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Background: We report clinical safety and biochemical efficacy from a dose-ranging study of intravenously administered AVI-4658 phosphorodiamidate morpholino oligomer (PMO) in patients with Duchenne muscular dystrophy., Method: We undertook an open-label, phase 2, dose-escalation study (0·5, 1·0, 2·0, 4·0, 10·0, and 20·0 mg/kg bodyweight) in ambulant patients with Duchenne muscular dystrophy aged 5-15 years with amenable deletions in DMD. Participants had a muscle biopsy before starting treatment and after 12 weekly intravenous infusions of AVI-4658. The primary study objective was to assess safety and tolerability of AVI-4658. The secondary objectives were pharmacokinetic properties and the ability of AVI-4658 to induce exon 51 skipping and dystrophin restoration by RT-PCR, immunohistochemistry, and immunoblotting. The study is registered, number NCT00844597., Findings: 19 patients took part in the study. AVI-4658 was well tolerated with no drug-related serious adverse events. AVI-4658 induced exon 51 skipping in all cohorts and new dystrophin protein expression in a significant dose-dependent (p=0·0203), but variable, manner in boys from cohort 3 (dose 2 mg/kg) onwards. Seven patients responded to treatment, in whom mean dystrophin fluorescence intensity increased from 8·9% (95% CI 7·1-10·6) to 16·4% (10·8-22·0) of normal control after treatment (p=0·0287). The three patients with the greatest responses to treatment had 21%, 15%, and 55% dystrophin-positive fibres after treatment and these findings were confirmed with western blot, which showed an increase after treatment of protein levels from 2% to 18%, from 0·9% to 17%, and from 0% to 7·7% of normal muscle, respectively. The dystrophin-associated proteins α-sarcoglycan and neuronal nitric oxide synthase were also restored at the sarcolemma. Analysis of the inflammatory infiltrate indicated a reduction of cytotoxic T cells in the post-treatment muscle biopsies in the two high-dose cohorts., Interpretation: The safety and biochemical efficacy that we present show the potential of AVI-4658 to become a disease-modifying drug for Duchenne muscular dystrophy., Funding: UK Medical Research Council; AVI BioPharma., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Mutations that disrupt the open reading frame and prevent full translation of DMD, the gene that encodes dystrophin, underlie the fatal X-linked disease Duchenne muscular dystrophy. Oligonucleotides targeted to splicing elements (splice switching oligonucleotides) in DMD pre-mRNA can lead to exon skipping, restoration of the open reading frame, and the production of functional dystrophin in vitro and in vivo, which could benefit patients with this disorder., Methods: We did a single-blind, placebo-controlled, dose-escalation study in patients with DMD recruited nationally, to assess the safety and biochemical efficacy of an intramuscular morpholino splice-switching oligonucleotide (AVI-4658) that skips exon 51 in dystrophin mRNA. Seven patients with Duchenne muscular dystrophy with deletions in the open reading frame of DMD that are responsive to exon 51 skipping were selected on the basis of the preservation of their extensor digitorum brevis (EDB) muscle seen on MRI and the response of cultured fibroblasts from a skin biopsy to AVI-4658. AVI-4658 was injected into the EDB muscle; the contralateral muscle received saline. Muscles were biopsied between 3 and 4 weeks after injection. The primary endpoint was the safety of AVI-4658 and the secondary endpoint was its biochemical efficacy. This trial is registered, number NCT00159250., Findings: Two patients received 0.09 mg AVI-4658 in 900 microL (0.9%) saline and five patients received 0.9 mg AVI-4658 in 900 microL saline. No adverse events related to AVI-4658 administration were reported. Intramuscular injection of the higher-dose of AVI-4658 resulted in increased dystrophin expression in all treated EDB muscles, although the results of the immunostaining of EDB-treated muscle for dystrophin were not uniform. In the areas of the immunostained sections that were adjacent to the needle track through which AVI-4658 was given, 44-79% of myofibres had increased expression of dystrophin. In randomly chosen sections of treated EDB muscles, the mean intensity of dystrophin staining ranged from 22% to 32% of the mean intensity of dystrophin in healthy control muscles (mean 26.4%), and the mean intensity was 17% (range 11-21%) greater than the intensity in the contralateral saline-treated muscle (one-sample paired t test p=0.002). In the dystrophin-positive fibres, the intensity of dystrophin staining was up to 42% of that in healthy muscle. We showed expression of dystrophin at the expected molecular weight in the AVI-4658-treated muscle by immunoblot., Interpretation: Intramuscular AVI-4658 was safe and induced the expression of dystrophin locally within treated muscles. This proof-of-concept study has led to an ongoing systemic clinical trial of AVI-4658 in patients with DMD., Funding: UK Department of Health.

