonstrated with great potential for targeted exon skipping and restoration of dystrophin production in cultured muscle cells and in muscles of Duchenne Muscular Dystrophy (DMD) patients. Therapeutic values of exon skipping critically depend on efficacy of the drugs, antisense oligomers (AOs). However, no animal model has been established to test AO targeting human dystrophin exon in vivo systemically. In this study, we applied Vivo-Morpholino to the hDMD mouse, a transgenic model carrying the full-length human dystrophin gene, and achieved for the first time more than 70% efficiency of targeted human dystrophin exon skipping in vivo systemically. We also established a GFP-reporter myoblast culture to screen AOs targeting human dystrophin exon 50. Antisense efficiency for most AOs is consistent between the reporter cells, human myoblasts and in the hDMD mice in vivo. However, variation in efficiency was also clearly observed. A combination of in vitro cell culture and a Vivo-Morpholino based evaluation in vivo systemically in the hDMD mice therefore may represent a prudent approach for selecting AO drug and to meet the regulatory requirement.     

An error in dystrophin mRNA processing in golden retriever muscular dystrophy, an animal homologue of Duchenne muscular dystrophy.

Golden retriever muscular dystrophy (GRMD) is a spontaneous, X-linked, progressively fatal disease of dogs and is also a homologue of Duchenne muscular dystrophy (DMD). Two-thirds of DMD patients carry detectable deletions in their dystrophin gene. The defect underlying the remaining one-third of DMD patients is undetermined. Analysis of the canine dystrophin gene in normal and GRMD dogs has failed to demonstrate any detectable loss of exons. Here, we have demonstrated a RNA processing error in GRMD that results from a single base change in the 3' consensus splice site of intron 6. The seventh exon is then skipped, which predicts a termination of the dystrophin reading frame within its N-terminal domain in exon 8. This is the first example of dystrophin deficiency caused by a splice-site mutation.     

Antisense-induced multiexon skipping for Duchenne muscular dystrophy makes more sense

Dystrophin deficiency, which leads to severe and progressive muscle degeneration in patients with Duchenne muscular dystrophy (DMD), is caused by frameshifting mutations in the dystrophin gene. A relatively new therapeutic strategy is based on antisense oligonucleotides (AONs) that induce the specific skipping of a single exon, such that the reading frame is restored. This allows the synthesis of a largely functional dystrophin, associated with a milder Becker muscular dystrophy phenotype. We have previously successfully targeted 20 different DMD exons that would, theoretically, be beneficial for >75% of all patients. To further enlarge this proportion, we here studied the feasibility of double and multiexon skipping. Using a combination of AONs, double skipping of exon 43 and 44 was induced, and dystrophin synthesis was restored in myotubes from one patient affected by a nonsense mutation in exon 43. For another patient, with an exon 46-50 deletion, the therapeutic double skipping of exon 45 and 51 was achieved. Remarkably, in control myotubes, the latter combination of AONs caused the skipping of the entire stretch of exons from 45 through 51. This in-frame multiexon skipping would be therapeutic for a series of patients carrying different DMD-causing mutations. In fact, we here demonstrate its feasibility in myotubes from a patient with an exon 48-50 deletion. The application of multiexon skipping may provide a more uniform methodology for a larger group of patients with DMD.     

A nonsense mutation-created intraexonic splice site is active in the lymphocytes,but not in the skeletal muscle of a DMD patient

Production of semi-functional dystrophin mRNA from the dystrophin gene encoding a premature stop codon has been shown to modify the severe phenotype of Duchenne muscular dystrophy (DMD). In this study, we report the tissue-specific production of semi-functional dystrophin mRNA via activation of a nonsense mutation-created intraexonic splice acceptor site. In a DMD patient a novel nonsense mutation was identified in exon 42. In his lymphocytes semi-functional dystrophin mRNA with a 63-nucleotide deletion in exon 42 (dys-63) was found to be produced. In vitro splicing assay using hybrid minigenes disclosed that the mutation-created intraexonic splice acceptor site was activated. In his skeletal muscle cells, however, only the authentically spliced dystrophin mRNA was found. This finding identifies the modulation of the splicing of muscle dystrophin mRNA in cases of DMD as a potential target for therapeutic strategies to generate a milder phenotype for this disease.     

