Deletion Analysis of 15 exons located outside and inside a “Hot Spot” in the Duchenne Muscular Dystrophy gene in Duchenne’s Muscular dystrophy patients

Introduction. Duchenne and Becker Muscular Dystrophies (DMD/DMB) are X-linked recessive diseases characterized by progressive muscle weakness and wasting, loss of motor skills and death after the second decade of life. Deletions are the most prevalent mutations that affect the dystrophin gene, which spans 79 exons.Objective: Identify deletions on the dystrophin gene in 58 patients affected with DMD.Methods: Through multiplex PCR identify deletions on the dystrophin gene in 58 patients with DMD and observe the frequency of this mutation in our population.Results: We found deletions in 1.72% of patients (1 of 58 persons). Deletions were not the principal cause of disease in our population. It is possible that duplications and point mutations caused this illness in our patients.Conclusions: The frequency of deletions in the 15 exons analyzed from the dystrophin gene was low. The predominant types of mutation in our patients` samples were not deletions as has been observed in the literature worldwide, therefore, it is important to determine other types of mutations as are duplications and point mutations.

Codon and mRNA sequence optimization of microdystrophin transgenes improves expression and physiological outcome in dystrophic mdx mice following AAV2/8 gene transfer

Duchenne muscular dystrophy is a fatal muscle-wasting disorder. Lack of dystrophin compromises the integrity of the sarcolemma and results in myofibers that are highly prone to contraction-induced injury. Recombinant adenoassociated virus (rAAV)-mediated dystrophin gene transfer strategies to muscle for the treatment of Duchenne muscular dystrophy (DMD) have been limited by the small cloning capacity of rAAV vectors and high titers necessary to achieve efficient systemic gene transfer. In this study, we assess the impact of codon optimization on microdystrophin (ΔAB/R3-R18/ΔCT) expression and function in the mdx mouse and compare the function of two different configurations of codon-optimized microdystrophin genes (ΔAB/R3-R18/ΔCT and ΔR4-R23/ΔCT) under the control of a muscle-restrictive promoter (Spc5-12). Codon optimization of microdystrophin significantly increases levels of microdystrophin mRNA and protein after intramuscular and systemic administration of plasmid DNA or rAAV2/8. Physiological assessment demonstrates that codon optimization of ΔAB/R3-R18/ΔCT results in significant improvement in specific force, but does not improve resistance to eccentric contractions compared with noncodon-optimized ΔAB/ R3-R18/ΔCT. However, codon-optimized microdystrophin ΔR4-R23/ΔCT completely restored specific force generation and provided substantial protection from contraction-induced injury. These results demonstrate that codon optimization of microdystrophin under the control of a muscle-specific promoter can significantly improve expression levels such that reduced titers of rAAV vectors will be required for efficient systemic administration.

Delivery of AAV2/9-microdystrophin genes incorporating helix 1 of the coiled-coil motif in the C-terminal domain of dystrophin improves muscle pathology and restores the level of α1-syntrophin and α-dystrobrevin in skeletal muscles of mdx mice. Level of α1-Syntrophin and α-Dystrobrevin in Skeletal Muscles of mdx Mice

