Publications by authors named "Lucia Morandi"

93 Publications

Clinical and Molecular Spectrum of Myotonia and Periodic Paralyses Associated With Mutations in in a Large Cohort of Italian Patients.

Front Neurol 2020 29;11:646. Epub 2020 Jul 29.

Neuroimmunology and Neuromuscular Diseases, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy.

Four main clinical phenotypes have been traditionally described in patients mutated in SCN4A, including sodium-channel myotonia (SCM), paramyotonia congenita (PMC), Hypokaliemic type II (HypoPP2), and Hyperkaliemic/Normokaliemic periodic paralysis (HyperPP/NormoPP); in addition, rare phenotypes associated with mutations in SCN4A are congenital myasthenic syndrome and congenital myopathy. However, only scarce data have been reported in literature on large patient cohorts including phenotypes characterized by myotonia and episodes of paralysis. We retrospectively investigated clinical and molecular features of 80 patients fulfilling the following criteria: (1) clinical and neurophysiological diagnosis of myotonia, or clinical diagnosis of PP, and (2) presence of a pathogenic SCN4A gene variant. Patients presenting at birth with episodic laryngospasm or congenital myopathy-like phenotype with later onset of myotonia were considered as neonatal SCN4A. PMC was observed in 36 (45%) patients, SCM in 30 (37.5%), Hyper/NormoPP in 7 (8.7%), HypoPP2 in 3 (3.7%), and neonatal SCN4A in 4 (5%). The median age at onset was significantly earlier in PMC than in SCM ( < 0.01) and in Hyper/NormoPP than in HypoPP2 ( = 0.02). Cold-induced myotonia was more frequently observed in PMC ( = 34) than in SCM ( = 23) ( = 0.04). No significant difference was found in age at onset of episodes of paralysis among PMC and PP or in frequency of permanent weakness between PP ( = 4), SCM ( = 5), and PMC ( = 10). PP was more frequently associated with mutations in the S4 region of the NaV1.4 channel protein compared to SCM and PMC ( < 0.01); mutations causing PMC were concentrated in the C-terminal region of the protein, while SCM-associated mutations were detected in all the protein domains. Our data suggest that skeletal muscle channelopathies associated with mutations in SCN4A represent a continuum in the clinical spectrum.
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http://dx.doi.org/10.3389/fneur.2020.00646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403394PMC
July 2020

A novel mutation causing total loss of RNA and protein expression in two NLSDM siblings with early onset but slowly progressive severe myopathy.

Genes Dis 2021 Jan 29;8(1):73-78. Epub 2019 Jul 29.

Laboratory of Cellular Biochemistry and Molecular Biology, CRIBENS, Università Cattolica del Sacro Cuore, pz Buonarroti 30, Milan, 20145, Italy.

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 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 mutation, localized in the splice site of intron 2. Molecular analyses revealed that neither aberrant 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 mutation in two siblings with clinical presentation characterized by an early onset, but a slowly evolution of severe myopathy.
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http://dx.doi.org/10.1016/j.gendis.2019.07.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7859421PMC
January 2021

Longitudinal evaluation of SMN levels as biomarker for spinal muscular atrophy: results of a phase IIb double-blind study of salbutamol.

J Med Genet 2019 05 28;56(5):293-300. Epub 2018 Dec 28.

Muscle Pathology and Neuroimmunology Unit, Neurological Institute Carlo Besta, Milano, Italy.

Background: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder, due to the loss of function of the survival motor neuron () 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 (). 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 levels.

Methods: We 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 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.

Results: Thirty-six patients completed the study. Salbutamol was safe and well tolerated. We observed a significant and progressive increase in full-length levels in peripheral blood of the salbutamol-treated patients (p<0.00001). The exploratory analysis of motor function showed an improvement in most patients.

Conclusions: Our 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: transcript dosage in peripheral blood is reliable and may be used as pharmacodynamic marker in clinical trials with systemic compounds modifying levels.

Trial Registration Number: EudraCT no. 2007-001088-32.
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http://dx.doi.org/10.1136/jmedgenet-2018-105482DOI Listing
May 2019

Long term follow-up and further molecular and histopathological studies in the LGMD1F sporadic TNPO3-mutated patient.

