Duchenne muscular dystrophy (DMD) is associated with mutations in the dystrophin gene that disrupt the open reading frame whereas the milder Becker’s form is associated with mutations which leave an in-frame mRNA transcript that can be translated into a protein that includes the N- and C- terminal functional domains. It has been shown that by excluding specific exons at, or adjacent to, frame-shifting mutations, open reading frame can be restored to an out-of-frame mRNA, leading to the production of a partially functional Becker-like dystrophin protein. Such targeted exclusion can be achieved by administration of oligonucleotides that are complementary to sequences that are crucial to normal splicing of the exon into the transcript. This principle has been validated in mouse and canine models of DMD with a number of variants of oligonucleotide analogue chemistries and by transduction with adeno-associated virus (AAV)-small nuclear RNA (snRNA) reagents encoding the antisense sequence. Two different oligonucleotide agents are now being investigated in human trials for splicing out of exon 51 with some early indications of success at the biochemical level.

OBJECTIVE: Mutations in the genes encoding the extracellular matrix protein collagen VI (ColVI) cause a spectrum of disorders with variable inheritance including Ullrich congenital muscular dystrophy, Bethlem myopathy, and intermediate phenotypes. We extensively characterized, at the clinical, cellular, and molecular levels, 49 patients with onset in the first 2 years of life to investigate genotype-phenotype correlations.

METHODS: Patients were classified into 3 groups: early-severe (18%), moderate-progressive (53%), and mild (29%). ColVI secretion was analyzed in patient-derived skin fibroblasts. Chain-specific transcript levels were quantified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and mutation identification was performed by sequencing of complementary DNA.

RESULTS: ColVI secretion was altered in all fibroblast cultures studied. We identified 56 mutations, mostly novel and private. Dominant de novo mutations were detected in 61% of the cases. Importantly, mutations causing premature termination codons (PTCs) or in-frame insertions strikingly destabilized the corresponding transcripts. Homozygous PTC-causing mutations in the triple helix domains led to the most severe phenotypes (ambulation never achieved), whereas dominant de novo in-frame exon skipping and glycine missense mutations were identified in patients of the moderate-progressive group (loss of ambulation).

INTERPRETATION: This work emphasizes that the diagnosis of early onset ColVI myopathies is arduous and time-consuming, and demonstrates that quantitative RT-PCR is a helpful tool for the identification of some mutation-bearing genes. Moreover, the clinical classification proposed allowed genotype-phenotype relationships to be explored, and may be useful in the design of future clinical trials. ANN NEUROL 2010;68:511-520.

Duchenne's muscular dystrophy (DMD) is a severe progressive myopathy caused by mutations in the DMD gene leading to a deficiency of the dystrophin protein. Due to ongoing muscle necrosis in respiratory muscles late-stage DMD is associated with respiratory insufficiency and chronic hypoxia (CH). To understand the effects of CH on dystrophin-deficient muscle in vivo, we exposed the Drosophila model for DMD (dmDys) to CH during a 16-day ascent to the summit of Mount Denali/McKinley (6194 meters above sea level). Additionally, dmDys and wild type (WT) flies were also exposed to CH in laboratory simulations of high altitude hypoxia. Expression profiling was performed using Affymetrix GeneChips® and validated using qPCR. Hypoxic dmDys differentially expressed 1281 genes, whereas the hypoxic WT flies differentially expressed 56 genes. Interestingly, a number of genes (e.g. heat shock proteins) were discordantly regulated in response to CH between dmDys and WT. We tested the possibility that the disparate molecular responses of dystrophin-deficient tissues to CH could adversely affect muscle by performing functional assays in vivo. Normoxic and CH WT and dmDys flies were challenged with acute hypoxia and time-to-recover determined as well as subjected to climbing tests. Impaired performance was noted for CH-dmDys compared to normoxic dmDys or WT flies (rank order: Normoxic-WT ? CH-WT> Normoxic-dmDys> CH-dmDys). These data suggest that dystrophin-deficiency is associated with a disparate, pathological hypoxic stress response(s) and is more sensitive to hypoxia induced muscle dysfunction in vivo. We hypothesize that targeting/correcting the disparate molecular response(s) to hypoxia may offer a novel therapeutic strategy in DMD.

STUDY DESIGN: A retrospective study.

OBJECTIVES: To evaluate clinical and functional success by all pedicle screw construct in paralytic neuromuscular scoliosis (NMS) with poor pulmonary functions (PFT).

SUMMARY OF BACKGROUND: Duchene muscular dystrophy and spinal muscular atrophy are often associated with poor PFT and the development of scoliosis simultaneously. Poor PFT often make surgeons reluctant to operate.