Duchenne muscular dystrophy is a severe X-linked inherited muscle wasting disorder caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vectors have been extensively used to deliver genes efficiently for dystrophin expression in skeletal muscles. To overcome limited packaging capacity of AAV vectors (<5 kb), truncated recombinant microdystrophin genes with deletions of most of rod and carboxyl-terminal (CT) domains of dystrophin have been developed. We have previously shown the efficiency of mRNA sequence–optimized microdystrophin (ΔR4-23/ΔCT, called MD1) with deletion of spectrin-like repeat domain 4 to 23 and CT domain in ameliorating the pathology of dystrophic mdx mice. However, the CT domain of dystrophin is thought to recruit part of the dystrophin-associated protein complex, which acts as a mediator of signalling between extracellular matrix and cytoskeleton in muscle fibers. In this study, we extended the ΔR4-23/ΔCT microdystrophin by incorporating helix 1 of the coiled-coil motif in the CT domain of dystrophin (MD2), which contains the α1-syntrophin and α-dystrobrevin binding sites. Intramuscular injection of AAV2/9 expressing CT domain–extended microdystrophin showed efficient dystrophin expression in tibialis anterior muscles of mdx mice. The presence of the CT domain of dystrophin in MD2 increased the recruitment of α1-syntrophin and α-dystrobrevin at the sarcolemma and significantly improved the muscle resistance to lengthening contraction–induced muscle damage in the mdx mice compared with MD1. These results suggest that the incorporation of helix 1 of the coiled-coil motif in the CT domain of dystrophin to the microdystrophins will substantially improve their efficiency in restoring muscle function in patients with Duchenne muscular dystrophy.

Dystrophin abnormalities in polymyositis and dermatomyositis.

The expression of dystrophin in muscle biopsies from nine cases of polymyositis, ten cases of juvenile dermatomyositis and three adults with dermatomyositis was studied by Western blot analysis and immunocytochemistry. Five antibodies corresponding to different N- and C-terminal regions of the dystrophin gene were used. Sixteen of the 22 cases (73%) showed an abnormality in the expression of dystrophin on Western blot analysis, either with a reduced molecular weight protein or a reduced amount. Immunostaining was abnormal in 11 out of 19 cases (58%) and showed varying degrees of discontinuity or loss of sarcolemmal staining. Immunolabelling of these areas with antibodies to beta-spectrin was normal implying that the changes were not caused by a loss of the sarcolemma. These results show that secondary changes in the expression of dystrophin can occur in the absence of an abnormality in the corresponding gene and that dystrophin cannot be used in isolation as a diagnostic marker for muscular dystrophy.

A dystrophin-immunoreactive protein in mammalian brain.

Dystrophin is expressed only in muscle and brain, but is absent from all tissues of the adult mdx mouse, a mutant with a single base substitution in the dystrophin gene. The brains of both normal and mdx mice contain a protein of approximately 230 kDa that is recognised by anti-dystrophin antibodies raised to the N-terminal region of the rod-like domain. Although the N-terminal and central rod regions of dystrophin share structural homologies with spectrin, the 230-kDa protein represents neither of the presently described forms of brain spectrin by a variety of criteria (molecular weight, cerebellar localisation, and developmental regulation) and is distinct from the product of the dystrophin gene. Studies of mdx and normal mouse brain show different postnatal developmental regulation of the 230-kDa dystrophin-immunoreactive protein.

Brain metabolite composition in relation to cognitive function and dystrophin mutations in boys with Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a hereditary X-linked recessive disorder affecting the synthesis of dystrophin, a protein essential for structural stability in muscle. Dystrophin also occurs in the central nervous system, particularly in the neocortex, hippocampus and cerebellum. Quantitative metabolic analysis by localized (1) H MRS was performed in the cerebellum (12 patients and 15 controls) and a temporo-parietal location (eight patients and 15 controls) in patients with DMD and healthy controls to investigate possible metabolic differences. In addition, the site of individual mutations on the dystrophin gene was analyzed and neuropsychological cognitive functions were examined. Cognitive deficits in the patient group were found in line with earlier investigations, mainly concerning verbal short-term memory, visuo-spatial long-term memory and verbal fluency, but also the full-scale IQ. Causal mutations were identified in all patients with DMD. Quantitative MRS showed consistent choline deficits, in both cerebellar white matter and temporo-parietal cortex, as well as small, but significant, metabolic abnormalities for glutamate and total N-acetyl compounds in the temporo-parietal region. Compartment water analysis did not reveal any abnormalities. In healthy subjects, choline levels were age related in the cerebellum. The choline deficit contrasts with earlier findings in DMD, where a surplus of choline was postulated for the cerebellum. In patients, total N-acetyl compounds in the temporo-parietal region were related to verbal IQ and verbal short-term memory. However, choline, the putative main metabolic abnormality, was not found to be associated with cognitive deficits. Furthermore, in contrast with the cognitive performance, the metabolic brain composition did not depend significantly on whether or not gene mutations concerned the expression of the dystrophin isoform Dp140, leading to the conclusion that the effect of the missing Dp140 isoform on cognitive performance is not mediated through the observed metabolite composition, or is caused by local effects beyond the resolution accessible to MRS investigations.