Antisense PMO Found in Dystrophic Dog Model Was Effective in Cells from Exon 7-Deleted DMD Patient

Background

Antisense oligonucleotide-induced exon skipping is a promising approach for treatment of Duchenne muscular dystrophy (DMD). We have systemically administered an antisense phosphorodiamidate morpholino oligomer (PMO) targeting dystrophin exons 6 and 8 to a dog with canine X-linked muscular dystrophy in Japan (CXMD_J) lacking exon 7 and achieved recovery of dystrophin in skeletal muscle. To date, however, antisense chemical compounds used in DMD animal models have not been directly applied to a DMD patient having the same type of exon deletion. We recently identified a DMD patient with an exon 7 deletion and tried direct translation of the antisense PMO used in dog models to the DMD patient''s cells.

Methodology/Principal Findings

We converted fibroblasts of CXMD_J and the DMD patient to myotubes by FACS-aided MyoD transduction. Antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 were designed. These antisense PMOs were mixed and administered as a cocktail to either dog or human cells in vitro. In the CXMD_J and human DMD cells, we observed a similar efficacy of skipping of exons 6 and 8 and a similar extent of dystrophin protein recovery. The accompanying skipping of exon 9, which did not alter the reading frame, was different between cells of these two species.

Conclusion/Significance

Antisense PMOs, the effectiveness of which has been demonstrated in a dog model, achieved multi-exon skipping of dystrophin gene on the FACS-aided MyoD-transduced fibroblasts from an exon 7-deleted DMD patient, suggesting the feasibility of systemic multi-exon skipping in humans.     

Restoration of human dystrophin following transplantation of exon-skipping-engineered DMD patient stem cells into dystrophic mice

Duchenne muscular dystrophy (DMD) is a hereditary disease caused by mutations that disrupt the dystrophin mRNA reading frame. In some cases, forced exclusion (skipping) of a single exon can restore the reading frame, giving rise to a shorter, but still functional, protein. In this study, we constructed lentiviral vectors expressing antisense oligonucleotides in order to induce an efficient exon skipping and to correct the initial frameshift caused by the DMD deletion of CD133+ stem cells. The intramuscular and intra-arterial delivery of genetically corrected CD133 expressing myogenic progenitors isolated from the blood and muscle of DMD patients results in a significant recovery of muscle morphology, function, and dystrophin expression in scid/mdx mice. These data demonstrate that autologous engrafting of blood or muscle-derived CD133+ cells, previously genetically modified to reexpress a functional dystrophin, represents a promising approach for DMD.     

Novel mutations of dystrophin gene in DMD patients detected by rapid scanning in biplex exons DHPLC analysis

Mutations in the dystrophin gene result in both Duchenne and Becher muscular dystrophies (DMD and BMD). Approximately 65% of all mutations causing DMD are deletions (60%) or duplications (5%) of large segments of this gene, spanning one exon or more. Due to the large size of the dystrophin gene (79 exons), finding point mutations has been prohibitively expensive and laborious. Recent studies confirm the utility of pre-screening methods, as denaturing high-performance liquid chromatography (DHPLC) analysis in the identification of point mutations in the dystrophin gene, with an increment of mutation detection rate from 65% to more than 92%. Here we suggest an alternative and convenient method of DHPLC analysis in order to find mutations in a more rapid and less expensive way by introducing the analysis of 16 couples of dystrophin amplicons, in biplex exons DHPLC runs. Using this new protocol of biplex exons DHPLC screening, new mutations were identified in four male patients affected by DMD who had tested negative for large DNA rearrangements.     

Gene therapy for muscle disease

The molecular mechanisms of Duchenne muscular dystrophy (DMD) have been extensively investigated since the discovery of the dystrophin gene in 1986. Nonetheless, there is currently no effective treatment for DMD. Recent reports, however, indicate that adenoassociated viral (AAV) vector-mediated transfer of a functional dystrophin cDNA into the affected muscle is a promising strategy. In addition, antisense-mediated exon skipping technology has been emerging as another promising approach to restore dystrophin expression in DMD muscle. Ongoing clinical trials show restoration of dystrophin in DMD patients without serious side effects. Here, we summarize the recent progress in gene therapy, with an emphasis on exon skipping for DMD.     

