Treatment of osteoporosis in a mouse model of Duchenne muscular dystrophy using black bear parathyroid hormone

Duchenne muscular dystrophy (DMD) is a progressive disease affecting skeletal and cardiac muscle, as well as bone. Long term disuse and glucocorticoid treatments cause progressive osteoporosis in DMD patients, leading to an increase in fracture incidence. Treatments for osteoporosis in these patients have not been widely explored. Parathyroid hormone (PTH), an anabolic treatment for post-menopausal osteoporosis, could benefit DMD patients by improving skeletal properties and reducing fracture risk. Other PTH analogues are not currently FDA approved to treat osteoporosis, but may have improved osteogenic effects compared to the human analogue. Black bear PTH is especially promising as an osteoporosis treatment for the DMD population. Black bears are unique models of bone maintenance during disuse, since during six months of inactivity (hibernation), they maintain skeletal properties, unlike other hibernators. Additionally, black bear PTH has been correlated to bone formation markers during hibernation, indicating it may be, at least in part, the mechanism by which bears maintain bone during disuse. Employing black bear PTH as a treatment for osteoporosis in DMD patients could greatly improve quality of life for these individuals, and reduce the pain and expense associated with frequent fractures.

Cardiac phenotype of Duchenne Muscular Dystrophy: Insights from cellular studies

Dilated cardiomyopathy is a serious and almost inevitable complication of Duchenne Muscular Dystrophy, a devastating and fatal disease of skeletal muscle resulting from the lack of functional dystrophin, a protein linking the cytoskeleton to the extracellular matrix. Ultimately, it leads to congestive heart failure and arrhythmias resulting from both cardiac muscle fibrosis and impaired function of the remaining cardiomyocytes. Here we summarize findings obtained in several laboratories, focusing on cellular mechanisms that result in degradation of cardiac functions in dystrophy. This article is part of a Special Issue entitled "Calcium Signaling in Heart".

The TREAT-NMD DMD Global Database: analysis of more than 7,000 Duchenne muscular dystrophy mutations.

Analyzing the type and frequency of patient-specific mutations that give rise to Duchenne muscular dystrophy (DMD) is an invaluable tool for diagnostics, basic scientific research, trial planning, and improved clinical care. Locus-specific databases allow for the collection, organization, storage, and analysis of genetic variants of disease. Here, we describe the development and analysis of the TREAT-NMD DMD Global database (http://umd.be/TREAT_DMD/). We analyzed genetic data for 7,149 DMD mutations held within the database. A total of 5,682 large mutations were observed (80% of total mutations), of which 4,894 (86%) were deletions (1 exon or larger) and 784 (14%) were duplications (1 exon or larger). There were 1,445 small mutations (smaller than 1 exon, 20% of all mutations), of which 358 (25%) were small deletions and 132 (9%) small insertions and 199 (14%) affected the splice sites. Point mutations totalled 756 (52% of small mutations) with 726 (50%) nonsense mutations and 30 (2%) missense mutations. Finally, 22 (0.3%) mid-intronic mutations were observed. In addition, mutations were identified within the database that would potentially benefit from novel genetic therapies for DMD including stop codon read-through therapies (10% of total mutations) and exon skipping therapy (80% of deletions and 55% of total mutations).

A second promoter provides an alternative target for therapeutic up-regulation of utrophin in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an inherited muscle-wasting disease caused by the absence of a muscle cytoskeletal protein, dystrophin. We have previously shown that utrophin, the autosomal homologue of dystrophin, is able to compensate for the absence of dystrophin in a mouse model of DMD; we have therefore undertaken a detailed study of the transcriptional regulation of utrophin to identify means of effecting its up-regulation in DMD muscle. We have previously isolated a promoter element lying within the CpG island at the 5′ end of the gene and have shown it to be synaptically regulated in vivo. In this paper, we show that there is an alternative promoter lying within the large second intron of the utrophin gene, 50 kb 3′ to exon 2. The promoter is highly regulated and drives transcription of a widely expressed unique first exon that splices into a common full-length mRNA at exon 3. The two utrophin promoters are independently regulated, and we predict that they respond to discrete sets of cellular signals. These findings significantly contribute to understanding the molecular physiology of utrophin expression and are important because the promoter reported here provides an alternative target for transcriptional activation of utrophin in DMD muscle. This promoter does not contain synaptic regulatory elements and might, therefore, be a more suitable target for pharmacological manipulation than the previously described promoter.

