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BMD is characterized by a marked heterogeneity of gene mutations resulting in many abnormal dystrophin proteins with different expression and residual functions. The smaller dystrophin molecules lacking a portion around exon 48 of the rod domain, named the D8 region, are related to milder phenotypes. The study aimed to determine which proteins might contribute to preserving muscle function in these patients. Patients were subdivided, based on the absence or presence of deletions in the D8 region, into two groups, BMD1 and BMD2. Muscle extracts were analyzed by 2-D DIGE, label-free LC-ESI-MS/MS, and Ingenuity pathway analysis (IPA). Increased levels of proteins typical of fast fibers and of proteins involved in the sarcomere reorganization characterize BMD2. IPA of proteomics datasets indicated in BMD2 prevalence of glycolysis and gluconeogenesis and a correct flux through the TCA cycle enabling them to maintain both metabolism and epithelial adherens junction. A 2-D DIGE analysis revealed an increase of acetylated proteoforms of moonlighting proteins aldolase, enolase, and glyceraldehyde-3-phosphate dehydrogenase that can target the nucleus promoting stem cell recruitment and muscle regeneration. In BMD2, immunoblotting indicated higher levels of myogenin and lower levels of PAX7 and SIRT1/2 associated with a set of proteins identified by proteomics as involved in muscle homeostasis maintenance.

Background:Spinal muscular atrophy (SMA) is a neuromuscular disorder characterized by the degeneration of the second motor neuron. The phenotype ranges from very severe to very mild forms. All patients have the homozygous loss of the SMN1 gene and a variable number of SMN2 (generally 2–4 copies), inversely related to the severity. The amazing results of the available treatments have made compelling the need of prognostic biomarkers to predict the progression trajectories of patients. Besides the SMN2 products, few other biomarkers have been evaluated so far, including some miRs.Methods:We performed whole miRNome analysis of muscle samples of patients and controls (14 biopsies and 9 cultures). The levels of muscle differentially expressed miRs were evaluated in serum samples (51 patients and 37 controls) and integrated with SMN2 copies, SMN2 full-length transcript levels in blood and age (SMA-score).Results:Over 100 miRs were differentially expressed in SMA muscle; 3 of them (hsa-miR-181a-5p, -324-5p, -451a; SMA-miRs) were significantly upregulated in the serum of patients. The severity predicted by the SMA-score was related to that of the clinical classification at a correlation coefficient of 0.87 (p-5).Conclusions:miRNome analyses suggest the primary involvement of skeletal muscle in SMA pathogenesis. The SMA-miRs are likely actively released in the blood flow; their function and target cells require to be elucidated. The accuracy of the SMA-score needs to be verified in replicative studies: if confirmed, its use could be crucial for the routine prognostic assessment, also in presymptomatic patients.Funding:Telethon Italia (grant #GGP12116).

International audience; Among rare diseases caused by mutations in LMNA gene, Emery-Dreifuss Muscular Dystrophy type 2 and Limb-Girdle muscular Dystrophy 1B are characterized by muscle weakness and wasting, joint contractures, cardiomyopathy with conduction system disorders. Circulating biomarkers for these pathologies have not been identified. Here, we analyzed the secretome of a cohort of patients affected by these muscular laminopathies in the attempt to identify a common signature. Multiplex cytokine assay showed that transforming growth factor beta 2 (TGF β2) and interleukin 17 serum levels are consistently elevated in the vast majority of examined patients, while interleukin 6 and basic fibroblast growth factor are altered in subgroups of patients. Levels of TGF β2 are also increased in fibroblast and myoblast cultures established from patient biopsies as well as in serum from mice bearing the H222P Lmna mutation causing Emery-Dreifuss Muscular Dystrophy in humans. Both patient serum and fibroblast conditioned media activated a TGF β2-dependent fibrogenic program in normal human myoblasts and tenocytes and inhibited myoblast differentiation. Consistent with these results, a TGF β2 neutralizing antibody avoided fibrogenic marker activation and myogenesis impairment. Cell intrinsic TGF β2-dependent mechanisms were also determined in laminopathic cells, where TGF β2 activated AKT/mTOR phosphorylation. These data show that TGF β2 contributes to the pathogenesis of Emery-Dreifuss Muscular Dystrophy type 2 and Limb-Girdle muscular Dystrophy 1B and can be considered a potential biomarker of those diseases. Further, the evidence of TGF β2 pathogenetic effects in tenocytes provides the first mechanistic insight into occurrence of joint contractures in muscular laminopathies.

Objective Myotonic dystrophy type 1 (DM1) is a hereditary myopathy characterized by an autosomal dominant inheritance with important cardiovascular and autonomic deregulation. DM1 patients have a high prevalence of obstructive sleep apnea (OSA), but the effects of this comorbidity on cardiovascular autonomic control (CAC) are unknown. The present study aimed to investigate CAC during sleep–wake cycle in DM1 patients, taking into account the effects of OSA comorbidity. Method Twenty-three patients with a diagnosis of DM1, and a control group, underwent a complete polysomnographic study (PSG). Electrocardiogram and respiration were extracted from PSG, divided according to the sleep stages, and analyzed using spectral analysis (SpA) of heart rate variability (HRV). SpA identified three components: very low frequency (VLF), low frequency (LF), a marker of sympathetic modulation, and high frequency (HF), a marker of vagal modulation. Results The results showed that in DM1 patients, the sympathovagal balance shifted towards a vagal predominance during non-rapid eye movement (NREM) sleep and a sympathetic predominance during rapid eye movement (REM) sleep. Second, this preserved cardiac autonomic modulation was not affected by the comorbidity with obstructive sleep apnea syndrome (OSAS). Third, in DM1 patients, OSAS comorbidity was associated with a reduction in HRV during the whole sleep–wake cycle. Lastly, in DM1 patients with OSA, cardiorespiratory coupling was reduced compared to controls. Conclusions DM1 patients had preserved cardiac autonomic dynamics during NREM and REM sleep, and this phenomenon was not affected by the presence of OSA. However, the comorbidity with OSA was characterized by a reduction in total HRV, which is a marker of the ability of autonomic control to respond to stressors stimuli.

Congenital myopathies are a group of genetic muscle disorders characterized clinically by hypotonia and weakness, usually from birth, and a static or slowly progressive clinical course. Historically, congenital myopathies have been classified on the basis of major morphological features seen on muscle biopsy. However, different genes have now been identified as associated with the various phenotypic and histological expressions of these disorders, and in recent years, because of their unexpectedly wide genetic and clinical heterogeneity, next-generation sequencing has increasingly been used for their diagnosis. We reviewed clinical and genetic forms of congenital myopathy and defined possible strategies to improve cost-effectiveness in histological and imaging diagnosis.

To date, only few mutations in the mitochondrial DNA (mtDNA)-encoded ND2 subunit of Complex I have been reported, usually presenting a severe phenotype characterized by early onset encephalomyopathy and early death. In this report, we describe a new mutation in the MTND2 gene in a 21-year-old man with a mild myopathic phenotype characterized by exercise intolerance and increased plasma lactate at rest. Electromyography and brain NMR were normal, and no cardiac involvement was present. Muscle biopsy showed a massive presence of ragged red – COX-positive fibres, with enlarged mitochondria containing osmiophilic inclusions. Biochemical assays revealed a severe isolated complex I deficiency. We identified a novel, heteroplasmic mutation m.4831G > A in the MTND2 gene, causing the p.Gly121Asp substitution in the ND2 protein. The mutation was present in the 95% of mitochondrial genomes from patient's muscle tissue, at a lower level in cells from the urinary tract and at a lowest level in lymphocytes from patient's blood; the base substitution was absent in fibroblasts and in the tissues from proband's healthy mother and brother. The specific skeletal muscle tissue involvement can explain the childhood-onset and the relatively benign, exclusively myopathic course of the disease.