METHODS: Eighteen paralytic NMS patients who had preoperative forced vital capacity (FVC) <30% were operated with all pedicle screw construct. Average preoperative, postoperative, and final follow-up Cobb angle, pelvic obliquity, thoracic kyphosis and lumbar lordosis, PFT (FVC% and forced expiratory volume 1%), and preoperative and follow-up functional status were analyzed. Perioperative and postoperative complications were also noted.

RESULTS: The average follow-up was 31.6±7.7 months. There was significant improvement in Cobb angle (61.7%) and pelvic obliquity (56.7%), postoperatively (P<0.001). All corrections were maintained at final follow-up. FVC was decreased from 25.2±4.7% preoperatively to 24.2±5.0%, 6 weeks postoperatively (P=0.067); and on follow-up it further decreased to 20.6±3.9% (P<0.0001) (1.8%/y). Forced Expiratory Volume 1 also decreased from 22.7±4.5% preoperatively to 21.8±4.2% postoperatively (P=0.037) and was 19.8±3.8% at final follow-up (P<0.0001) (1.1%/y). However, none of the patients had any respiratory complications postoperatively. Functional status was improved in 6 patients and they were able to sit without support (P=0.027). Eight (44.4%) perioperative complications (5 pulmonary, 1 intraoperative death, and 2 others) were noticed. Postoperatively, 4 patients (23.5%) had complications; coccygodynia, back sore because of screw prominence, impingement of iliac screw, and loosening of the rod from L5 screw. All the patients were satisfied with the treatment. There were no major pulmonary complications requiring admission postoperatively.

CONCLUSIONS: Although complications are associated with the treatment of paralytic NMS, a good clinical and function outcome suggests that poor PFT should not be considered as a contraindication of scoliosis surgery.

OBJECTIVE: In some 5% of patients with facioscapulohumeral muscular dystrophy (FSHD), no D4Z4 repeat contraction on chromosome 4q35 is observed. Such patients, termed patients with FSHD2, show loss of DNA methylation and heterochromatin markers at the D4Z4 repeat that are similar to patients with D4Z4 contractions (FSHD1). This commonality suggests that a change in D4Z4 chromatin structure unifies FSHD1 and FSHD2. The aim of our study was to critically evaluate the clinical features in patients with FSHD2 in order to establish whether these patients are phenotypically identical to FSHD1 and to establish the effects of the (epi-) genotype on the phenotype.

METHODS: This cross-sectional study studied 33 patients with FSHD2 from 27 families, the largest cohort described to date. All patients were clinically assessed using a standardized clinical evaluation form. Genotype analysis was performed by pulsed field gel electrophoresis and PCR; D4Z4 methylation was studied by methylation-sensitive Southern blot analysis.

RESULTS: FSHD2 is identical to FSHD1 in its clinical presentation. Notable differences include a higher incidence (67%) of sporadic cases and the absence of gender differences in disease severity in FSHD2. Overall, average disease severity in FSHD2 was similar to that reported in FSHD1 and was not influenced by D4Z4 repeat size. In FSHD2, a small effect of the degree of hypomethylation on disease severity was observed.

CONCLUSIONS: Clinically, patients with FSHD2 are indistinguishable from patients with FSHD1. The present data suggest that FSHD1 and FSHD2 are the result of the same pathophysiologic process.

X-linked muscular dystrophy of the mouse (mdx) has been reported to progressively remodel skeletal muscle to preferentially reduce fast fiber composition. Despite this, mdx muscle displays normal levels of posttetanic potentiation (PTP). Since PTP may primarily depend on phosphorylation of the myosin regulatory light chain (RLC) in fast muscle fibers, maintenance of PTP with mdx disease progression is paradoxical and may represent an adaptation of the diseased muscle. This study assesses the role of RLC phosphorylation during PTP of mdx muscle. Extensor digitorum longus muscles were isolated from mdx and from C57BL/10 (control) mice at ~50 (young) and ~300 (adult) days and stimulated in vitro (25°C) to induce PTP. During potentiation, muscles were harvested for subsequent determination of RLC phosphorylation levels. Immunofluorescence was used to assess muscle fiber type composition and no age effects were found. The magnitude of PTP was higher (P < 0.05) in mdx than control muscles at both young (mdx: 21.9 ± 1.6%; control: 17.7 ± 1.2%) and adult (mdx: 30.4 ± 1.8%; control: 23.2 ± 2.2%) ages. However, RLC phosphate content was similar between all groups both at rest and following stimulation. Our results are consistent with a model where the sensitivity of mdx muscle to RLC phosphorylation-induced force potentiation is increased by disease- and age-dependent alterations in excitation-contraction coupling noted for mdx and aging muscle.