Neuropsychological impairments and the impact of dystrophin mutations on general cognitive functioning of patients with Duchenne muscular dystrophy

Mutations in the dystrophin gene have long been recognised as a cause of mental retardation. However, for reasons that are unclear, some boys with dystrophin mutations do not show general cognitive deficits. To investigate the relationship between dystrophin mutations and cognition, the general intellectual abilities of a group of 25 boys with genetically confirmed Duchenne muscular dystrophy were evaluated. Furthermore, a subgroup underwent additional detailed neuropsychological assessment. The results showed a mean full scale intelligence quotient (IQ) of 88 (standard deviation 24). Patients performed very poorly on various neuropsychological tests, including arithmetics, digit span tests and verbal fluency. No simple relationship between dystrophin mutations and cognitive functioning could be detected. However, our analysis revealed that patients who lack the dystrophin isoform Dp140 have significantly greater cognitive problems.

U7 snRNAs induce correction of mutated dystrophin pre-mRNA by exon skipping.

Most cases of Duchenne muscular dystrophy are caused by dystrophin gene mutations that disrupt the mRNA reading frame. Artificial exclusion (skipping) of a single exon would often restore the reading frame, giving rise to a shorter, but still functional dystrophin protein. Here, we analyzed the ability of antisense U7 small nuclear (sn)RNA derivatives to alter dystrophin pre-mRNA splicing. As a proof of principle, we first targeted the splice sites flanking exon 23 of dystrophin pre-mRNA in the wild-type muscle cell line C2C12 and showed precise exon 23 skipping. The same strategy was then successfully adapted to dystrophic immortalized mdx muscle cells where exon-23-skipped dystrophin mRNA rescued dystrophin protein synthesis. Moreover, we observed a stimulation of antisense U7 snRNA expression by the murine muscle creatine kinase enhancer. These results demonstrate that alteration of dystrophin pre-mRNA splicing could correct dystrophin gene mutations by expression of specific U7 snRNA constructs.

Rescue of dystrophin expression in mdx mouse muscle by RNA/DNA oligonucleotides

Chimeric RNA/DNA oligonucleotides (“chimeraplasts”) have been shown to induce single base alterations in genomic DNA both in vitro and in vivo. The mdx mouse strain has a point mutation in the dystrophin gene, the consequence of which is a muscular dystrophy resulting from deficiency of the dystrophin protein in skeletal muscle. To test the feasibility of chimeraplast-mediated gene therapy for muscular dystrophies, we used a chimeraplast (designated “MDX1”) designed to correct the point mutation in the dystrophin gene in mdx mice. After direct injection of MDX1 into muscles of mdx mice, immunohistochemical analysis revealed dystrophin-positive fibers clustered around the injection site. Two weeks after single injections into tibialis anterior muscles, the maximum number of dystrophin-positive fibers (approximately 30) in any muscle represented 1–2% of the total number of fibers in that muscle. Ten weeks after single injections, the range of the number of dystrophin-positive fibers was similar to that seen after 2 wk, suggesting that the expression was stable, as would be predicted for a gene-conversion event. Staining with exon-specific antibodies showed that none of these were “revertant fibers.” Furthermore, dystrophin from MDX1-injected muscles was full length by immunoblot analysis. No dystrophin was detectable by immunohistochemical or immunoblot analysis after control chimeraplast injections. Finally, reverse transcription–PCR analysis demonstrated the presence of transcripts with the wild-type dystrophin sequence only in mdx muscles injected with MDX1 chimeraplasts. These results provide the foundation for further studies of chimeraplast-mediated gene therapy as a therapeutic approach to muscular dystrophies and other genetic disorders of muscle.