Acta Neuropathol Commun 2018 12 19;6(1):141. Epub 2018 Dec 19.

Neuromuscular Diseases and Neuroimmunology Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Via Temolo 4, 20126, Milan, Italy.

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http://dx.doi.org/10.1186/s40478-018-0648-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6299540PMC
December 2018

A checklist for clinical trials in rare disease: obstacles and anticipatory actions-lessons learned from the FOR-DMD trial.

Trials 2018 May 10;19(1):291. Epub 2018 May 10.

John Walton Muscular Dystrophy Research Centre, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, NE1 3BZ, UK.

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.
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http://dx.doi.org/10.1186/s13063-018-2645-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5968578PMC
May 2018

Elevated TGF β2 serum levels in Emery-Dreifuss Muscular Dystrophy: Implications for myocyte and tenocyte differentiation and fibrogenic processes.

Nucleus 2018 01;9(1):292-304

j Institute of Molecular Genetics (IGM)-CNR, Unit of Bologna , Bologna , Italy.

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.
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http://dx.doi.org/10.1080/19491034.2018.1467722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5973167PMC
January 2018

Botulinum toxin type A affects the transcriptome of cell cultures derived from muscle biopsies of controls and spastic patients.

Toxicol In Vitro 2018 Aug 6;50:124-136. Epub 2018 Mar 6.

Division of Neuromuscular Diseases and Neuroimmunology, Fondazione IRCCS Istituto Neurologico "C. Besta", Milano, Italy.

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.
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http://dx.doi.org/10.1016/j.tiv.2018.02.008DOI Listing
August 2018

Cardiac autonomic control during sleep in patients with myotonic dystrophy type 1: the effects of comorbid obstructive sleep apnea.

Sleep Med 2017 Nov 5;39:32-37. Epub 2017 Sep 5.

Department of Internal Medicine, Fondazione IRCSS Ca' Granda, Ospedale Maggiore Policlinico, Milan, Italy; Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.sleep.2017.07.023DOI Listing
November 2017

Transcriptional and epigenetic analyses of the DMD locus reveal novel cis‑acting DNA elements that govern muscle dystrophin expression.

Biochim Biophys Acta Gene Regul Mech 2017 Nov 1;1860(11):1138-1147. Epub 2017 Sep 1.

Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy; CIRI Health Sciences & Technologies (HST), Bologna, Italy. Electronic address:

The dystrophin gene (DMD) is the largest gene in the human genome, mapping on the Xp21 chromosome locus. It spans 2.2Mb 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 16h, 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 2.2Mb 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.
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http://dx.doi.org/10.1016/j.bbagrm.2017.08.010DOI Listing
November 2017

Neutral Lipid Storage Diseases: clinical/genetic features and natural history in a large cohort of Italian patients.

Orphanet J Rare Dis 2017 05 12;12(1):90. Epub 2017 May 12.

IRCCS Fondazione Ospedale S. Camillo, Venice, Italy.

Background: A small number of patients affected by Neutral Lipid Storage Diseases (NLSDs: NLSD type M with Myopathy and NLSD type I with Ichthyosis) have been described in various ethnic groups worldwide. However, relatively little is known about the progression and phenotypic variability of the disease in large specific populations. The aim of our study was to assess the natural history, disability and genotype-phenotype correlations in Italian patients with NLSDs. Twenty-one patients who satisfied the criteria for NLSDs were enrolled in a retrospective cross-sectional study to evaluate the genetic aspects, clinical signs at onset, disability progression and comorbidities associated with this group of diseases.

Results: During the clinical follow-up (range: 2-44 years, median: 17.8 years), two patients (9.5%, both with NLSD-I) died of hepatic failure, and a further five (24%) lost their ability to walk or needed help when walking after a mean period of 30.6 years of disease. None of the patients required mechanical ventilation. No patient required a heart transplant, one patient with NLSD-M was implanted with a cardioverter defibrillator for severe arrhythmias.

