X-linked muscular dystrophy in a Labrador Retriever strain: phenotypic and molecular characterisation.

BACKGROUND: Canine models of Duchenne muscular dystrophy (DMD) are a valuable tool to evaluate potential therapies because they faithfully reproduce the human disease. Several cases of dystrophinopathies have been described in canines, but the Golden Retriever muscular dystrophy (GRMD) model remains the most used in preclinical studies. Here, we report a new spontaneous dystrophinopathy in a Labrador Retriever strain, named Labrador Retriever muscular dystrophy (LRMD). METHODS: A colony of LRMD dogs was established from spontaneous cases. Fourteen LRMD dogs were followed-up and compared to the GRMD standard using several functional tests. The disease causing mutation was studied by several molecular techniques and identified using RNA-sequencing. RESULTS: The main clinical features of the GRMD disease were found in LRMD dogs; the functional tests provided data roughly overlapping with those measured in GRMD dogs, with similar inter-individual heterogeneity. The LRMD causal mutation was shown to be a 2.2-Mb inversion disrupting the DMD gene within intron 20 and involving the TMEM47 gene. In skeletal muscle, the Dp71 isoform was ectopically expressed, probably as a consequence of the mutation. We found no evidence of polymorphism in either of the two described modifier genes LTBP4 and Jagged1. No differences were found in Pitpna mRNA expression levels that would explain the inter-individual variability. CONCLUSIONS: This study provides a full comparative description of a new spontaneous canine model of dystrophinopathy, found to be phenotypically equivalent to the GRMD model. We report a novel large DNA mutation within the DMD gene and provide evidence that LRMD is a relevant model to pinpoint additional DMD modifier genes.

[The Frozen Man and the Chinese Alphabet]. / L'Homme Gelé (渐冻人) et le déficit en cristalline αB.

The Confucian philosophy teaches us that the search for truth does not always follow a straight line. The clinical observation presented here illustrates this perfectly and is about a child afflicted by a rare neuromuscular disorder (in Chinese, the word 'myopathy' is translated to meaning 'frozen man') in whom was suspected a deficit in αB crystallin. The authors take the opportunity to put the spotlight on China, this great country which did not wait for Alain Peyrefitte to wake up or, more precisely, to rewake. In the light of past and recent missions in the former Middle Kingdom, an update is made on the medico-scientific but also societal issues of this country on the verge of becoming, perhaps, a giant in the field of neuromuscular diseases.

TITLE: L'Homme Gelé (渐冻人) et le déficit en cristalline αB. ABSTRACT: La philosophie confucéenne nous enseigne que la recherche de la vérité n'emprunte pas toujours un chemin rectiligne. L'observation clinique présentée ici l'illustre parfaitement. Il y est question d'un enfant souffrant d'une maladie neuromusculaire rare (en chinois, le mot myopathie se traduit par 渐冻人 soit « homme gelé ¼) chez qui fut suspecté un déficit en cristalline αB. Les auteurs profitent de l'occasion pour mettre le projecteur sur la Chine, ce grand pays qui n'a pas attendu Alain Peyrefitte pour s'éveiller ou, plus exactement, se réveiller. à la lumière de missions passées et récentes dans l'ex-Empire du Milieu, le point est fait sur les enjeux médico-scientifiques mais aussi sociétaux de ce pays en passe de devenir, peut-être, un géant dans le domaine des maladies neuromusculaires.

Innovating therapies for muscle diseases.

The neuromuscular disorders (NMDs) involve many different genetic and acquired diseases. Corticosteroids (e.g., prednisone and deflazacort) are prescribed for some NMDs as a palliative treatment to slow down disease progression to some extent. For the vast majority of NMDs, no specific therapy is currently available that stops progression or reverses the clinical deficits of the diseases. However, recent progress with different therapeutic approaches is now resulting in numerous clinical trials. In this chapter, we give an overview of the current state of the art, opportunities and challenges for gene therapy, cell therapy, antisense-mediated modulation of splicing, and numerous drug therapies for NMDs in general, and Duchenne muscular dystrophy as a paradigm in particular. Although none of the proposed strategies has yet proven to be of therapeutic value in patients, it is reasonable to expect that clinical efficacy will soon be demonstrated for some of the more advanced approaches.

Assessment of the structural and functional impact of in-frame mutations of the DMD gene, using the tools included in the eDystrophin online database.

BACKGROUND: Dystrophin is a large essential protein of skeletal and heart muscle. It is a filamentous scaffolding protein with numerous binding domains. Mutations in the DMD gene, which encodes dystrophin, mostly result in the deletion of one or several exons and cause Duchenne (DMD) and Becker (BMD) muscular dystrophies. The most common DMD mutations are frameshift mutations resulting in an absence of dystrophin from tissues. In-frame DMD mutations are less frequent and result in a protein with partial wild-type dystrophin function. The aim of this study was to highlight structural and functional modifications of dystrophin caused by in-frame mutations. METHODS AND RESULTS: We developed a dedicated database for dystrophin, the eDystrophin database. It contains 209 different non frame-shifting mutations found in 945 patients from a French cohort and previous studies. Bioinformatics tools provide models of the three-dimensional structure of the protein at deletion sites, making it possible to determine whether the mutated protein retains the typical filamentous structure of dystrophin. An analysis of the structure of mutated dystrophin molecules showed that hybrid repeats were reconstituted at the deletion site in some cases. These hybrid repeats harbored the typical triple coiled-coil structure of native repeats, which may be correlated with better function in muscle cells. CONCLUSION: This new database focuses on the dystrophin protein and its modification due to in-frame deletions in BMD patients. The observation of hybrid repeat reconstitution in some cases provides insight into phenotype-genotype correlations in dystrophin diseases and possible strategies for gene therapy. The eDystrophin database is freely available: http://edystrophin.genouest.org/.