Neutral lipid storage disease with myopathy (NLSDM) is a rare autosomal recessive disorder, due to an enzymatic error of lipid metabolism. Patients present always with skeletal muscle myopathy and variable cardiac and hepatic involvement. NLSDM is caused by mutations in the PNPLA2 gene, which encodes the adipose triglyceride lipase (ATGL). Here we report the molecular characterization and clinical findings of two NLSDM siblings carrying the novel c.187+1G > C homozygous PNPLA2 mutation, localized in the splice site of intron 2. Molecular analyses revealed that neither aberrant PNPLA2 mRNA isoforms, nor ATGL mutated protein were detectable in patient's cells. Clinically, both patients presented early onset muscle weakness, in particular of proximal upper limb muscles. In almost 15 years, muscle damage affected also distal upper limbs. This is a NLSDM family, displaying a severe PNPLA2 mutation in two siblings with clinical presentation characterized by an early onset, but a slowly evolution of severe myopathy.

Neutral lipid storage disease with myopathy (NLSDM) is a rare autosomal recessive disorder, due to an enzymatic error of lipid metabolism. Patients present always with skeletal muscle myopathy and variable cardiac and hepatic involvement. NLSDM is caused by mutations in the PNPLA2 gene, which encodes the adipose triglyceride lipase (ATGL). Here we report the molecular characterization and clinical findings of two NLSDM siblings carrying the novel c.187+1G > C homozygous PNPLA2 mutation, localized in the splice site of intron 2. Molecular analyses revealed that neither aberrant PNPLA2 mRNA isoforms, nor ATGL mutated protein were detectable in patient's cells. Clinically, both patients presented early onset muscle weakness, in particular of proximal upper limb muscles. In almost 15 years, muscle damage affected also distal upper limbs. This is a NLSDM family, displaying a severe PNPLA2 mutation in two siblings with clinical presentation characterized by an early onset, but a slowly evolution of severe myopathy.

Introduction: The aim of this study was to assess the perceived effect of salbutamol in adult patients with spinal muscular atrophy and to evaluate the usefulness of the World Health Organization Disability Assessment Schedule II (WHODAS II) and Fatigue Severity Scale (FSS) for its measurement. Methods: A longitudinal mixed methods study was performed. Ten patients were interviewed and completed WHODAS II and FSS questionnaires to assess disability and fatigue at 2 time-points. Inductive thematic analysis was used for qualitative data. The non-parametric Wilcoxon test was performed for quantitative analysis. Results: All participants reported an improvement in their condition after salbutamol consumption. WHODAS II and FSS reliably captured changes in patients' disability and fatigue. Conclusions: The mixed methods design allowed us to identify the functional domains in which participants experienced effects of salbutamol. Patients were satisfied with the treatment as shown by decreased fatigue, improved functioning, and infrequent side effects. Muscle Nerve, 2016 Muscle Nerve54: 843-849, 2016

Highlights • At baseline, the PedsQLTM inventories correlated with almost all the functional measures. • There was a significant decrease between baseline and 12 months on PedsQLTM GCS. • This decrement paralleled with the decrement in the functional outcome measures. • PedsQLTM correlates with the level of impairment. • This correlations were not confirmed when 12 month changes are considered., In Duchenne muscular dystrophy (DMD) little has been reported on the association between clinical outcome measures and patient health-related quality of life (HRQOL) tools. Our study evaluated the relationship between 12 month changes on the Generic Core Scales (GCS), the Multidimensional Fatigue Scale and the Neuromuscular Module of the PedsQLTM with several outcome measures (6 minute walk test, North Star Ambulatory Assessment and timed items) in ambulatory DMD. Ninety-eight ambulatory DMD in a multicentric setting were included in the study. At baseline, the PedsQLTM inventories correlated with almost all the functional measures On the Child Self-Report there was a significant decrease between baseline and 12 months on the PedsQLTM GCS and its first domain, in parallel with the decrement in the functional outcome measures. Correlation between the 12 month changes on the PedsQLTM inventories and functional measures were almost all negligible. Similar results were obtained on the Parent Proxy-Report. In conclusion, PedsQLTM correlates with the level of impairment at baseline, but this does not hold true when 12 month changes are considered. Further studies comparing different tools are needed to better elucidate the complexity of the relationship between HRQOL and functional performances.

Background Trials in rare diseases have many challenges, among which are the need to set up multiple sites in different countries to achieve recruitment targets and the divergent landscape of clinical trial regulations in those countries. Over the past years, there have been initiatives to facilitate the process of international study set-up, but the fruits of these deliberations require time to be operationally in place. FOR-DMD (Finding the Optimum Steroid Regimen for Duchenne Muscular Dystrophy) is an academic-led clinical trial which aims to find the optimum steroid regimen for Duchenne muscular dystrophy, funded by the National Institutes of Health (NIH) for 5 years (July 2010 to June 2015), anticipating that all sites (40 across the USA, Canada, the UK, Germany and Italy) would be open to recruitment from July 2011. However, study start-up was significantly delayed and recruitment did not start until January 2013. Method The FOR-DMD study is used as an example to identify systematic problems in the set-up of international, multi-centre clinical trials. The full timeline of the FOR-DMD study, from funding approval to site activation, was collated and reviewed. Systematic issues were identified and grouped into (1) study set-up, e.g. drug procurement; (2) country set-up, e.g. competent authority applications; and (3) site set-up, e.g. contracts, to identify the main causes of delay and suggest areas where anticipatory action could overcome these obstacles in future studies. Results Time from the first contact to site activation across countries ranged from 6 to 24 months. Reasons of delay were universal (sponsor agreement, drug procurement, budgetary constraints), country specific (complexity and diversity of regulatory processes, indemnity requirements) and site specific (contracting and approvals). The main identified obstacles included (1) issues related to drug supply, (2) NIH requirements regarding contracting with non-US sites, (3) differing regulatory requirements in the five participating countries, (4) lack of national harmonisation with contracting and the requirement to negotiate terms and contract individually with each site and (5) diversity of languages needed for study materials. Additionally, as with many academic-led studies, the FOR-DMD study did not have access to the infrastructure and expertise that a contracted research organisation could provide, organisations often employed in pharmaceutical-sponsored studies. This delay impacted recruitment, challenged the clinical relevance of the study outcomes and potentially delayed the delivery of the best treatment to patients. Conclusion Based on the FOR-DMD experience, and as an interim solution, we have devised a checklist of steps to not only anticipate and minimise delays in academic international trial initiation but also identify obstacles that will require a concerted effort on the part of many stakeholders to mitigate.