Conclusion: The genotype/phenotype correlation analysis in our population showed that the same gene mutations were associated with a varying clinical onset and course. This study highlights peculiar aspects of Italian NLSD patients that differ from those observed in Japanese patients, who were found to be affected by a marked hypertrophic cardiopathy. Owing to the varying phenotypic expression of the same mutations, it is conceivable that some additional genetic or epigenetic factors affect the symptoms and progression in this group of diseases.
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http://dx.doi.org/10.1186/s13023-017-0646-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5427600PMC
May 2017

Pure myopathy with enlarged mitochondria associated to a new mutation in gene.

Mol Genet Metab Rep 2017 Mar 15;10:24-27. Epub 2016 Dec 15.

Unit of Molecular Neurogenetics, Fondazione IRCCS Istituto Neurologico 'Carlo Besta', 20126 Milan, Italy.

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 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 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.
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http://dx.doi.org/10.1016/j.ymgmr.2016.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217772PMC
March 2017

The italian limb girdle muscular dystrophy registry: Relative frequency, clinical features, and differential diagnosis.

Muscle Nerve 2017 01 28;55(1):55-68. Epub 2016 Oct 28.

Dino Ferrari Centre, Department of Pathophysiology and Transplantation, University of Milan, Neurology Unit, IRCCS Foundation Ca' Granda, Ospedale Maggiore Policlinico, Via Francesco Sforza 35, 20122, Milan, Italy.

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.
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http://dx.doi.org/10.1002/mus.25192DOI Listing
January 2017

Perceived efficacy of salbutamol by persons with spinal muscular atrophy: A mixed methods study.

Muscle Nerve 2016 11 15;54(5):843-849. Epub 2016 Jul 15.

Department of Neuroimmunology and Neuromuscular Diseases, SOD Day Hospital/Day Service, Neurological Institute C. Besta IRCCS Foundation, Via Celoria 11, 20133, Milan, Italy.

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 Nerve 54: 843-849, 2016.
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http://dx.doi.org/10.1002/mus.25102DOI Listing
November 2016

Home-based aerobic exercise training improves skeletal muscle oxidative metabolism in patients with metabolic myopathies.

J Appl Physiol (1985) 2016 09 21;121(3):699-708. Epub 2016 Jul 21.

Institute of Molecular Bioimaging and Physiology, National Research Council, Segrate, Italy; Department of Medical and Biological Sciences, University of Udine, Udine, Italy.

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.
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http://dx.doi.org/10.1152/japplphysiol.00885.2015DOI Listing
September 2016

Imaging alterations in skeletal muscle channelopathies: a study in 15 patients.

Acta Myol 2015 Dec;34(2-3):109-15

Neuromuscular Diseases and Neuroimmunology Unit, Fondazione IRCCS Istituto Neurologico "Carlo Besta", Milan, Italy;

Skeletal muscle channelopathies (SMC), including non dystrophic myotonias (NDM) and periodic paralyses (PP), are characterized by considerable clinical overlap and clinical features not always allow addressing molecular diagnosis. Muscle imaging has been shown to be useful for differential diagnosis in neuromuscular disorders, however it has been relatively poorly investigated in SMC. We studied 15 patients affected by genetically confirmed SMC (NDM = 9, PP = 6) through muscle MRI or CT of thighs and legs, including 11 patients mutated in SCN4A gene, 2 in CACNA1S and 2 in CLCN1. Mean age at muscle imaging was 45.2 ± 18 years (range 22-70). Overall, fatty infiltration was found in thigh muscles in 8 (53%) patients and in leg muscles in 10 (60%). All patients mutated in CLCN1 and CACNA1S had abnormal thigh and/or leg muscle MRI, regardless the disease duration. On the contrary normal thigh and leg muscle MRI or CT scans were observed in 4/15 (27%) patients, all mutated in SCN4A. Variable degrees of fatty changes were found in patients mutated in SCN4A, CACNA1S and CLCN1. No differences on overall score of fatty infiltration were detected between NDM and PP (p-value = 0.953) neither between presence or absence of permanent weakness (p-value = 0.951). Our data confirm the presence of muscle fatty changes in the majority of SMC patients, although without any specific pattern of involvement. However muscle MRI may be a useful tool for longitudinal follow-up of SMC patients, in particular to evaluate the occurrence and the progression of fixed myopathy.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4859077PMC
December 2015

A novel clinical tool to classify facioscapulohumeral muscular dystrophy phenotypes.