G-utrophin, the autosomal homologue of dystrophin Dp116, is expressed in sensory ganglia and brain.

The utrophin gene is closely related to the dystrophin gene in both sequence and genomic structure. The Duchenne muscular dystrophy (DMD) locus encodes three 14-kb dystrophin transcripts in addition to several smaller isoforms, one of which, Dp116, is specific to peripheral nerve. We describe here the corresponding 5.5-kb mRNA from the utrophin locus. This transcript, designated G-utrophin, is of particular interest because it is specifically expressed in the adult mouse brain and appears to be the predominant utrophin transcript in this tissue. G-utrophin is expressed in brain sites generally different from the regions expressing beta-dystroglycan. During mouse embryogenesis G-utrophin is also seen in the developing sensory ganglia. Our data confirm the close evolutionary relationships between the DMD and utrophin loci; however, the functions for the corresponding proteins probably differ.

Ringo: Discordance between the molecular and clinical manifestation in a Golden Retriever Muscular Dystrophy dog

Of the various genetic homologues to Duchenne Muscular Dystrophy (DMD), the Golden Retriever Muscular Dystrophy (GRMD) dog, which presents a variable but usually severe and progressive muscle weakness, has the closest relevance to DMD in both clinical severity and histopathological change. Among 77 GRMD dogs born in our colony in Brazil, we have identified a very mildly affected dog, Ringo, born July 2003. Among his descendants, at least one male, Suflair, is also showing a mild course. In an attempt to better characterize these two dogs, we studied the pattern of muscle proteins expression in Ringo and Suflair, as compared to severely affected and normal control dogs. Dystrophin was absent in both and utrophin was overexpressed in a pattern similar to the observed in severely affected dogs. Understanding the mechanism that is protecting Ringo and Suflair from the deleterious effect of the dystrophin gene mutation is of utmost interest, In addition it points out that the clinical impact of therapeutic trials should be interpreted with caution. (C) 2009 Elsevier B.V. All rights reserved.

Marked hemiatrophy in carriers of Duchenne muscular dystrophy.

OBJECTIVE: To describe the clinical and molecular genetic findings in 2 carriers of Duchenne muscular dystrophy (DMD) who exhibited marked hemiatrophy. Duchenne muscular dystrophy is an X-linked disorder in which affected male patients harbor mutations in the dystrophin gene. Female patients with heterozygous mutations may be manifesting carriers. DESIGN: Case study. SETTING: Neurology clinic. PATIENTS: Two manifesting carriers of DMD. INTERVENTIONS: Clinical and radiologic examinations along with histologic and molecular investigations. RESULTS: Both patients had marked right-sided hemiatrophy on examination with radiologic evidence of muscle atrophy and fatty replacement on the affected side. In each case, histologic analysis revealed a reduction in dystrophin staining on the right side. Genetic analysis of the dystrophin gene revealed a tandem exonic duplication in patient 1 and a multiexonic deletion in patient 2 with no further point mutations identified on the other chromosome. CONCLUSIONS: Marked hemiatrophy can occur in DMD manifesting carriers. This is likely to result from a combination of skewed X-inactivation and somatic mosaicism.

Essais thérapeutiques dans la dystrophie musculaire de Duchenne: entre espoirs et désespoirs [Therapeutic trials for Duchenne muscular dystrophy: between hopes and disappointments].