Background: The commonest pathogenic DMD changes are intragenic deletions/duplications which make up to 78% of all cases and point mutations (roughly 20%) detectable through direct sequencing. The remaining mutations (about 2%) are thought to be pure intronic rearrangements/mutations or 5'-3' UTR changes. In order to screen the huge DMD gene for all types of copy number variation mutations we designed a novel custom high density comparative genomic hybridisation array which contains the full genomic region of the DMD gene and spans from 100 kb upstream to 100 kb downstream of the 2.2 Mb DMD gene., Results: We studied 12 DMD/BMD patients who either had no detectable mutations or carried previously identified quantitative pathogenic changes in the DMD gene. We validated the array on patients with previously known mutations as well as unaffected controls, we identified three novel pure intronic rearrangements and we defined all the mutation breakpoints both in the introns and in the 3' UTR region. We also detected a novel polymorphic intron 2 deletion/duplication variation. Despite the high resolution of this approach, RNA studies were required to confirm the functional significance of the intronic mutations identified by CGH. In addition, RNA analysis identified three intronic pathogenic variations affecting splicing which had not been detected by the CGH analysis., Conclusion: This novel technology represents an effective high throughput tool to identify both common and rarer DMD rearrangements. RNA studies are required in order to validate the significance of the CGH array findings. The combination of these tools will fully cover the identification of causative DMD rearrangements in both coding and non-coding regions, particularly in patients in whom standard although extensive techniques are unable to detect a mutation.

Dystrophin mutations occurring at the 5' end of the gene frequently behave as exceptions to the "frame rule," their clinical severity being variable and often not related to the perturbation of the translation reading frame. The molecular mechanisms underlying the phenotypic variability of 5' dystrophin mutations have not been fully clarified. We have characterized the genomic breakpoints within introns 2, 6 and 7 and identified the splicing profiles in a cohort of DMD/BMD patients with deletion of dystrophin exons 3-7, 3-6 and duplication of exons 2-4. Our findings indicate that the occurrence of intronic cryptic promoter as well as corrective splicing events are unlikely to play a role in exons 3-7 deleted patients phenotypic variability. Our data suggest that re-initiation of translation could represent a major mechanism responsible for the production of a residual dystrophin in some patients with exons 3-7 deletion. Furthermore, we observed that the out-of-frame exon 2a is almost constantly spliced into a proportion of the dystrophin transcripts in the analysed patients. In the exons 2-4 duplicated DMD patient, producing both in-frame and out-of-frame transcripts, this splicing behaviour might represent a critical factor contributing to the severe phenotype. In conclusion, we suggest that multiple mechanisms may have a role in modulating the outcome of 5' dystrophin mutations, including recoding mechanisms and unusual splicing choices.