J Neurol 2016 Jun 28;263(6):1204-14. Epub 2016 Apr 28.

Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy.

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.
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http://dx.doi.org/10.1007/s00415-016-8123-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4893383PMC
June 2016

Health-related quality of life and functional changes in DMD: A 12-month longitudinal cohort study.

Neuromuscul Disord 2016 Mar 2;26(3):189-96. Epub 2016 Feb 2.

Department of Paediatric Neurology, Catholic University, Rome, Italy. Electronic address:

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 PedsQL(TM) 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 PedsQL(TM) 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 PedsQL(TM) GCS and its first domain, in parallel with the decrement in the functional outcome measures. Correlation between the 12 month changes on the PedsQL(TM) inventories and functional measures were almost all negligible. Similar results were obtained on the Parent Proxy-Report. In conclusion, PedsQL(TM) 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.
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http://dx.doi.org/10.1016/j.nmd.2016.01.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4819956PMC
March 2016

Severe cardiomyopathy in a young patient with complete deficiency of adipose triglyceride lipase due to a novel mutation in PNPLA2 gene.

Int J Cardiol 2016 Mar 9;207:165-7. Epub 2016 Jan 9.

Laboratory of Cellular Biochemistry and Molecular Biology, CRIBENS, Catholic University of the Sacred Heart, Milan, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.ijcard.2016.01.137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4766941PMC
March 2016

SEPN1-related myopathy in three patients: novel mutations and diagnostic clues.

Eur J Pediatr 2016 Aug 16;175(8):1113-8. Epub 2016 Jan 16.

Unit of Child Neurology, Foundation IRCCS C.Besta Neurological Institute, Milan, Italy.

Unlabelled: Mutations in SEPN1 cause selenoprotein N (SEPN)-related myopathy (SEPN-RM) characterized by early-onset axial and neck weakness, spinal rigidity, respiratory failure and histopathological features, ranging from mild dystrophic signs to a congenital myopathy pattern with myofibrillar disorganization. We report on clinical and instrumental features in three patients affected with a congenital myopathy characterized by prevalent neck weakness starting at different ages and mild myopathy, in whom we performed diagnosis of SEPN-RM. The patients presented myopathic signs since their first years of life, but the disease remained unrecognized because of a relatively benign myopathic course. In two cases, myopathic features were stable after 2 years of follow-up, but respiratory involvement worsened. The muscle MRI and muscle biopsy showed a typical pattern of SEPN-RM. Molecular diagnosis revealed two novel homozygous mutations in SEPN1, c.1176delA and c.726_727InsTCC.

Conclusion: This report underlines the clinical diagnostic clues of early neck and axial weakness to suspect a SEPN-RM and the usefulness of muscle MRI in conjunction with clinical features to achieve the diagnosis. Our data confirm the slow progression of respiratory involvement in spite of the relatively stable course of myopathy. We report two previously undescribed mutations in SEPN1.

What Is Known: • Mutations in SEPN1 cause myopathy characterized by early-onset axial and neck weakness spinal rigidity and respiratory failure. • SEPN-related myopathies have been initially associated with four distinct histopathological entities that however appear more mixed in recently described cases. What is New: • SEPN-related myopathies can remain unrecognized because of the normal early motor development and relatively benign myopathic course of the disease. • Our study adds two novel homozygous mutations to the number of reported pathogenic SEPN1 variants.
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http://dx.doi.org/10.1007/s00431-015-2685-3DOI Listing
August 2016

Clinical expression of facioscapulohumeral muscular dystrophy in carriers of 1-3 D4Z4 reduced alleles: experience of the FSHD Italian National Registry.

BMJ Open 2016 Jan 5;6(1):e007798. Epub 2016 Jan 5.