Sleep-related breathing disorder in Duchenne muscular dystrophy: Disease spectrum in the paediatric population

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease with death usually occurring because of respiratory failure. Signs of early respiratory insufficiency are usually first detectable in sleep. Objective: To study the presentation of sleep-related breathing disorder (SRBD) in patients with DMD. Method:> A retrospective review of patients with DMD attending a tertiary paediatric sleep disorder clinic over a 5-year period. Symptoms, lung function and polysomnographic indices were reviewed. Results: A total of 34 patients with DMD were referred for respiratory assessment (1-15 years). Twenty-two (64%) reported sleep-related symptomatology. Forced vital capacity (FVC) was between 12 and 107% predicted (n = 29). Thirty-two progressed to have polysomnography of which 15 were normal studies (median age: 10 years) and 10 (31%) were diagnostic of obstructive sleep apnoea (OSA) (median age: 8 years). A total of 11 patients (32%) showed hypoventilation (median age: 13 years) during the 5-year period and non-invasive ventilation (NIV) was offered to them. The median FVC of this group was 27% predicted. There was a significant improvement in the apnoea/hypopnoea index (AHI) (mean difference = 11.31, 95% CI = 5.91-16.70, P = 0.001) following the institution of NIV. Conclusions: The prevalence of SRBD in DMD is significant. There is a bimodal presentation of SRBD, with OSA found in the first decade and hypoventilation more commonly seen at the beginning of the second decade. Polysomnography is recommended in children with symptoms of OSA, or at the stage of becoming wheelchair-bound. In patients with the early stages of respiratory failure, assessment with polysomnography-identified sleep hypoventilation and assisted in initiating NIV.

Cognitive dysfunction in Duchenne muscular dystrophy: a possible role for neuromodulatory immune molecules

Duchenne muscular dystrophy (DMD) is an X chromosome-linked disease characterized by progressive physical disability, immobility, and premature death in affected boys. Underlying the devastating symptoms of DMD is the loss of dystrophin, a structural protein that connects the extracellular matrix to the cell cytoskeleton and provides protection against contraction-induced damage in muscle cells, leading to chronic peripheral inflammation. However, dystrophin is also expressed in neurons within specific brain regions, including the hippocampus, a structure associated with learning and memory formation. Linked to this, a subset of boys with DMD exhibit nonprogressing cognitive dysfunction, with deficits in verbal, short-term, and working memory. Furthermore, in the genetically comparable dystrophin-deficient mdx mouse model of DMD, some, but not all, types of learning and memory are deficient, and specific deficits in synaptogenesis and channel clustering at synapses has been noted. Little consideration has been devoted to the cognitive deficits associated with DMD compared with the research conducted into the peripheral effects of dystrophin deficiency. Therefore, this review focuses on what is known about the role of full-length dystrophin (Dp427) in hippocampal neurons. The importance of dystrophin in learning and memory is assessed, and the potential importance that inflammatory mediators, which are chronically elevated in dystrophinopathies, may have on hippocampal function is also evaluated.

Assisted standing for Duchenne muscular dystrophy

This is the protocol for a review and there is no abstract. The objectives are as follows: To assess the effects of standing devices and orthoses on musculoskeletal impairments (such as pain, contracture, scoliosis development and bone density) in people with DMD, and secondarily to determine their effect on quality of life, participation in activities, and patient experience.