Limb girdle muscular dystrophies (LGMD) are a large group of muscular disorders, with progressive shoulder and pelvic muscle weakness as the most relevant feature. They are classified as autosomal dominant (LGMD1) and autosomal recessive (LGMD2) forms. Up to now, eight genetically defined LGMD1 subtypes (LGMD1 A-H) have been identified [1, 9]. In 2001, the clinical and morphological phenotype of a novel form of LGMD type 1, affecting 32 subjects in a large Spanish family, was described [5]. According to subsequent molecular studies, the disease was demonstrated to be linked to the novel chromosomal locus 7q32.1–32.2. This genetically distinct form of autosomal dominant-LGMD was classified as LGMD1F [10] (OMIM #608423). Recently, using a whole genome sequencing approach, the causative mutation of the LGMD1F was identified in the termination codon of TNPO3, the gene coding for transportin 3. Molecular results at DNA, RNA and protein levels as well as morphological findings supported the pathogenic role of this mutation in LGMD1F [8]. Investigation by next-generation sequencing in further 4 members of the Spanish family, originating from Italy, confirmed the mutation in TNPO3 [13]. Up to now, beside this Italo-Spanish family, only one sporadic LGMD patient has been identified with a heterozygous point mutation in the TNPO3 gene [13]. In this patient we now report the long term clinical and radiological follow-up, morphological and immunochemical studies on patient muscle biopsy, and molecular studies by Real Time PCR and by cell transfection with the mutant cDNA.

BackgroundSpinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder, due to the loss of function of the survival motor neuron (SMN1) gene. The first treatment for the condition, recently approved, is based on the reduction of exon 7 skipping in mRNAs produced by a highly homologous gene (SMN2). The primary objective of the present study was to evaluate the applicability of the dosage of SMN gene produts in blood, as biomarker for SMA, and the safety of oral salbutamol, a beta2-adrenergic agonist modulating SMN2 levels.MethodsWe have performed a 1-year multicentre, double-blind, placebo-controlled study with salbutamol in 45 adult patients with SMA. Patients assumed 4 mg of salbutamol or placebo/three times a day. Molecular tests were SMN2 copy number, SMN transcript and protein levels. We have also explored the clinical effect, by the outcome measures available at the time of study design.ResultsThirty-six patients completed the study. Salbutamol was safe and well tolerated. We observed a significant and progressive increase in SMN2 full-length levels in peripheral blood of the salbutamol-treated patients (pConclusionsOur data demonstrate safety and molecular efficacy of salbutamol. We provide the first longitudinal evaluation of SMN levels (both transcripts and protein) in placebo and in response to a compound modulating the gene expression: SMN transcript dosage in peripheral blood is reliable and may be used as pharmacodynamic marker in clinical trials with systemic compounds modifying SMN2levels.Trial registration numberEudraCT no. 2007-001088-32.

Aims Pompe disease is an autosomal recessive lysosomal storage disorder resulting from deficiency of acid α-glucosidase (GAA) enzyme. Histopathological hallmarks in skeletal muscle tissue are fiber vacuolization and autophagy. Since 2006, ERT is the only approved treatment with human recombinant GAA alglucosidase alfa. We designed a study to examine ERT-related skeletal muscle changes in 18 modestly to moderately affected LOPD patients along with the relationship between morphological/biochemical changes and clinical outcomes. Treatment duration was short-to-long term. Methods We examined muscle biopsies from 18 LOPD patients at both histopathological and biochemical level. All patients underwent two muscle biopsies, before and after ERT administration respectively. The study is partially retrospective because the first biopsies were taken before the study was designed whereas the second biopsy was always performed after at least six months of ERT administration. Results After ERT, 15 out of 18 patients showed improved 6MWT (p=0.0007) and most of them achieved respiratory stabilization. Pre-treatment muscle biopsies disclosed marked histopathological variability, ranging from an almost normal pattern to a severe vacuolar myopathy. After treatment, we detected morphological improvement in 15 patients and worsening in 3 patients. Post-ERT GAA enzymatic activity was mildly increased compared with pre-treatment levels in all patients. Protein levels of the mature enzyme increased in 14 of the 18 patients (mean increase = +35%; p

Duchenne muscular dystrophy (DMD) is a lethal disease. The outcome measures used in numerous therapeutic trials include skeletal muscle biopsy. We studied the natural history of DMD from the standpoint of muscle histology with the aim of providing a reproducible tool for use in evaluating and comparing any histologic changes occurring in patients with DMD undergoing treatment and hence be able to determine how therapy modulates the histologic evolution of the disease.Three independent operators analyzed 56 muscle biopsies from 40 patients not treated with steroids, aged 1 to 10 years and 16 individuals treated with steroids, aged 7 to 10 years. We analyzed morphologic measures, normalized every measure for the average number of fibers observed for each year of age, and calculated intraclass correlation coefficients.The average proportion of connective tissue in patients not treated with steroids was 16.98% from ages 1 to 6 years and 30% from ages 7 to 10 years (p0.0001). The average proportion in patients treated with steroids was 24.90%. Muscle fiber area mirrored that of connective tissue in both groups.Having provided a reproducible tool for evaluation and comparison of histologic changes occurring in patients undergoing clinical trials, it was observed that at ages 6 to 7 years, fibrotic tissue rapidly peaks to 29.85%; this is a crucial moment when muscle tissue loses its self-regeneration ability, veering toward fibrotic degeneration. These data should be considered when deciding the most suitable time to begin therapy.

Spinal Muscular Atrophy (SMA) is an autosomal recessive neuromuscular disease, leading to progressive denervation atrophy in the involved skeletal muscles. Bone status has been poorly studied. We assessed bone metabolism, bone mineral density (BMD) and fractures in 30 children (age range 15-171 months) affected by SMA types 2 and 3. Eighteen children (60%) had higher than normal levels of CTx (bone resorption marker); 25-OH vitamin D was in the lower range of normal (below 20 ng/ml in 9 children and below 12 ng/ml in 2). Lumbar spine BMAD (bone mineral apparent density) Z-score was below -1.5 in 50% of children. According to clinical records, four children had sustained four peripheral fractures; on spine X-rays, we observed 9 previously undiagnosed vertebral fractures in 7 children. There was a significant inverse regression between PTH and 25-OH D levels, and a significant regression between BMC and BMAD values and the scores of motor-functional tests. Even if this study could not establish the pathogenesis of bone derangements in SMA, its main findings - reduced bone density, low 25OH vitamin D levels, increased bone resorption markers and asymptomatic vertebral fractures also in very young patients - strongly suggest that even young subjects affected by SMA should be considered at risk of osteopenia and even osteoporosis and fractures.