Department of Science of Life, Institute of Biology, University of Modena and Reggio Emilia, Modena, Italy Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

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.
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http://dx.doi.org/10.1136/bmjopen-2015-007798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4716236PMC
January 2016

Anti-fibrotic effect of pirfenidone in muscle derived-fibroblasts from Duchenne muscular dystrophy patients.

Life Sci 2016 Jan 8;145:127-36. Epub 2015 Dec 8.

Neuromuscular Diseases and Neuroimmunology Unit, Foundation IRCCS Neurological Institute C. Besta, Milano, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.lfs.2015.12.015DOI Listing
January 2016

Histologic muscular history in steroid-treated and untreated patients with Duchenne dystrophy.

Neurology 2015 Nov 23;85(21):1886-93. Epub 2015 Oct 23.

From the Neuromuscular and Rare Diseases Unit (L.P., S.T., L.V., M.S., M. Moggio), Department of Neuroscience, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milan; Laboratory of Molecular Medicine for Muscular and Neurodegenerative Diseases (A.D.), Research Center, Confocal Microscopy Facility (S.P.), and Department of Neurosciences, Unit of Neuromuscular and Neurodegenerative Disorders (E.B.), Bambino Gesù Children's Hospital, Rome; Center of Molecular and Genetic Epidemiology (C.F.), Department of Clinical Sciences and Community Health, Università degli Studi di Milano, Milan; Dino Ferrari Center (F.M., G.P.C.), Neuroscience Section, Department of Pathophysiology and Transplantation, University of Milan, Neurology Unit, IRCCS Foundation Ca' Granda, Ospedale Maggiore Policlinico, Milan; U.O. Neuromuscular Diseases and Neuroimmunology (L.M., M. Mora), Fondazione IRCCS Istituto Neurologico C. Besta, Milan; and Department of Neurosciences Rita Levi Montalcini (T.M.), University of Turin, Italy.

Objective: 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.

Methods: 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.

Results: 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 (p < 0.0001). The average proportion in patients treated with steroids was 24.90%. Muscle fiber area mirrored that of connective tissue in both groups.

Conclusions: 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.
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http://dx.doi.org/10.1212/WNL.0000000000002147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4662699PMC
November 2015

Complete loss of the DNAJB6 G/F domain and novel missense mutations cause distal-onset DNAJB6 myopathy.

Acta Neuropathol Commun 2015 Jul 25;3:44. Epub 2015 Jul 25.

The Centre for Applied Genomics and Program in Genetics and Genome Biology, The Hospital for Sick Children and University of Toronto, Toronto, Canada.

Introduction: Protein aggregation is a common cause of neuropathology. The protein aggregation myopathy Limb-Girdle Muscular Dystrophy 1D (LGMD1D) is caused by mutations of amino acids Phe89 or Phe93 of DNAJB6, a co-chaperone of the HSP70 anti-aggregation protein. Another DNAJB6 mutation, Pro96Arg, was found to cause a distal-onset myopathy in one family.

Results: We detail the mutational, neuropathological, neurophysiological, neurological and radiological features of five new DNAJB6-myopathy families. One has the known Phe93Leu mutation and classic late-onset slowly progressive LGMD1D. Two have different mutations of Phe91 causing a variant childhood-onset severe limb-girdle myopathy. One has a Phe100Val mutation and distal-onset myopathy, unique early bulbar involvement, and a gender-modified wide age-of-onset range. The last has childhood-onset severe distal-onset myopathy and the first non-missense DNAJB6 mutation, c.346 + 5G > A, causing a splicing defect that entirely eliminates DNAJB6's G/F domain (ΔG/F), the domain that harbours all other mutations. Clinical and imaging examinations reveal that muscles considered uninvolved in DNAJB6-myopathy, e.g. lateral gastrocnemii, are affected in our patients with new mutations. Mutational modelling based on the known structure of the bacterial DNAJ2 protein indicates that all past and present mutated residues cluster within 15 Å in the G/F domain and all disturb the interface of this domain with the protein's J domain that confers the interaction with HSP70.