Variable phenotype of del45-55 Becker patients correlated with nNOSµ mislocalization and RYR1 hypernitrosylation.

Duchenne and Becker muscular dystrophies (DMD and BMD) are muscle-wasting diseases caused by mutations in the DMD gene-encoding dystrophin. Usually, out-of-frame deletions give rise to DMD, whereas in-frame deletions result in BMD. BMD patients exhibit a less severe disease because an abnormal but functional dystrophin is produced. This is the rationale for attempts to correct the reading frame by using an exon-skipping strategy. In order to apply this approach to a larger number of patients, a multi-exon skipping strategy of exons 45-55 has been proposed, because it should correct the mRNA reading frame in almost 75% of DMD patients with a deletion. The resulting dystrophin lacks part of the binding site for the neuronal nitric oxide synthase (nNOSµ), which normally binds to spectrin-like repeats 16 and 17 of the dystrophin. Since these domains are encoded by exons 42-45, we investigated the nNOSµ status in muscle biopsies from 12 BMD patients carrying spontaneous deletions spaning exons 45-55. We found a wide spectrum of nNOSµ expression and localization. The strictly cytosolic mislocalization of nNOSµ was associated with the more severe phenotypes. Cytosolic NO production correlated with both hypernitrosylation of the sarcoplasmic reticulum calcium-release-channel ryanodine receptor type-1 (RyR1) and release of calstabin-1, a central hub of Ca(2+) signaling and contraction in muscle. Finally, this study shows that the terminal truncation of the nNOS-binding domain in the 'therapeutic' del45-55 dystrophin is not innocuous, since it can perturb the nNOS-dependent stability of the RyR1/calstabin-1 complex.

Does the severity of the LGMD2A phenotype in compound heterozygotes depend on the combination of mutations?

INTRODUCTION: Limb-girdle muscular dystrophy type 2A (LGMD2A) is caused by a deficiency of calpain-3/p94. Although the symptoms in most LGMD2A patients are generally homogeneous, some variation in the severity and progression of the disease has been reported. METHODS: We describe 2 patients who carry the same combination of compound heterozygous mutations (pG222R/pR748Q) and whose symptoms are exceptionally benign compared to homozygotes with each missense mutation. RESULTS: The benign phenotype observed in association with the combined pG222R and pR748Q mutations suggested that it may result from a compensatory effect of compound heterozygosity rather than the individual mutations themselves. Our analyses revealed that these two mutations exert different effects on the protease activity of calpain-3, suggesting "molecular complementation" in these patients. CONCLUSION: We propose several hypotheses to explain how this specific combination of mutations may rescue the normal proteolytic activity of calpain-3, resulting in an exceptionally benign phenotype.

Analysis of Dp71 contribution in the severity of mental retardation through comparison of Duchenne and Becker patients differing by mutation consequences on Dp71 expression.

The presence of variable degrees of cognitive impairment, extending from severe mental retardation to specific deficits, in patients with dystrophinopathies is a well-recognized problem. However, molecular basis underlying mental retardation and its severity remain poorly understood and still a matter of debate. Here, we report one of the largest study based on the comparison of clinical, cognitive, molecular and expression data in a large cohort of 81 patients affected with Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) bearing mutations predicted to affect either all dystrophin products, including Dp71 or all dystrophin products, except Dp71. In addition to the consistent data defining molecular basis underlying mental retardation in DMD, we show that BMD patients with MR have mutations that significantly affect Dp71 expression or with mutations located in exons 75 and 76. We also show that mutations upstream to exon 62, with DMD phenotype, predicted to lead to a loss-of-function of all dystrophin products, except Dp71 isoform, are associated, predominantly, with normal or borderline cognitive performances. Altogether, these reliable phenotype-genotype correlations in combination with Dp71 mRNA and protein expression studies, strongly indicate that loss-of-function of all dystrophin products is systematically associated with severe form of MR, and Dp71 deficit is a factor that contributes in the severity of MR and may account for a shift of 2 SD downward of the intelligence quotient.

Genotype-phenotype analysis in 2,405 patients with a dystrophinopathy using the UMD-DMD database: a model of nationwide knowledgebase.

UMD-DMD France is a knowledgebase developed through a multicenter academic effort to provide an up-to-date resource of curated information covering all identified mutations in patients with a dystrophinopathy. The current release includes 2,411 entries consisting in 2,084 independent mutational events identified in 2,046 male patients and 38 expressing females, which corresponds to an estimated number of 39 people per million with a genetic diagnosis of dystrophinopathy in France. Mutations consist in 1,404 large deletions, 215 large duplications, and 465 small rearrangements, of which 39.8% are nonsense mutations. The reading frame rule holds true for 96% of the DMD patients and 93% of the BMD patients. Quality control relies on the curation by four experts for the DMD gene and related diseases. Data on dystrophin and RNA analysis, phenotypic groups, and transmission are also available. About 24% of the mutations are de novo events. This national centralized resource will contribute to a greater understanding of prevalence of dystrophinopathies in France, and in particular, of the true frequency of BMD, which was found to be almost half (43%) that of DMD. UMD-DMD is a searchable anonymous database that includes numerous newly developed tools, which can benefit to all the scientific community interested in dystrophinopathies. Dedicated functions for genotype-based therapies allowed the prediction of a new multiexon skipping (del 45-53) potentially applicable to 53% of the deleted DMD patients. Finally, such a national database will prove to be useful to implement the international global DMD patients' registries under development.