Best Practice Guidelines on molecular diagnostics in Duchenne/Becker muscular dystrophies

Meeting participants: Rosário dos Santos, Porto, Portugal

Développement d'un promoteur efficace et muscle spécifique pour la thérapie génique de la dystrophie musculaire de Duchenne

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Optimisation du vecteur adénoviral pour la thérapie génique de la dystrophie musculaire de Duchenne

La dystrophie musculaire de Duchenne (DMD) est une maladie très sévère, progressive et sans traitement vraiment efficace. Elle est caractérisée par l’absence fonctionnelle de la dystrophine, une protéine essentielle au maintien des muscles squelettiques. La thérapie génique est actuellement envisagée comme approche thérapeutique pour livrer la dystrophine dans les muscles. Les vecteurs adénoviraux de troisième génération (Helper-dependent adenoviral vector, HD) sont des véhicules de transfert génique très prometteurs pour traiter la DMD. Puisque les gènes adénoviraux ont été enlevés complètement du HD, ils sont peu toxiques, faiblement immunogéniques et ils possèdent un espace cargo suffisant pour transporter l’ADN codant complet de la dystrophine. Bien que le HD puisse fournir la dystrophine de façon thérapeutique chez des souris dystrophiques (mdx), l’expression du gène thérapeutique est progressivement perdue plusieurs mois suivant l’injection intramusculaire. Deux stratégies innovantes furent explorées dans cette thèse dans le but de stabiliser l’expression de la dystrophine. La première stratégie vise à l’intégration de l’ADN du HD dans les chromosomes cellulaires, ce qui pourrait le protéger contre son élimination progressive des muscles. Une intégrase site-spécifique issue du phage ΦC31 a été utilisée pour catalyser l’intégration d’un HD transportant un marqueur de sélection. Dans les cellules humaines et les myoblastes murins, l’activité de l’intégrase a été évaluée d’après son efficacité d’intégration (après sélection) et sa spécificité (dans les clones résistants). L’efficacité atteint jusqu’à 0,5 % par cellule et jusqu’à 76 % des événements d’intégration ont été réalisés de façon site-spécifique. Bien que des délétions aient été trouvées aux extrémités du vecteur, 70 % des clones analysés montraient une seule copie du vecteur intégré (le nombre attendu). Seulement une petite augmentation du nombre de brisures double-brin a été mesurée dans les myoblastes exprimant l’intégrase. En conclusion, l’intégration du HD est relativement efficace, spécifique et sécuritaire. Cette méthode est très prometteuse, car la dystrophine peut être livrée dans le muscle avec l’aide du HD et l’intégration de l’ADN du HD pourrait stabiliser son expression in vivo. La deuxième stratégie implique l’utilisation d’un nouveau promoteur musculospécifique (ΔUSEx3) pour réduire la toxicité induite liée à une expression trop étendue de la dystrophine. Dans cette étude, nous avons investigué l’effet du contexte viral sur l’activité du promoteur. Un HD et un vecteur lentiviral (LV) ont été construits avec le promoteur ΔUSEx3 pour contrôler l’expression d’un gène rapporteur. Les résultats démontrent que ΔUSEx3 confère une expression puissante, musculospécifique et stable (via le LV) in vitro. L’injection intramusculaire du HD a conduit à une expression puissante du transgène. Ces résultats contrastent avec ceux du LV, car après l’injection de ce dernier, l’expression était faible. La livraison du HD dans le muscle, mais aussi dans plusieurs organes démontre la musculospécificité de ΔUSEx3. Par conséquent, le contexte du vecteur et l’environnement musculaire modulent tous les deux l’activité de ΔUSEx3. Bien que ΔUSEx3 soit musculospécifique, d’autres études sont requises pour déterminer si le promoteur peut stabiliser l’expression de la dystrophine in vivo.

Dystrophin immunoreactivity in normal and Duchenne human fetal neurons in culture.