Conclusions: Our patients expand the phenotypic spectrum of DNAJB6-myopathy and allow tentative genotype-phenotype specifications. Combining with previous studies, the clinical severity spectrum is as follows: ΔG/F and Phe91 mutations, most severe; Phe100, Pro96, Phe89 mutations, intermediate; and Phe93, least severe. As it stands presently, proximal G/F domain mutations (Phe89, Phe91, Phe93) cause proximal limb-girdle myopathy, while distal G/F mutations (Pro96, Phe100) cause distal-onset myopathy. While all mutations affect the G/F-J interaction, each likely does so in different unknown extents or ways. One mutation, ΔG/F, causes its associated severe distal-onset myopathy phenotype in a clear way, through generation of a G/F domain-lacking DNAJB6 protein.
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http://dx.doi.org/10.1186/s40478-015-0224-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4513909PMC
July 2015

A CASQ1 founder mutation in three Italian families with protein aggregate myopathy and hyperCKaemia.

J Med Genet 2015 Sep 1;52(9):617-26. Epub 2015 Jul 1.

Neuromuscular Diseases and Neuroimmunology Unit, Foundation IRCCS Neurological Institute C. Besta, Milano, Italy.

Background: Protein aggregate myopathies are increasingly recognised conditions characterised by a surplus of endogenous proteins. The molecular and mutational background for many protein aggregate myopathies has been clarified with the discovery of several underlying mutations. Familial idiopathic hyperCKaemia is a benign genetically heterogeneous condition with autosomal dominant features in a high proportion of cases.

Methods: In 10 patients from three Italian families with autosomal dominant benign vacuolar myopathy and hyperCKaemia, we performed linkage analysis and exome sequencing as well as morphological and biochemical investigations.

Results And Conclusions: We show, by Sanger and exome sequencing, that the protein aggregate myopathy with benign evolution and muscle inclusions composed of excess CASQ1, affecting three Italian families, is due to the D244G heterozygous missense mutation in the CASQ1 gene. Investigation of microsatellite markers revealed a common haplotype in the three families indicating consanguinity and a founder effect. Results from immunocytochemistry, electron microscopy, biochemistry and transfected cell line investigations contribute to our understanding of pathogenetic mechanisms underlining this defect. The mutation is common to other Italian patients and is likely to share a founder effect with them. HyperCKaemia in the CASQ1-related myopathy is common and sometimes the sole overt manifestation. It is likely that CASQ1 mutations may remain undiagnosed if a muscle biopsy is not performed, and the condition could be more common than supposed.
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http://dx.doi.org/10.1136/jmedgenet-2014-102882DOI Listing
September 2015

Muscle MRI findings in facioscapulohumeral muscular dystrophy.

Eur Radiol 2016 Mar 27;26(3):693-705. Epub 2015 Jun 27.

Neuromuscular Diseases and Neuroimmunology Unit, Fondazione IRCCS Istituto Neurologico "Carlo Besta", Milan, Italy.

Objectives: 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.

Methods And Materials: 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).

Results: 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.

Conclusions: 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.

Key Points: 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.
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http://dx.doi.org/10.1007/s00330-015-3890-1DOI Listing
March 2016

Bone and Spinal Muscular Atrophy.

Bone 2015 Oct 5;79:116-20. Epub 2015 Jun 5.

Developmental Neurology Unit, Carlo Besta Neurological Institute Foundation, Milan, Italy.

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.
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http://dx.doi.org/10.1016/j.bone.2015.05.039DOI Listing
October 2015

Centronuclear myopathies: genotype-phenotype correlation and frequency of defined genetic forms in an Italian cohort.

J Neurol 2015 Jul 10;262(7):1728-40. Epub 2015 May 10.

Unit for Neuromuscular and Neurodegenerative Disorders, Bambino Gesù Children's Hospital, Viale San Paolo 15, 00146, Rome, Italy.