Duchenne muscular dystrophy is an X-linked progressive muscle disease. Since the discovery of the dystrophin gene responsible for the condition, various therapeutic strategies have been elaborated. In this paper we introduce three of them, which are well into clinical trials. The first is based on the ability to read through premature stop codons, the second is based on the technique of exon skipping. Both strategies are examples of "personalized medicines", tailored for specific mutation types. The third approach is a pharmacological one, potentially useful for all Duchenne patients, regardless of their mutation type. These first clinical trials raise many questions for researchers as well as for patients and their families, some of which are discussed.

Lihasperäiselle kreatiinikinaasille spesifinen immunomääritys Duchennen lihasdystrofian seulontaa varten

Duchennen lihasdystrofia (engl. Duchenne muscular dystrophy, DMD) on lähes pelkästään pojilla ilmenevä perinnöllinen lihasrappeumatauti, joka johtaa kuolemaan noin 25 vuoden iässä. Noin yksi 3500–6000 pojasta sairastaa DMD:tä. Taudin aiheuttaa X-kromosomissa sijaitsevan dystrofiinigeenin mutaatio, jonka seurauksena toimivaa, lihaksia koossapitävää dystrofiinia ei tuotu. Kliinisissä testeissä on lupaavia hoitoja, joten DMD:n vastasyntyneiden seulonnan aloittamista harkitaan. DMD:n seulonnassa analyyttina olisi mahdollista käyttää lihasperäistä kreatiinikinaasia (engl. muscle-type creatine kinase tai creatine kinase MM isoform, CK-MM), jota päätyy vereen lihassolujen vaurioituessa. DMD:tä sairastavilla vastasyntyneillä CK-MM:n määrä veressä on moninkertainen terveisiin vastasyntyneisiin verrattuna lihasten rappeutumisesta johtuen. Perinteisesti kreatiinikinaasia on mitattu entsyymiaktiivisuusmäärityksillä, jotka mittaavat kaikkia kreatiinikinaasimuotoja eli myös sydänperäistä ja aivoperäistä kreatiinikinaasia (CK-MB ja CK-BB). Työn tarkoituksena oli kehittää kuivatuista veritäplistä tehtävä CK-MM:lle spesifinen kaksipuoleinen immunomääritys, joka olisi siirrettävissä PerkinElmerin automaattiselle GSP® Genetic Screening Processor -analysaattorille. Työ suoritettiin kolmessa vaiheessa. Ensimmäiseksi vertailtiin kaupallisesti saatavilla olevien CK-MM-vasta-aineiden affiniteetteja biosensorilla. Seuraavassa vaiheessa pystytettiin manuaalinen kaksipuoleinen immunomääritys käyttäen ensimmäisessä vaiheessa valittuja vasta-aineita ja optimoitiin immunomäärityksen parametreja. Lopuksi immunomääritys sovitettiin GSP-laitteelle. Biosensorimittausten ja manuaalisten immunomääritysten tulosten perusteella valittiin kaksi potentiaalista leimavasta-ainetta ja yksi sitojavasta-aineeksi sopiva vasta-aine. Niitä käytettäessä määritys on melko spesifinen CK-MM:lle, sillä CK-BB ei tuottanut lainkaan signaalia ja CK-MB:n ristireaktiivisuus oli noin 7 %. GSP-laitteella mitattaessa DMD:tä sairastavien (n = 10) CK-MM-pitoisuuksien mediaani (vaihteluväli) oli 7590 ng/ml (1490–13400 ng/ml) ja terveiden vastasyntyneiden (n = 8) 165 ng/ml (108–263 ng/ml). Määrityksen dynaamista mittausaluetta ei vielä selvitetty, mutta alustavien mittausten perusteella se kattaa terveiden vastasyntyneiden pitoisuudet ja sairaiden pitoisuudet ainakin 8770 ng/ml asti, mikä mahdollistaa sairaiden erottumisen. Työssä kehitetty määritys vaikuttaa siis sopivalta DMD:n seulontaan vastasyntyneiltä.

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.