Aims: Susceptibility of cardiomyocytes to stress-induced damage has been implicated in the development of cardiomyopathy in Duchenne muscular dystrophy (DMD), a disease caused by lack of the cytoskeletal protein dystrophin in which heart failure is frequent. However, the factors underlying disease progression are unclear and treatments limited. Here, we tested the hypothesis of a greater susceptibility to opening of the mitochondrial permeability transition pore (PTP) in hearts from young dystrophic (mdx mice) (prior to development of overt cardiomyopathy) when subjected to a stress protocol, and determined whether prevention of PTP opening is involved in the cardio protective effect of sildenafil, which we have previously reported in mdx mice. Methods and results: Using the [(3)H]-DOG method to quantify PTP opening in ex vivo perfused hearts, we demonstrate that, compared to controls, hearts from young mdx mice subjected to ischemia-reperfusion (I-R) display excessive PTP opening as well as enhanced activation of cell death signaling, mitochondrial oxidative stress, cardiomyocyte damage and poorer recovery of contractile function. Functional analyses in permeabilized cardiac fibers from non-ischemic hearts revealed that in vitro mitochondria from mdx hearts display normal respiratory function and ROS handling, but enhanced Ca2+ uptake velocity, and premature opening of the PTP, which may predispose to I-R-induced injury. Administration of a single dose of sildenafil to mdx mice, prior to I-R prevented excessive PTP opening and its downstream consequences, and reduced tissue Ca(2+) levels. Furthermore, mitochondrial Ca(2+) uptake velocity was reduced following sildenafil treatment. Conclusion: Beyond documenting that an increased susceptibility to opening of the mitochondrial PTP in mdx heart occurs well before clinical signs of overt cardiomyopathy, our results demonstrate that sildenafil, which is already administered in other pediatric populations, and is reported safe and well-tolerated, provides efficient protection against this deleterious event, likely by reducing cellular Ca(2+) loading, and mitochondrial Ca(2+) uptake.

Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) is a candidate gene for mediating FSHD pathophysiology, however, very little is known about the endogenous FRG1 protein. This study uses immunocytochemistry (ICC) and histology to provide insight into FRG1′s role in vertebrate muscle development and address its potential involvement in FSHD pathophysiology. In cell culture, primary myoblast/myotube cultures, and mouse and human muscle sections, FRG1 showed distinct nuclear and cytoplasmic localizations and nuclear shuttling assays indicated the subcellular pools of FRG1 are linked. During myoblast differentiation, FRG1′s subcellular distribution changed dramatically with FRG1 eventually associating with the matured Z-discs. This Z-disc localization was confirmed using isolated mouse myofibers and found to be maintained in adult human skeletal muscle biopsies. Thus, FRG1 is not likely involved in the initial assembly and alignment of the Z-disc but may be involved in sarcomere maintenance or signaling. Further analysis of human tissue showed FRG1 is strongly expressed in arteries, veins, and capillaries, the other prominently affected tissue in FSHD. Overall, we show that in mammalian cells, FRG1 is a dynamic nuclear and cytoplasmic protein, however in muscle, FRG1 is also a developmentally regulated sarcomeric protein suggesting FRG1 may perform a muscle-specific function. Thus, FRG1 is the only FSHD candidate protein linked to the muscle contractile machinery and may address why the musculature and vasculature are specifically susceptible in FSHD.

Copyright © 2010 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Summary? The objective was to explore quantitative methods for the measurement of lip mobility and lip force and to relate these to qualitative assessments of lip function. Fifty healthy adults (mean age 45?years) and 23 adults with diagnoses affecting the facial muscles (mean age 37?years) participated in the study. Diagnoses were Möbius syndrome (n?=?5), Facioscapulohumeral muscular dystrophy (n?=?6) and Myotonic dystrophy type 1 (n?=?12). A system for computerised 3D analysis of lip mobility and a lip force meter were tested, and the results were related to results from qualitative assessments of lip mobility, speech (articulation), eating ability and saliva control. Facial expressions studied were open mouth smile and lip pucker. Normative data and cut-off values for adults on lip mobility and lip force were proposed, and the diagnostic value of these thresholds was tested. The proposed cut-off values could identify all inviduals with moderate or severe impairment of lip mobility but not always the milder cases. There were significant correlations between the results from quantitative measurements and qualitative assessments. The examined instruments for measuring lip function were found to be reliable with an acceptable measuring error. The combination of quantitative and qualitative ways to evaluate lip function made it possible to show the strong relation between lip contraction, lip force, eating ability and saliva control. The same combination of assessments can be used in the future to study if oral motor exercises aimed at improving lip mobility and strength could have a positive effect on lip function.

© 2010 Blackwell Publishing Ltd.