Key points Loss-of-function mutations of the skeletal muscle ClC-1 channel cause myotonia congenita with variable phenotypes. Using patch clamp we show that F484L, located in the conducting pore, probably induces mild dominant myotonia by right-shifting the slow gating of ClC-1 channel, without exerting a dominant-negative effect on the wild-type (WT) subunit. Molecular dynamics simulations suggest that F484L affects the slow gate by increasing the frequency and the stability of H-bond formation between E232 in helix F and Y578 in helix R. Three other myotonic ClC-1 mutations are shown to produce distinct effects on channel function: L198P shifts the slow gate to positive potentials, V640G reduces channel activity, while L628P displays a WT-like behaviour (electrophysiology data only). Our results provide novel insight into the molecular mechanisms underlying normal and altered ClC-1 function. Abstract Myotonia congenita is an inherited disease caused by loss-of-function mutations of the skeletal muscle ClC-1 chloride channel, characterized by impaired muscle relaxation after contraction and stiffness. In the present study, we provided an in-depth characterization of F484L, a mutation previously identified in dominant myotonia, in order to define the genotype–phenotype correlation, and to elucidate the contribution of this pore residue to the mechanisms of ClC-1 gating. Patch-clamp recordings showed that F484L reduced chloride currents at every tested potential and dramatically right-shifted the voltage dependence of slow gating, thus contributing to the mild clinical phenotype of affected heterozygote carriers. Unlike dominant mutations located at the dimer interface, no dominant-negative effect was observed when F484L mutant subunits were co-expressed with wild type. Molecular dynamics simulations further revealed that F484L affected the slow gate by increasing the frequency and stability of the H-bond formation between the pore residue E232 and the R helix residue Y578. In addition, using patch-clamp electrophysiology, we characterized three other myotonic ClC-1 mutations. We proved that the dominant L198P mutation in the channel pore also right-shifted the voltage dependence of slow gating, recapitulating mild myotonia. The recessive V640G mutant drastically reduced channel function, which probably accounts for myotonia. In contrast, the recessive L628P mutant produced currents very similar to wild type, suggesting that the occurrence of the compound truncating mutation (Q812X) or other muscle-specific mechanisms accounted for the severe symptoms observed in this family. Our results provide novel insight into the molecular mechanisms underlying normal and altered ClC-1 function.

Excessive extracellular matrix deposition progressively replacing muscle fibres is the endpoint of most severe muscle diseases. Recent data indicate major involvement of microRNAs in regulating pro- and anti-fibrotic genes. To investigate the roles of miR-21 and miR-29 in muscle fibrosis in Duchenne muscle dystrophy, we evaluated their expression in muscle biopsies from 14 patients, and in muscle-derived fibroblasts and myoblasts. In Duchenne muscle biopsies, miR-21 expression was significantly increased, and correlated directly with COL1A1 and COL6A1 transcript levels. MiR-21 expression was also significantly increased in Duchenne fibroblasts, more so after TGF-β1 treatment. In Duchenne fibroblasts the expression of miR-21 target transcripts PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SPRY-1 (Sprouty homolog 1) was significantly reduced; while collagen I and VI transcript levels and soluble collagen production were significantly increased. MiR-29a and miR-29c were significantly reduced in Duchenne muscle and myoblasts, and miR-29 target transcripts, COL3A1, FBN1 and YY1, significantly increased. MiR-21 silencing in mdx mice reduced fibrosis in the diaphragm muscle and in both Duchenne fibroblasts and mdx mice restored PTEN and SPRY-1 expression, and significantly reduced collagen I and VI expression; while miR-29 mimicking in Duchenne myoblasts significantly decreased miR-29 target transcripts. These findings indicate that miR-21 and miR-29 play opposing roles in Duchenne muscle fibrosis and suggest that pharmacological modulation of their expression has therapeutic potential for reducing fibrosis in this condition. copy; 2015 .

X-linked Myopathy with Excessive Autophagy (XMEA) affects proximal muscles of the lower extremities and follows a progressive course reminiscent of muscular dystrophy. It is caused by mutations in VMA21 whose protein product assembles lysosomes' proton pumps. All XMEA mutations to date have been single-nucleotide substitutions that reduce VMA21 expression, which leads to modest lysosomal pH increase, the first step in the disease's pathogenesis. We now report a new class of XMEA mutations. We identified two VMA21 non-coding microdeletions, one intronic (c.54-16_54-8del), the other in the 3′UTR (c.*13_*104del). Both resulted in a relatively more severe (early ambulation loss), diffuse (extra-ocular and upper extremity involvement), and early (neonatal) onset disease compared to previously reported patients. Our cases highlight the importance of including non-coding regions of VMA21 in genetic testing panels of dystrophies and myopathies. Specific diagnosis of XMEA will be particularly important as therapies aimed at correcting the modest rise in lysosomal pH at the root of this disease are developed.

Botulin toxin (BTX) is widely used for treating skeletal muscle spasticity. Experimental reports on BTX treatment were mainly focused on the neuromuscular junction, while relatively little is known about toxin effects on the muscle cell itself. We investigated possible impact of BTX type A on skeletal muscle cell transcriptome by microarray analysis in muscle-derived cell cultures (fibroblasts, myoblasts and myotubes) from controls and spastic patients, and results were then validated at transcript and protein level. BTX-A treatment of control cells induced major changes in the myogenic component of the transcriptome, whereas the same treatment had a negligible effect in the fibrogenic component. BTX-A treatment of cell cultures from spastic patients induced an increased number of genes differentially expressed both in the fibrogenic and myogenic components. Specifically, BTX-A had a major effect on cell cycle-related genes in myoblasts, on muscle contraction-related genes in myotubes, and on extracellular matrix-related genes in fibroblasts from spastic patients. Our findings show that in vitro BTX-A treatment differentially affects transcript expression in muscle cells from spastic patients compared to those from controls suggesting a direct effect of BTX-A on muscle-specific functional pathways.

Introduction Limb girdle muscular dystrophies (LGMDs) are characterized by high molecular heterogeneity, clinical overlap, and a paucity of specific biomarkers. However, their molecular definition is fundamental for prognostic and therapeutic purposes. Methods We created an Italian LGMD registry that included 370 molecularly defined patients. We reviewed detailed retrospective and prospective data and compared each LGMD subtype for differential diagnosis purposes. Results LGMD2A and 2B are the most frequent forms in Italy. The ages at disease onset, clinical progression, and cardiac and respiratory involvement can vary greatly between each LGMD subtype. In a set of extensively studied patients, targeted next-generation sequencing (NGS) identified mutations in 36.5% of cases. Introduction: Limb girdle muscular dystrophies (LGMDs) are characterized by high molecular heterogeneity, clinical overlap, and a paucity of specific biomarkers. Their molecular definition is fundamental for prognostic and therapeutic purposes. Methods: We created an Italian LGMD registry that included 370 molecularly defined patients. We reviewed detailed retrospective and prospective data and compared each LGMD subtype for differential diagnosis purposes. Results: LGMD types 2A and 2B are the most frequent forms in Italy. The ages at disease onset, clinical progression, and cardiac and respiratory involvement can vary greatly between each LGMD subtype. In a set of extensively studied patients, targeted next-generation sequencing (NGS) identified mutations in 36.5% of cases. Conclusion: Detailed clinical characterization combined with muscle tissue analysis is fundamental to guide differential diagnosis and to address molecular tests. NGS is useful for diagnosing forms without specific biomarkers, although, at least in our study cohort, several LGMD disease mechanisms remain to be identified. Muscle Nerve 55: 55–68, 2017.