Centronuclear myopathies (CNMs) are a group of clinically and genetically heterogeneous muscle disorders. To date, mutation in 7 different genes has been reported to cause CNMs but 30 % of cases still remain genetically undefined. Genetic investigations are often expensive and time consuming. Clinical and morphological clues are needed to facilitate genetic tests and to choose the best approach for genetic screening. We aimed to describe genotype-phenotype correlation in an Italian cohort of patients affected by CNMs, to define the relative frequencies of its defined genetic forms and to draw a diagnostic algorithm to address genetic investigations. We recruited patients with CNMs from all the Italian tertiary neuromuscular centers following clinical and histological criteria. All selected patients were screened for the four 'canonical' genes related to CNMs: MTM1, DNM2, RYR1 and BIN1. Pathogenetic mutations were found in 38 of the 54 screened patients (70 %), mostly in patients with congenital onset (25 of 30 patients, 83 %): 15 in MTM1, 6 in DNM2, 3 in RYR1 and one in TTN. Among the 13 patients with a childhood-adolescence onset, mutations were found in 6 patients (46 %), all in DNM2. In the group of the 11 patients with adult onset, mutations were identified in 7 patients (63 %), again in DNM2, confirming that variants in this gene are relatively more common in late-onset phenotypes. The present study provides the relative molecular frequency of centronuclear myopathy and of its genetically defined forms in Italy and also proposes a diagnostic algorithm to be used in clinical practice to address genetic investigations.
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http://dx.doi.org/10.1007/s00415-015-7757-9DOI Listing
July 2015

Opposing roles of miR-21 and miR-29 in the progression of fibrosis in Duchenne muscular dystrophy.

Biochim Biophys Acta 2015 Jul 17;1852(7):1451-64. Epub 2015 Apr 17.

Neuromuscular Diseases and Neuroimmunology Unit, Foundation IRCCS Neurological Institute C. Besta, Milano, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbadis.2015.04.013DOI Listing
July 2015

Impaired Muscle Mitochondrial Biogenesis and Myogenesis in Spinal Muscular Atrophy.

JAMA Neurol 2015 Jun;72(6):666-75

Neuromuscular Unit, Dino Ferrari Centre, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Foundation Ca' Granda Ospedale Maggiore Policlinico, University of Milan, Milan, Italy.

Importance: The important depletion of mitochondrial DNA (mtDNA) and the general depression of mitochondrial respiratory chain complex levels (including complex II) have been confirmed, implying an increasing paucity of mitochondria in the muscle from patients with types I, II, and III spinal muscular atrophy (SMA-I, -II, and -III, respectively).

Objective: To investigate mitochondrial dysfunction in a large series of muscle biopsy samples from patients with SMA.

Design, Setting, And Participants: We studied quadriceps muscle samples from 24 patients with genetically documented SMA and paraspinal muscle samples from 3 patients with SMA-II undergoing surgery for scoliosis correction. Postmortem muscle samples were obtained from 1 additional patient. Age-matched controls consisted of muscle biopsy specimens from healthy children aged 1 to 3 years who had undergone analysis for suspected myopathy. Analyses were performed at the Neuromuscular Unit, Istituto di Ricovero e Cura a Carattere Scientifico Foundation Ca' Granda Ospedale Maggiore Policlinico-Milano, from April 2011 through January 2015.

Exposures: We used histochemical, biochemical, and molecular techniques to examine the muscle samples.

Main Outcomes And Measures: Respiratory chain activity and mitochondrial content.

Results: Results of histochemical analysis revealed that cytochrome-c oxidase (COX) deficiency was more evident in muscle samples from patients with SMA-I and SMA-II. Residual activities for complexes I, II, and IV in muscles from patients with SMA-I were 41%, 27%, and 30%, respectively, compared with control samples (P < .005). Muscle mtDNA content and cytrate synthase activity were also reduced in all 3 SMA types (P < .05). We linked these alterations to downregulation of peroxisome proliferator-activated receptor coactivator 1α, the transcriptional activators nuclear respiratory factor 1 and nuclear respiratory factor 2, mitochondrial transcription factor A, and their downstream targets, implying depression of the entire mitochondrial biogenesis. Results of Western blot analysis confirmed the reduced levels of the respiratory chain subunits that included mitochondrially encoded COX1 (47.5%; P = .004), COX2 (32.4%; P < .001), COX4 (26.6%; P < .001), and succinate dehydrogenase complex subunit A (65.8%; P = .03) as well as the structural outer membrane mitochondrial porin (33.1%; P < .001). Conversely, the levels of expression of 3 myogenic regulatory factors-muscle-specific myogenic factor 5, myoblast determination 1, and myogenin-were higher in muscles from patients with SMA compared with muscles from age-matched controls (P < .05).