Analysis of a dystrophin gene deletion by amplification of mRNA isolated from DMD myotubes cultured in vitro

The most frequent causes for the X-linked muscular dystrophy of the allelic Duchenne (DMD) or Becker (BMD) type are partial deletions of the dystrophin gene. These mutations are accompanied either by disrupted or by preserved translational reading frames in mRNAs derived from the deleted genes. As a rule, the reading frame is destroyed in the more severe DMD, whereas it is preserved in the less severe BMD (M. Koenig et al., 1989, Am. J. Hum. Genet. 45, 498-506). We have analyzed in detail a deletion that was detected in a fetus at risk of DMD. The analysis of this mutation included the delineation of the altered subregion in the dystrophin mRNA. mRNA was isolated from myotubes derived from embryonic DMD myoblasts propagated in vitro. This study was based on enzymatic amplification by the polymerase chain reaction (PCR) of dystrophin mRNA and direct sequencing of the amplified cDNA. Exons 47 to 50 were found to be missing in the mRNA. The splicing of exon 46 to exon 51 resulted in a reading frameshift, indicating that this mutation is likely to be responsible for a DMD type of dystrophy. The clinical diagnosis of DMD for a 10-year-old patient in this family was compatible with the "reading frame" assumption.     

Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications.

We have studied 34 Becker and 160 Duchenne muscular dystrophy (DMD) patients with the dystrophin cDNA, using conventional blots and FIGE analysis. One hundred twenty-eight mutations (65%) were found, 115 deletions and 13 duplications, of which 106 deletions and 11 duplications could be precisely mapped in relation to both the mRNA and the major and minor mutation hot spots. Junction fragments, ideal markers for carrier detection, were found in 23 (17%) of the 128 cases. We identified eight new cDNA RFLPs within the DMD gene. With the use of cDNA probes we have completed the long-range map of the DMD gene, by the identification of a 680-kb SfiI fragment containing the gene's 3' end. The size of the DMD gene is now determined to be about 2.3 million basepairs. The combination of cDNA hybridizations with long-range analysis of deletion and duplication patients yields a global picture of the exon spacing within the dystrophin gene. The gene shows a large variability of intron size, ranging from only a few kilobases to 160-180 kb for the P20 intron.     

Spectrum of small mutations in the dystrophin coding region.

Duchenne and Becker muscular dystrophies (DMD and BMD) are caused by defects in the dystrophin gene. About two-thirds of the affected patients have large deletions or duplications, which occur in the 5' and central portion of the gene. The nondeletion/duplication cases are most likely the result of smaller mutations that cannot be identified by current diagnostic screening strategies. We screened approximately 80% of the dystrophin coding sequence for small mutations in 158 patients without deletions or duplications and identified 29 mutations. The study indicates that many of the DMD and the majority of the BMD small mutations lie in noncoding regions of the gene. All of the mutations identified were unique to single patients, and most of the mutations resulted in protein truncation. We did not find a clustering of small mutations similar to the deletion distribution but found > 40% of the small mutations 3' of exon 55. The extent of protein truncation caused by the 3' mutations did not determine the phenotype, since even the exon 76 nonsense mutation resulted in the severe DMD phenotype. Our study confirms that the dystrophin gene is subject to a high rate of mutation in CpG sequences. As a consequence of not finding any hotspots or prevalent small mutations, we conclude that it is presently not possible to perform direct carrier and prenatal diagnostics for many families without deletions or duplications.     

Fluorescent multiplex linkage analysis and carrier detection for Duchenne/Becker muscular dystrophy.

We have developed a fast and accurate PCR-based linkage and carrier detection protocol for families of Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) patients with or without detectable deletions of the dystrophin gene, using fluorescent PCR products analyzed on an automated sequencer. When a deletion is found in the affected male DMD/BMD patient by standard multiplex PCR, fluorescently labeled primers specific for the deleted and nondeleted exon(s) are used to amplify the DNA of at-risk female relatives by using multiplex PCR at low cycle number (20 cycles). The products are then quantitatively analyzed on an automatic sequencer to determine whether they are heterozygous for the deletion and thus are carriers. As a confirmation of the deletion data, and in cases in which a deletion is not found in the proband, fluorescent multiplex PCR linkage is done by using four previously described polymorphic dinucleotide sequences. The four (CA)n repeats are located throughout the dystrophin gene, making the analysis highly informative and accurate. We present the successful application of this protocol in families who proved refractory to more traditional analyses.