The dystrophin gene (DMD) is the largest gene in the human genome, mapping on the Xp21 chromosome locus. It spans 2.2 Mb and accounts for approximately 0,1% of the entire human genome. Mutations in this gene cause Duchenne and Becker Muscular Dystrophy, X-linked Dilated Cardiomyopathy, and other milder muscle phenotypes. Beside the remarkable number of reports describing dystrophin gene expression and the pathogenic consequences of the gene mutations in dystrophinopathies, the full scenario of the DMD transcription dynamics remains however, poorly understood. Considering that the full transcription of the DMD gene requires about 16 h, we have investigated the activity of RNA Polymerase II along the entire DMD locus within the context of specific chromatin modifications using a variety of chromatin-based techniques. Our results unveil a surprisingly powerful processivity of the RNA polymerase II along the entire 2.2 Mb of the DMD locus with just one site of pausing around intron 52. We also discovered epigenetic marks highlighting the existence of four novel cis‑DNA elements, two of which, located within intron 34 and exon 45, appear to govern the architecture of the DMD chromatin with implications on the expression levels of the muscle dystrophin mRNA. Overall, our findings provide a global view on how the entire DMD locus is dynamically transcribed by the RNA pol II and shed light on the mechanisms involved in dystrophin gene expression control, which can positively impact on the optimization of the novel ongoing therapeutic strategies for dystrophinopathies.

Contains fulltext : 137503.pdf (Publisher’s version ) (Open Access) Cytochrome c oxidase (COX) deficiency is a frequent biochemical abnormality in mitochondrial disorders, but a large fraction of cases remains genetically undetermined. Whole-exome sequencing led to the identification of APOPT1 mutations in two Italian sisters and in a third Turkish individual presenting severe COX deficiency. All three subjects presented a distinctive brain MRI pattern characterized by cavitating leukodystrophy, predominantly in the posterior region of the cerebral hemispheres. We then found APOPT1 mutations in three additional unrelated children, selected on the basis of these particular MRI features. All identified mutations predicted the synthesis of severely damaged protein variants. The clinical features of the six subjects varied widely from acute neurometabolic decompensation in late infancy to subtle neurological signs, which appeared in adolescence; all presented a chronic, long-surviving clinical course. We showed that APOPT1 is targeted to and localized within mitochondria by an N-terminal mitochondrial targeting sequence that is eventually cleaved off from the mature protein. We then showed that APOPT1 is virtually absent in fibroblasts cultured in standard conditions, but its levels increase by inhibiting the proteasome or after oxidative challenge. Mutant fibroblasts showed reduced amount of COX holocomplex and higher levels of reactive oxygen species, which both shifted toward control values by expressing a recombinant, wild-type APOPT1 cDNA. The shRNA-mediated knockdown of APOPT1 in myoblasts and fibroblasts caused dramatic decrease in cell viability. APOPT1 mutations are responsible for infantile or childhood-onset mitochondrial disease, hallmarked by the combination of profound COX deficiency with a distinctive neuroimaging presentation.

To test the efficacy and tolerability of clenbuterol in patients with spinal and bulbar muscular atrophy (SBMA).Twenty patients with a diagnosis of SBMA were given oral clenbuterol (0.04 mg/d) for 12 months. The primary efficacy end point was the change from baseline of the walking distance covered in 6 minutes at 12 months. Secondary end points included the change over time in muscle strength assessed with the Medical Research Council scale, the revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R), and forced vital capacity values. Safety was assessed by a series of laboratory and instrumental tests, as well as reporting of adverse events.Sixteen patients completed the study. There was a significant and sustained increase in walking distance covered in 6 minutes and forced vital capacity between the baseline and the 12-month assessments (p0.001). No differences were recorded in Medical Research Council or ALSFRS-R scores between baseline and follow-up assessments. Serious side effects, including those on heart function, were absent. A significant increase in serum creatine kinase levels was observed.Our findings suggest a positive effect of clenbuterol on SBMA disease progression.This study provides Class IV evidence that clenbuterol is effective in improving motor function in SBMA.

Aims Tissue fibrosis, characterized by excessive deposition of extracellular matrix proteins, is the end point of diseases affecting the kidney, bladder, liver, lung, gut, skin, heart and muscle. In Duchenne muscular dystrophy (DMD), connective fibrotic tissue progressively substitutes muscle fibers. So far no specific pharmacological treatment is available for muscle fibrosis. Among promising anti-fibrotic molecules, pirfenidone has shown anti-fibrotic and anti-inflammatory activity in animal and cell models, and has already been employed in clinical trials. Therefore we tested pirfenidone anti-fibrotic properties in an in vitro model of muscle fibrosis. Main methods We evaluated effect of pirfenidone on fibroblasts isolated from DMD muscle biopsies. These cells have been previously characterized as having a pro-fibrotic phenotype. We tested cell proliferation and migration, secretion of soluble collagens, intracellular levels of collagen type I and fibronectin, and diameter of 3D fibrotic nodules. Key findings We found that pirfenidone significantly reduced proliferation and cell migration of control and DMD muscle-derived fibroblasts, decreased extracellular secretion of soluble collagens by control and DMD fibroblasts, as well as levels of collagen type I and fibronectin, and, in DMD fibroblasts only, reduced synthesis and deposition of intracellular collagen. Furthermore, pirfenidone was able to reduce the diameter of fibrotic-nodules in our 3D model of in vitro fibrosis. Significance These pre-clinical results indicate that pirfenidone has potential anti-fibrotic effects also in skeletal muscle fibrosis, urging further studies in in vivo animal models of muscular dystrophy in order to translate the drug into the treatment of muscle fibrosis in DMD patients.

Objectives Facioscapulohumeral muscular dystrophy type 1 (FSHD1) has been genetically linked to reduced numbers (≤8) of D4Z4 repeats at 4q35. Particularly severe FSHD cases, characterised by an infantile onset and presence of additional extra-muscular features, have been associated with the shortest D4Z4 reduced alleles with 1–3 repeats (1–3 DRA). We searched for signs of perinatal onset and evaluated disease outcome through the systematic collection of clinical and anamnestic records of de novo and familial index cases and their relatives, carrying 1–3 DRA. Setting Italy. Participants 66 index cases and 33 relatives carrying 1–3 DRA. Outcomes The clinical examination was performed using the standardised FSHD evaluation form with validated inter-rater reliability. To investigate the earliest signs of disease, we designed the Infantile Anamnestic Questionnaire (IAQ). Comparison of age at onset was performed using the non-parametric Wilcoxon rank-sum or Kruskal-Wallis test. Comparison of the FSHD score was performed using a general linear model and Wald test. Kaplan-Meier survival analysis was used to estimate the age-specific cumulative motor impairment risk. Results No patients had perinatal onset. Among index cases, 36 (54.5%) showed the first signs by 10 years of age. The large majority of patients with early disease onset (26 out of 36, 72.2%) were de novo; whereas the majority of patients with disease onset after 10 years of age were familial (16, 53.3%). Comparison of the disease severity outcome between index cases with age at onset before and over 10 years of age, failed to detect statistical significance (Wald test p value=0.064). Of 61 index cases, only 17 (27.9%) presented extra-muscular conditions. Relatives carrying 1–3 DRA showed a large clinical variability ranging from healthy subjects, to patients with severe motor impairment. Conclusions The size of the D4Z4 allele is not always predictive of severe clinical outcome. The high degree of clinical variability suggests that additional factors contribute to the phenotype complexity.