Almost all of the thousands of pathogenic mutations which have been described in the dystrophin gene either reduce protein production or remove large regions of the protein. This has severely limited the use of mutational information for the functional dissection of the dystrophin protein and increases the value of rare subtle mutations. We report a 3-bp deletion which removes a single highly conserved residue (glutamic acid 3367) adjacent to the dystrophin ZZ domain. This results in a phenotype of Duchenne muscular dystrophy with substantial retention of a presumably functionally compromised dystrophin protein. Two missense mutations (both affecting nearby residues) have been previously reported to result in this unusual combination of severe phenotype and high protein level. We discuss the functional implications of this and other mutations in the light of the predicted structure of the region. The pathogenicity of E3367del serves to emphasise the functional importance of this region of the dystrophin protein., (Copyright 2003 Wiley-Liss, Inc.)

The region of the dystrophin gene containing introns 45-50 is characterized by a high rate of recombination events that give rise to large deletions causing dystrophinopathy. The nucleotide sequence of this intronic region has recently been released in GenBank. With the aim of further understanding the mechanism favoring the occurrence of these deletions, we have characterized the distribution of introns 47 and 48 deletion endpoints in 39 dystrophinopathy patients. In 14 of these patients we were able to sequence the break junction. On these sequences we were able to identify several intronic motifs that could predispose to DNA double-strand breaks. Our results, combined with other literature data, show that unequal homologous recombination is a very poorly represented event in the dystrophin gene, whereas junction features are suggestive of a model of recombination in which DNA double-strand breaks are incorrectly repaired by a nonhomologous end-joining mechanism. The correlation among recombination rate, deletion frequency, and percentage of repetitive elements is discussed.

We report here the first use of a multiplexed protein truncation test for the high throughput screening of dystrophin point mutations. We have developed a substantially more robust and efficient procedure incorporating large savings in cost which uses muscle biopsy or lymphocyte total RNA as the template. The entire dystrophin open reading frame is screened in only five overlapping fragments using a long RT-PCR strategy to amplify dystrophin cDNA in excess of 3.7 kb. These five fragments are uniquely transcribed and translated in vitro in a single multiplexed reaction containing magnesium ions to reduce nonspecific internal initiation of translation. We have used this system to analyze mutations in 11 Duchenne muscular dystrophy patients (10 unrelated) with previously uncharacterized mutations. A single truncating mutation was identified in all patients, which was confirmed at the genomic level. Multiplex PTT provides the most efficient method for point mutation screening in this large gene and has potential applications to several disease genes with a significant proportion of truncating mutations.

Deletions in the dystrophin gene give rise to both Duchenne and Becker muscular dystrophies. Good correlation is generally found between the severity of the phenotype and the effect of the deletion on the reading frame: deletions that disrupt the reading frame result in a severe phenotype, while in frame deletions are associated with a milder disease course. Rare exceptions to this rule, mainly owing to frameshift mutations in the 5' region of the gene (in particular deletions involving exons 3 to 7) which are associated with a milder than expected phenotype, have been reported previously. In order to characterise better the relationship between genotype and phenotype as a result of mutations arising in the 5' region of the gene, we have studied a large cohort of patients with small in frame and out of frame deletions in the first 13 exons of the dystrophin gene. Fifty-five patients with a deletion in this area were identified; approximately one third of them had a phenotype different from that theoretically expected. Patients were divided into two groups: (1) patients with a severe clinical phenotype despite the presence of a small, in frame deletion and (2) patients with a mild phenotype and an out of frame deletion. Noticeable examples observed in the first group were Duchenne boys with a deletion of exon 5, of exon 3, and of exons 3-13. In the second group we observed several patients with an intermediate or Becker phenotype and out of frame deletions involving not only the usual exons 3-7 but also 5-7 and 3-6. These data indicate that a high proportion of patients with a deletion in the 5' end of the gene have a phenotype that is not predictable on the basis of the effect of the deletion on the reading frame. The N-terminus of dystrophin has at least one actin binding domain that might be affected by the small, in frame deletions in this area. The effect of the in frame deletions of exon 3, 5, and 3-13 on this domain might account for the severe phenotype observed in these patients. Other mechanisms, such as unexpected effect of the deletion on splicing behaviour, might, however, also be implicated in determining the phenotype outcome.