Conclusions And Relevance: Our results strongly support the conclusion that an altered regulation of myogenesis and a downregulated mitochondrial biogenesis contribute to pathologic change in the muscle of patients with SMA. Therapeutic strategies should aim at counteracting these changes.
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http://dx.doi.org/10.1001/jamaneurol.2015.0178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4944827PMC
June 2015

Prevalence of congenital muscular dystrophy in Italy: a population study.

Neurology 2015 Mar 4;84(9):904-11. Epub 2015 Feb 4.

From the Departments of Paediatrics and Neurology (A.G., F.B., G.T., M. Monforte, E.R., M.P., E.M.), Catholic University, Rome; Unit of Neuromuscular and Neurodegenerative Disorders (A.D., G.T., E.B.), Department of Neurosciences, Bambino Gesù Children's Hospital, Rome; Pediatric Neurology and Neuromuscular Disease and Immunology Unit (I.M., M. Mora, L.M.), Istituto Neurologico Besta, Milan; Department of Neurosciences, Psychiatry and Anaesthesiology (S.M.), University of Messina; Neuromuscular Disease Unit (C.B., C.M.), G. Gaslini Institute, Genoa; Department of Neurosciences (E.P.), University of Padua, Italy; Department of Developmental Neuroscience and Molecular Medicine Neuromuscular Unit (G.A., R.B., F.M.S.), Stella Maris Institute, Pisa; Department of Neurological Sciences (F. Magri, G.P.C.), IRCCS Ospedale Maggiore Policlinico, University of Milan; Child Neurology and Psychiatry Unit (A.B.), IRCCS C. Mondino Foundation; Neuromuscular and Rare Diseases Unit (M. Moggio), IRCCS Foundation Ca' Granda Ospedale Maggiore Policlinico, Dino Ferrari Center, Milano; Child Neurology and Psychiatry Unit (A.P.), IRCCS Istituto delle Scienze Neurologiche di Bologna, Bellaria Hospital, Bologna; Cardiomiologia e Genetica Medica (R.P., L.P.), Dipartimento di Medicina Sperimentale Seconda Università di Napoli; Neuromuscular Center (T.M.), S.G. Battista Hospital, University of Turin; Centro Nemo (K.G.), Milan; Neuromuscular Unit (M.V.), Nigrisoli Hospital, Bologna; Section of Medical Genetics (F.G., A.F.), Department of Experimental and Diagnostic Medicine, Ferrara, Italy; and Dubowitz Neuromuscular Centre (F. Muntoni), UCL Institute of Child Health & Great Ormond Street Hospital for Children, London, UK.

Objective: We provide a nationwide population study of patients with congenital muscular dystrophy in Italy.

Methods: Cases were ascertained from the databases in all the tertiary referral centers for pediatric neuromuscular disorders and from all the genetic diagnostic centers in which diagnostic tests for these forms are performed.

Results: The study includes 336 patients with a point prevalence of 0.563 per 100,000. Mutations were identified in 220 of the 336 (65.5%). The cohort was subdivided into diagnostic categories based on the most recent classifications on congenital muscular dystrophies. The most common forms were those with α-dystroglycan glycosylation deficiency (40.18%) followed by those with laminin α2 deficiency (24.11%) and collagen VI deficiency (20.24%). The forms of congenital muscular dystrophy related to mutations in SEPN1 and LMNA were less frequent (6.25% and 5.95%, respectively).

Conclusions: Our study provides for the first time comprehensive epidemiologic information and point prevalence figures for each of the major diagnostic categories on a large cohort of congenital muscular dystrophies. The study also reflects the diagnostic progress in this field with an accurate classification of the cases according to the most recent gene discoveries.
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http://dx.doi.org/10.1212/WNL.0000000000001303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4351663PMC
March 2015