Facioscapulohumeral muscular dystrophy (FSHD) is characterized by extremely variable degrees of facial, scapular and lower limb muscle involvement. Clinical and genetic determination can be difficult, as molecular analysis is not always definitive, and other similar muscle disorders may have overlapping clinical manifestations. Whole-body muscle MRI examination for fat infiltration, atrophy and oedema was performed to identify specific patterns of muscle involvement in FSHD patients (30 subjects), and compared to a group of control patients (23) affected by other myopathies (NFSHD). In FSHD patients, we detected a specific pattern of muscle fatty replacement and atrophy, particularly in upper girdle muscles. The most frequently affected muscles, including paucisymptomatic and severely affected FSHD patients, were trapezius, teres major and serratus anterior. Moreover, asymmetric muscle involvement was significantly higher in FSHD as compared to NFSHD patients. In conclusion, muscle MRI is very sensitive for identifying a specific pattern of involvement in FSHD patients and in detecting selective muscle involvement of non-clinically testable muscles. Muscle MRI constitutes a reliable tool for differentiating FSHD from other muscular dystrophies to direct diagnostic molecular analysis, as well as to investigate FSHD natural history and follow-up of the disease. • Muscle MRI identifies a specific pattern of muscle involvement in FSHD patients. • Muscle MRI may predict FSHD in asymptomatic and severely affected patients. • Muscle MRI of upper girdle better predicts FSHD. • Muscle MRI may differentiate FSHD from other forms of muscular dystrophy. • Muscle MRI may show the involvement of non-clinical testable muscles.

Mutations in the adipose triglyceride lipase (ATGL), an enzyme that hydrolyzes fatty acids from triacylglycerol (TG) stored in multiple tissues into cytoplasmic lipid droplets (LDs), causes the autosomal recessive disorder Neutral Lipid Storage Disease with myopathy (NLSDM) [2]. ATGL protein is coded by PNPLA2 gene. NLSDM patients are mainly affected by progressive myopathy, cardiomyopathy and hepatomegaly. Their clinical severity appears to be highly variable with particular reference to cardiac involvement [1], [6], [7], [8]. Cardiomyopathy was reported in 36% of the patients. In some of them, it was lethal (25%), or manifested with severe heart condition (31%). Patients with cardiac involvement have adult-onset progressive heart failure, mimicking dilated or hypertrophic cardiomyopathy. The analyses of explanted and autopsied hearts revealed that the defective intracellular hydrolysis of TG results in congestive heart failure with cardiomyocyte steatosis as well as a novel type of coronary atherosclerosis with TG-deposit smooth muscle cells [3], [4], [5]. Molecular mechanisms underlying pathophysiology of heart damage are unknown; moreover, no therapy is available for NLSDM patients. We report the case of a 26 year-old male patient affected by neutral lipid storage myopathy with severe cardiac involvement. Patient parents were first cousins; a brother died at 3 years of age, during surgery. They referred that the child had always walked on toes, but he never presented weakness or difficulties in physical activity, compared to peers. The patient was first evaluated when he was 11 years-old and was reported to walk on toes, with difficulty to walk on heels, and to have mild calves hypertrophy and reduced tendon reflexes. Blood test revealed high values of CK (1657 U/L), while total and free carnitine levels were normal. Electromyography was normal; an effort test revealed excessive increase in lactic acid levels. He underwent a muscle biopsy that showed abnormal lipid storage. He was diagnosed to suffer from a lipid storage myopathy and therapy with riboflavin was started with some benefit to the patient. At 20 years of age, the patient complained of walking difficulties and of exercise intolerance. CK levels remained very high (> 2000 U/L). On neurological evaluation he revealed normal strength, mild fatigability, reduced tendon reflexes, walking on forefoot and impossibility to walk on heels. He performed squats without limitation, but with pain at lower limbs. The analysis of plasma free and esterified carnitine resulted normal. A muscle CT detected atrophy and fat infiltration of lower limb muscles. A muscle biopsy revealed numerous vacuoles (Fig. 1A) positive to lipid staining in most fibers (Fig. 1B). Respiratory chain complexes showed normal activity. Electron microscopy showed numerous lipid droplets between myofibrils in most fibers, often causing sarcomere disorganization; while mitochondria were normal (Fig 1C). A neutral lipid storage myopathy was hypothesized and molecular analysis of the PNPLA2 gene revealed a homozygous novel deletion of seven nucleotides in exon 2 (c.41_47delGCTGCGG) (Fig 1H). The mutated protein was predicted to consist of only fourteen amino acids (p.G14Afs75X). This mutation was submitted to GenBank (accession number KU057409). Fig. 1 Histochemical, clinical and molecular characterization of NLSDM patient. During most recent years clinical features of the patient have progressively deteriorated with mild involvement of upper limbs and worsening of distal lower limb weakness; moreover walking difficulties become more evident. Whole-body MRI detected mild fatty infiltration of bilateral trapezius, deltoid and infraspinatus; severe fatty degeneration of pelvic girdle muscles, hamstring, semimembranosus and semitendinosus; diffuse fatty infiltration of leg muscles with partial saving of anterior and posterior tibialis (Fig 1D–G). The patient has undergone cardiological evaluation at 24 years of age. Cardiac MRI detected left ventricular dilation with diffused hypokinesia, moderate–severe reduction of left ventricular systolic function (ejection fraction FE 36%) and fibrosis of cardiac wall, without fatty infiltration (Fig. 2). A CT excluded coronary pathology, and no innervation defects were seen by myocardial scintigraphy. Holter-ECG revealed sinus rhythm without arrhythmia or conduction defects. A cardiopulmonary exercise test showed a severe reduction in functional capacity mainly due to cardiogenic and extraction defect. After those exams, the patient started a therapy with enalapril 5 mg bid and ivabradine 7.5 mg/die. During the subsequent follow up both exercise capacity and echocardiography ejection fraction improved significantly, indeed oxygen consumption increased from 48% to 57% of the predicted value and a recent echocardiography, performed a year and a half after starting treatment, confirmed the reduction in left ventricle volume and detected a recovery of ventricular function (FE 53%). No indication to implantable-cardioverter-defibrillator (ICD) implantation has been given by the cardiologist so far. Fig. 2 Left ventricle fibrosis detected by cardiovascular magnetic resonance. To the best of our knowledge, 44 NLSDM patients have been clinically and genetically characterized until now [3], [6]; about one third of them presented cardiac myopathy. More than 90% of NLSDM patients (with cardiac involvement) harbor highly deleterious PNPLA2 mutations, causing no ATGL protein production or non-functional ATGL truncated proteins. By contrast, patients with missense mutations – partially preserving lipase activity – have been generally described without cardiac involvement [8]. The PNPLA2 mutation identified in our patient, determining total loss of ATGL protein, caused early onset and severe myocardial damage. Noteworthy, the patient showed an improvement of cardiac function after therapy with enalapril and ivabradine, in spite of the severe molecular defect. These drugs did not counteract neutral lipid accumulation and yet determined a significant recovery of ventricular function. Informed consent for genetic and clinical analyses were obtained from the patient. Moreover, written informed consent was obtained for publication of this Case report and any accompanying images. It has already been suggested that molecular and functional analysis of ATGL may improve NLSDM prognosis, paving the way towards personalized therapy. Nevertheless, intra-familial distinct cardiac phenotype has been recently reported in NLSDM [3], underlining that molecular mechanisms regulating the disease development could be influenced by other factors in addition to genotype. Since ATGL deficiency is a very rare disease, the international registry for NLSDM has been established (www.tgcv.org/r/home.html) with the purpose to compare different clinical phenotypes and elucidate NLSDM pathophysiology.