A direct, non-radioactive method of quantitative PCR amplification has been investigated for the diagnosis of deletion and duplication carriers in the dystrophin gene. The simultaneous amplification of two loci, or several loci using multiplex PCR, allows for the direct comparison of relative amounts of products from normal homozygous loci and potentially heterozygous deleted/duplicated loci. Sufficient cycles of PCR are performed to enable visual analysis or densitometric quantification of products on ethidium bromide stained gels. The method has been verified in blind trials performed on known genotypes and by showing that under the conditions used the assay remains within the exponential phase of amplification.

Knowledge about the parental origin of new mutations and the occurrence of germline mosaicism is important for estimating recurrence risks in Duchenne (DMD) and Becker muscular dystrophy (BMD). However, there are problems in resolving these issues partly because not all mutations can as yet be directly detected, and additionally because genetic ratios are very sensitive to ascertainment bias. In the present study, therefore, analysis was restricted to currently detectable mutations (deletions and duplications) in particular types of families which tend to be rare. In order to obtain sufficient data we pooled results from 25 European centers. In mothers of affected patients who were the first in their family with a dystrophin gene deletion or duplication, the ratio between the paternal and the maternal origin of this new mutation was 32:49 (binomial test P = 0.075) for DMD. In five BMD families the ratio between paternal and maternal origin of new mutations was 3:2. Recurrence risk because of maternal germline mosaicism was studied in sisters or subsequent sibs of isolated cases with an apparently new detectable mutation. In 12 out of 59 (0.20; 95% CI 0.10-0.31) transmissions of the risk haplotype the DMD mutation was transmitted as well. No recurrences were found in nine BMD families.

We report the results of screening for molecular deletions in 164 boys with DMD and BMD and correlation of deletions with clinical features. A deletion was detected in 100 cases (61%) by Southern blot hybridization analysis with cDNA probes. Thirty-eight different deletions and two duplications were identified. All deletions except one (deletion of exons 48-53) found in males with DMD disrupted the translational reading frame of the gene; however, six deletions in boys with BMD were out of frame. The same deletion in different individuals was found to occur with or without mental impairment, and many different deletions were associated with mental retardation. We were able to ascertain a series of boys [from this study and a previous one (Hodgson S V, Hart K, Abbs S, et al. Correlation of clinical and deletion data in Duchenne and Becker muscular dystrophy. J Med Genet 1989; 26: 682-693)] without significant mental retardation who had deletions which, when combined, covered the whole region of the gene in which deletions are commonly found, and within which region individual deletions can be associated with mental retardation.

Using whole cosmids as probes, we have mapped 242 DMD/BMD deletion breakpoints located in the major deletion hot spot of the DMD gene. Of these, 113 breakpoints were mapped more precisely to individual restriction enzyme fragments in the distal 80 kb of the 170-kb intron 44. An additional 12 breakpoints are distributed over the entire region, with no significant local variation in frequency. Furthermore, deletion sizes vary and are not influenced by the positions of the breakpoints. This argues against a predominant role of one or a few specific sequences in causing frequent rearrangements. It suggests that structural characteristics or a more widespread recombinogenic sequence makes this region so susceptible to deletion. Our study revealed several RFLPs, one of which is a 300-bp insertion/deletion polymorphism. Abnormally migrating junction fragments are found in 81% of the precisely mapped deletions and are highly valuable in the diagnosis of carrier females.

Existing reactions for the multiplex PCR amplification of exons in the dystrophin gene have been modified to produce two multiplex reactions which separately cover the 5' and 3' major deletion 'hotspots' in the gene, and together detect approximately 98% of all deletions detectable by Southern cDNA hybridisation. A comparative study of 148 patients showed mistypings in both the cDNA hybridisation data (4%) and the PCR analysis (1.2%). We suggest means of circumventing the underlying problems in order to avoid mistyping and subsequent misdiagnosis, and conclude that, with appropriate precautions, multiplex PCR amplification can be the method of choice for detecting deletions in the dystrophin gene.