Dystrophin, the protein product defective in Duchenne muscular dystrophy (DMD), is present in all types of muscle and in the brain. The function of the protein is unknown and its role in the brain is unclear, although 30% of DMD patients show nonprogressive mental retardation. We have therefore studied the localisation of dystrophin in cultures of normal and DMD human fetal neurons using antibodies raised to different regions of the protein. Dystrophin immunoreactivity was demonstrated in the soma and axon hillock of normal neurons and appeared to be associated with the inner part of the cell membrane, although some intracellular staining was also observed. Positive dystrophin staining was present only in cells with fully developed neuronal features, although not all the neurons were positive. Glial cells were always negative for the antigen. Immunostaining with antibodies to the brain spectrins indicate that the dystrophin antibodies did not crossreact with these proteins. The possibility of cross-reactivity with other proteins is discussed. Studies of cells cultured from a DMD fetus also showed specific dystrophin immunostaining in neurons, although the muscle was generally negative for dystrophin. However, the localisation of dystrophin immunostaining and that of the brain spectrins and neurofilaments appeared abnormal, as did the overall morphology of the cells. This suggests that dystrophin may play a role during brain development and dystrophin deficiency results in abnormal neuronal features. This would be consistent with the nonprogressive nature of the mental retardation observed in DMD patients.

Manifesting carriers of Xp21 muscular dystrophy; lack of correlation between dystrophin expression and clinical weakness.

Ten females presenting with muscle weakness and a raised serum creatine kinase revealed abnormalities in the expression of dystrophin in their muscle biopsies and were diagnosed as manifesting carriers of Xp21 Duchenne/Becker muscular dystrophy. Seven cases, aged 3-22 yr at the time of biopsy, had a variable proportion of dystrophin-deficient fibres and an abnormal expression on immunoblot. These were confidently diagnosed as manifesting carriers. Results in the remaining three cases, aged 8-10 yr, were less clear-cut. Dystrophin expression on immunoblots was slightly reduced and some unevenness and reduction of immunolabelling was seen on sections, but dystrophin-deficient fibres were not a feature of these cases. The weakness in the ten carriers ranged from minimal to severe and there was no correlation between the degree of weakness and the number of dystrophin-deficient fibres. Two minimally weak girls had a high proportion of dystrophin-deficient fibres. Our results show that analysis of dystrophin expression is useful for the differential diagnosis of carriers of Xp21 dystrophy and autosomal muscular dystrophy, but that dystrophin expression does not correlate directly with the degree of clinical weakness.

Muscular Dystrophy in Dogs: Does the Crossing of Breeds Influence Disease Phenotype?

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Involvement of organic systems in golden retriever X-linked muscular dystrophy

Duchenne muscular dystrophy is a lethal genetic disease characterized by progressive muscle degeneration that usually had been used the Golden Retriever as a model for studying the disease (GRMD - Golden Retriever Muscular Dystrophy). A total of 16 male dystrophic Golden Retrievers dogs between 5 to 51 months of age were examined in the present study. The animals were classified as dystrophic according to two simultaneous complementary criteria: genotypic analysis and serum creatine kinase levels. The macroscopic abnormalities of the different organs and tissues and histopathological features were described using hematoxylin-eosin. The lesions in the skeletal muscles associated with the digestive problems resulted in cachexia with different intensities in all the dystrophic dogs. Cardiac muscle involvement was found in 87,5% of the GRMD dogs resulting, however, in cardiac failure in only 18,8% of the animals. The musculature of the diaphragm was hypertrophic in all affected animals resulting in progressive respiratory muscle weakness and at later stages in respiratory failure (81,25%). The liver abnormalities found in dystrophic dogs were originated mainly from heart disease and developed progressively. Hyperemia of mucosa and granular material indicated changes in the functioning and emptying of bladder. The germinative lineage cells presented moderate to severe degeneration probably due to degeneration of the scrotum and cremaster muscle which prevented the proper thermo-regulation of the testicle. Our results highlight the fact that there is significant impairment of the cardiac, respiratory and skeletal muscle systems in GRMD dogs since the age of five months. In addition, significant alterations of the gastrointestinal tract, urinary and reproductive systems are indicating the presence of degenerative lesions in the smooth musculature.