Aerobic training can be effective in patients with mitochondrial myopathies (MM) and McArdle's disease (McA). The aim of the study was to use noninvasive functional evaluation methods, specifically aimed at skeletal muscle oxidative metabolism, to evaluate the effects of an aerobic exercise training (cycle ergometer, 12 wk, 4 days/wk, ∼65-70% of maximal heart rate) in 6 MM and 7 McA. Oxygen uptake and skeletal muscle vastus lateralis fractional O2 extraction by near-infrared spectroscopy were assessed during incremental and low-intensity constant work rate (CWR) exercises before (BEFORE) and at the end (AFTER) of training. Peak O2 uptake increased significantly with training both in MM [14.7 ± 1.2 vs. 17.6 ± 1.4 ml·kg−1·min−1 (mean ± SD)] and in McA (18.5 ± 1.8 ml·kg−1·min−1 vs. 21.6 ± 1.9). Peak skeletal muscle fractional O2 extraction increased with training both in MM (22.0 ± 6.7 vs. 32.6 ± 5.9%) and in McA (18.5 ± 6.2 vs. 37.2 ± 7.2%). During low-intensity CWR in both MM and McA: V̇o2 kinetics became faster in AFTER, but only in the patients with slow V̇o2 kinetics in BEFORE; the transient overshoot in fractional O2 extraction kinetics disappeared. The level of habitual physical activity was not higher 3 mo after training (FOLLOW-UP vs. PRE). In MM and McA patients a home-based aerobic training program significantly attenuated the impairment of skeletal muscle oxidative metabolism and improved variables associated with exercise tolerance. Our findings indicate that in MM and McA patients near-infrared spectroscopy and V̇o2 kinetics can effectively detect the functional improvements obtained by training.

Based on the 7-year experience of the Italian Clinical Network for FSHD, we revised the FSHD clinical form to describe, in a harmonized manner, the phenotypic spectrum observed in FSHD. The new Comprehensive Clinical Evaluation Form (CCEF) defines various clinical categories by the combination of different features. The inter-rater reproducibility of the CCEF was assessed between two examiners using kappa statistics by evaluating 56 subjects carrying the molecular marker used for FSHD diagnosis. The CCEF classifies: (1) subjects presenting facial and scapular girdle muscle weakness typical of FSHD (category A, subcategories A1–A3), (2) subjects with muscle weakness limited to scapular girdle or facial muscles (category B subcategories B1, B2), (3) asymptomatic/healthy subjects (category C, subcategories C1, C2), (4) subjects with myopathic phenotype presenting clinical features not consistent with FSHD canonical phenotype (D, subcategories D1, D2). The inter-rater reliability study showed an excellent concordance of the final four CCEF categories with a κ equal to 0.90; 95 % CI (0.71; 0.97). Absolute agreement was observed for categories C and D, an excellent agreement for categories A [κ = 0.88; 95 % CI (0.75; 1.00)], and a good agreement for categories B [κ = 0.79; 95 % CI (0.57; 1.00)]. The CCEF supports the harmonized phenotypic classification of patients and families. The categories outlined by the CCEF may assist diagnosis, genetic counseling and natural history studies. Furthermore, the CCEF categories could support selection of patients in randomized clinical trials. This precise categorization might also promote the search of genetic factor(s) contributing to the phenotypic spectrum of disease. Electronic supplementary material The online version of this article (doi:10.1007/s00415-016-8123-2) contains supplementary material, which is available to authorized users.

Proximal spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations of the SMN1 gene. Based on severity, three forms of SMA are recognised (types I-III). All patients usually have 2-4 copies of a highly homologous gene (SMN2) which produces insufficient levels of functional survival motor neuron (SMN) protein, due to the alternative splicing of exon 7. The availability of potential candidates to the treatment of SMA has raised a number of issues, including the availability of biomarkers. This study was aimed at evaluating whether the quantification of SMN2 products in peripheral blood is a suitable biomarker for SMA. Forty-five adult type III patients were evaluated by Manual Muscle Testing, North Star Ambulatory Assessment scale, 6-Minute Walk Test, myometry, forced vital capacity, and dual energy X-ray-absorptiometry. Molecular assessments included SMN2 copy number, levels of full length SMN2 (SMN2-fl) transcripts and those lacking exon 7, and SMN protein. Clinical outcome measures strongly correlated to each other. Lean body mass correlated inversely with years from diagnosis and with several aspects of motor performance. SMN2 copy number and SMN protein levels were not associated with motor performance or transcript levels. SMN2-fl levels correlated with motor performance in ambulant patients. Our results indicate that SMN2-fl levels correlate with motor performance only in patients preserving higher levels of motor function, while motor performance was strongly influenced by disease duration and lean body mass. If not taken into account, the confounding effect of disease duration may impair the identification of potential SMA biomarkers.

Congenital muscular dystrophies due to defects in genes encoding proteins involved in α-dystroglycan (α-DG) glycosylation are a heterogeneous group of muscle disorders variably associated with central nervous system and eye abnormalities. One of the more severe is muscle-eye-brain disease (MEB). Mutations in genes coding for proven or putative glycosyltransferases (POMT1, POMT2, POMGnT1, fukutin, FKRP, and LARGE), the DPM3 gene encoding a DOL-P-Man synthase subunit, and the DAG1 gene encoding α-dystroglycan, have been associated with altered α-DG glycosylation. We report new POMGnT1 mutations and evaluate protein expression in 3 patients and 2 foetuses with variably severe MEB features. We identify two new point mutations (c.643 C > T, c.1863delC), one new intragenic rearrangement (deletion of exons 2–8), and a new intron retention (between exons 21 and 22) resulting from a known point mutation c.1895 + 1 G > T. Our study provides further evidence that rearrangements of the POMGnT1 gene are relatively common. Importantly, if heterozygous, they can be missed on standard genomic DNA sequencing. POMGNT1 protein analysis in 3 patients showed that the severity of the phenotype does not correlate with protein expression. Cerebral MRI is important for identifying MEB and α-dystroglycanopathy phenotypes in children and foetuses, and hence for directing the genetic analysis.

E. Colombo, S. Romaggi, F. Blasevich, M. Mora, C. Falcone, H. Lochmuller, L. Morandi and C. Farina (2012) Neuropathology and Applied Neurobiology38, 367–378 The neurotrophin receptor p75NTR is induced on mature myofibres in inflammatory myopathies and promotes myotube survival to inflammatory stress Aims: Recent studies propose the neurotrophin receptor p75NTR as a marker for muscle satellite cells and a key regulator of regenerative processes after injury. Here, we investigated the contribution of cellular compartments other than satellite cells and regenerating myofibres to p75NTR signal in diseased skeletal muscle. Methods: We checked regulation of p75NTR expression in muscle biopsies from patients with inflammatory myopathies (polymyositis, dermatomyositis and inclusion body myositis), or Becker muscular dystrophy, and in nonmyopathic tissues. Quantitative PCR, immunohistochemistry, immunofluorescence or electron microscopy were used. RNA interference approaches were applied to myotubes to explore p75NTR function. Results: We found p75NTR transcript and protein upregulation in all inflammatory myopathies but not in dystrophic muscle, suggesting a role for inflammatory mediators in induction of p75NTR expression. In inflamed muscle p75NTR was localized on distinct cell types, including immune cells and mature myofibres. In vitro assays on human myotubes confirmed that inflammatory factors such as IL-1 could induce p75NTR. Finally, RNA interference experiments in differentiated cells showed that, in the absence of p75NTR, myotubes were more susceptible to apoptosis when exposed to inflammatory stimuli. Conclusions: Our observations that p75NTR is upregulated on skeletal myofibres in inflammatory myopathies in vivo and promotes resistance to inflammatory mediators in vitro suggest that neurotrophin signalling through p75NTR may mediate a tissue-protective response to inflammation in skeletal myofibres.

PURPOSE: Enzyme replacement therapy (ERT) has recently became available for Pompe's disease. Data on the effects of ERT on physiological variables related to exercise tolerance have never been published. METHODS: Pulmonary gas exchange, cardiac output (by impedance cardiography), vastus lateralis muscle O2 extraction (by near-infrared spectroscopy) were determined during cycle ergometer exercise in a 50-year-old patient BEFORE and after 1, 12 and 24 months of ERT. RESULTS: At the same constant-workload submaximal exercise, rates of self-perceived exertion and values of respiratory exchange ratio, pulmonary ventilation and heart rate were lower during ERT vs.. BEFORE, suggesting an increased exercise tolerance. Peak oxygen uptake (VÙO2peak) increased by ~35% from BEFORE (0.64 L/min, or 11.4 ml/kg/min) to 1 month (0.88 L/min, or 15.7 ml/kg/min) of treatment, and did not significantly change thereafter. Also peak cardiac output significantly increased during ERT, whereas peak skeletal muscle fractional O2 extraction was unchanged compared to BEFORE. CONCLUSIONS: Improvements of peak exercise capacity and exercise tolerance at submaximal workloads were observed in a patient with Pompe's disease after 1 month of ERT, with no further changes during the ensuing treatment period (up to 24 months).

An 80-year-old woman (PI) has been suffering of late onset progressive weakness and wasting of lower-limb muscles, accompanied by high creatine kinase levels in blood. A muscle biopsy, performed at 63 years, showed myopathic features with partial deficiency of cytochrome c oxidase. A second biopsy taken 7 years later confirmed the presence of a mitochondrial myopathy but also of vacuolar degeneration and other morphological features resembling inclusion body myopathy. Her 46-year-old daughter (PII) and 50-year-old son (PIII) are clinically normal, but the creatine kinase levels were moderately elevated and the EMG was consistently myopathic in both. Analysis of mitochondrial DNA sequence revealed in all three patients a novel, homoplasmic 15 bp tandem duplication adjacent to the 5' end of mitochondrial tRNA(Phe) gene, encompassing the first 11 nucleotides of this gene and the four terminal nucleotides of the adjacent D-loop region. Both mutant fibroblasts and cybrids showed low oxygen consumption rate, reduced mitochondrial protein synthesis, and decreased mitochondrial tRNA(Phe) amount. These findings are consistent with an unconventional pathogenic mechanism causing the tandem duplication to interfere with the maturation of the mitochondrial tRNA(Phe) transcript.

Zanotti S, Gibertini S, Di Blasi C, Cappelletti C, Bernasconi P, Mantegazza R, Morandi L & Mora M (2011) Histopathology 59, 1215–1228 Osteopontin is highly expressed in severely dystrophic muscle and seems to play a role in muscle regeneration and fibrosis Aims: To increase our understanding of profibrotic mechanisms in dystrophic muscle. Methods and results: Extracellular matrix, fibrosis-related molecules and histopathology were assessed in skeletal muscle of patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and congenital muscular dystrophy type 1A (MDC1A).Osteopontin expression was much higher in DMD and MDC1A than in BMD and control muscle. Osteopontin was expressed in mononuclear cell infiltrates, on some muscle fibre surfaces, in regenerating fibres, and in calcified fibres. In all pathological muscles, matrix metalloproteinase (MMP)-1 was increased around groups of fibres that were also characterized by absence of collagen 1. The amounts of MMP-2, MMP-9 and tissue inhibitor of MMP -1 transcripts were also increased, whereas their proteins were variably expressed in muscle fibres (surface or cytoplasm) and at foci of necrosis and regeneration. Inflammatory cells, fibroblasts and myofibroblasts were more numerous in DMD and MDC1A than in BMD muscle. Conclusions: Several fibrosis-related factors are greatly altered in severely dystrophic skeletal muscle. Osteopontin was the most conspicuously upregulated, both as transcript and as protein, in muscle fibres and infiltrating cells, indicating an intimate involvement in fibrosis, and also in inflammation and muscle regeneration, although its precise roles in these processes remain to be elucidated.

Patients affected by Duchenne muscular dystrophy (DMD) and dystrophic MDX mice were investigated in this study for their bone phenotype and systemic regulators of bone turnover. Micro–computed tomographic (µCT) and histomorphometric analyses showed reduced bone mass and higher osteoclast and bone resorption parameters in MDX mice compared with wild-type mice, whereas osteoblast parameters and mineral apposition rate were lower. In a panel of circulating pro-osteoclastogenic cytokines evaluated in the MDX sera, interleukin 6 (IL-6) was increased compared with wild-type mice. Likewise, DMD patients showed low bone mineral density (BMD) Z-scores and high bone-resorption marker and serum IL-6. Human primary osteoblasts from healthy donors incubated with 10% sera from DMD patients showed decreased nodule mineralization. Many osteogenic genes were downregulated in these cultures, including osterix and osteocalcin, by a mechanism blunted by an IL-6-neutralizing antibody. In contrast, the mRNAs of osteoclastogenic cytokines IL6, IL11, inhibin-βA, and TGFβ2 were increased, although only IL-6 was found to be high in the circulation. Consistently, enhancement of osteoclastogenesis was noted in cultures of circulating mononuclear precursors from DMD patients or from healthy donors cultured in the presence of DMD sera or IL-6. Circulating IL-6 also played a dominant role in osteoclast formation because ex vivo wild-type calvarial bones cultured with 10% sera of MDX mice showed increase osteoclast and bone-resorption parameters that were dampen by treatment with an IL-6 antibody. These results point to IL-6 as an important mediator of bone loss in DMD and suggest that targeted anti-IL-6 therapy may have a positive impact on the bone phenotype in these patients. © 2011 American Society for Bone and Mineral Research

Satellite cells are resident stem cells of adult skeletal muscle that have roles in tissue repair. Although several efforts have led to the functional characterization of distinct myogenic populations in animal models, the translation of these findings to humans has been limited. Here, we analyzed the expression and function of the neurotrophin receptor p75NTR in human skeletal muscle precursor cells. We combined histological investigations of muscle biopsies with molecular and cellular analyses of primary muscle precursor cells. p75NTR is expressed by most satellite cells in vivo and is a marker for regenerating fibers in inflamed and dystrophic muscle. p75NTR mRNA and protein are also detectable in primary myoblasts, and these levels increase transiently when cell differentiation is triggered. Transcriptome analyses of p75NTR high versus p75NTR low muscle cells showed that p75NTR is the prototype marker for a precursor cell population that has a broad transcriptional repertoire associated with muscle development and maturation. Several in vitro experiments, including receptor blockade and gene silencing in myoblasts, proved that p75NTR specifically regulates myogenesis and dystrophin expression. Taken together, the results indicate that p75NTR is a novel marker of human differentiation-prone muscle precursor cells that is involved in myogenesis in vivo and in vitro.