Publications by authors named "Marina Mora"

98 Publications

Muscle Proteomic Profile before and after Enzyme Replacement Therapy in Late-Onset Pompe Disease.

Int J Mol Sci 2021 Mar 11;22(6). Epub 2021 Mar 11.

Department of Biomedical Sciences for Health, University of Milan, 20090 Milano, Italy.

Mutations in the acidic alpha-glucosidase (GAA) coding gene cause Pompe disease. Late-onset Pompe disease (LOPD) is characterized by progressive proximal and axial muscle weakness and atrophy, causing respiratory failure. Enzyme replacement therapy (ERT), based on recombinant human GAA infusions, is the only available treatment; however, the efficacy of ERT is variable. Here we address the question whether proteins at variance in LOPD muscle of patients before and after 1 year of ERT, compared withhealthy age-matched subjects (CTR), reveal a specific signature. Proteins extracted from skeletal muscle of LOPD patients and CTR were analyzed by combining gel based (two-dimensional difference gel electrophoresis) and label-free (liquid chromatography-mass spectrometry) proteomic approaches, and ingenuity pathway analysis. Upstream regulators targeting autophagy and lysosomal tethering were assessed by immunoblotting. 178 proteins were changed in abundance in LOPD patients, 47 of them recovered normal level after ERT. Defects in oxidative metabolism, muscle contractile protein regulation, cytoskeletal rearrangement, and membrane reorganization persisted. Metabolic changes, ER stress and UPR (unfolded protein response) contribute to muscle proteostasis dysregulation with active membrane remodeling (high levels of LC3BII/LC3BI) and accumulation of p62, suggesting imbalance in the autophagic process. Active lysosome biogenesis characterizes both LOPD PRE and POST, unparalleled by molecules involved in lysosome tethering (VAMP8, SNAP29, STX17, and GORASP2) and BNIP3. In conclusion this study reveals a specific signature that suggests ERT prolongation and molecular targets to ameliorate patient's outcome.
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http://dx.doi.org/10.3390/ijms22062850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8001152PMC
March 2021

Gain-of-Function STIM1 L96V Mutation Causes Myogenesis Alteration in Muscle Cells From a Patient Affected by Tubular Aggregate Myopathy.

Front Cell Dev Biol 2021 26;9:635063. Epub 2021 Feb 26.

Department of Pharmacy-Drug Sciences, University of Bari, Bari, Italy.

Tubular Aggregate Myopathy (TAM) is a hereditary ultra-rare muscle disorder characterized by muscle weakness and cramps or myasthenic features. Biopsies from TAM patients show the presence of tubular aggregates originated from sarcoplasmic reticulum due to altered Ca homeostasis. TAM is caused by gain-of-function mutations in STIM1 or ORAI1, proteins responsible for Store-Operated-Calcium-Entry (SOCE), a pivotal mechanism in Ca signaling. So far there is no cure for TAM and the mechanisms through which or gene mutation lead to muscle dysfunction remain to be clarified. It has been established that post-natal myogenesis critically relies on Ca influx through SOCE. To explore how Ca homeostasis dysregulation associated with TAM impacts on muscle differentiation cascade, we here performed a functional characterization of myoblasts and myotubes deriving from patients carrying STIM1 L96V mutation by using fura-2 cytofluorimetry, high content imaging and real-time PCR. We demonstrated a higher resting Ca concentration and an increased SOCE in STIM1 mutant compared with control, together with a compensatory down-regulation of genes involved in Ca handling (. Differentiating STIM1 L96V myoblasts persisted in a mononuclear state and the fewer multinucleated myotubes had distinct morphology and geometry of mitochondrial network compared to controls, indicating a defect in the late differentiation phase. The alteration in myogenic pathway was confirmed by gene expression analysis regarding early () and late () differentiation markers together with mitochondrial markers (. We provided evidences of mechanisms responsible for a defective myogenesis associated to TAM mutant and validated a reliable cellular model usefull for TAM preclinical studies.
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http://dx.doi.org/10.3389/fcell.2021.635063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7952532PMC
February 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Novel mutation identification and copy number variant detection via exome sequencing in congenital muscular dystrophy.

Mol Genet Genomic Med 2020 11 16;8(11):e1387. Epub 2020 Sep 16.

Department of Pharmacology and Physiology, Institute for Neuroscience, The George Washington University, Washington, DC, USA.

Background: Congenital muscular dystrophy type 1A (MDC1A), also termed merosin-deficient congenital muscular dystrophy (CMD), is a severe form of CMD caused by mutations in the laminin α2 gene (LAMA2). Of the more than 300 likely pathogenic variants found in the Leiden Open Variant Database, the majority are truncating mutations leading to complete LAMA2 loss of function, but multiple copy number variants (CNVs) have also been reported with variable frequency.

Methods: We collected a cohort of individuals diagnosed with likely MDC1A and sought to identify both single nucleotide variants and small and larger CNVs via exome sequencing by extending the analysis of sequencing data to detect splicing changes and CNVs.

Results: Standard exome analysis identified multiple novel LAMA2 variants in our cohort, but only four cases carried biallelic variants. Since likely truncating LAMA2 variants are often found in heterozygosity without a second allele, we performed additional splicing and CNV analysis on exome data and identified one splice change outside of the canonical sequences and three CNVs, in the remaining four cases.

Conclusions: Our findings support the expectation that a portion of MDC1A cases may be caused by at least one CNV allele and show how these changes can be effectively identified by additional analysis of existing exome data.
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http://dx.doi.org/10.1002/mgg3.1387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667317PMC
November 2020

Myopathic changes associated with psychomotor delay and seizures caused by a novel homozygous mutation in TBCK.

Muscle Nerve 2020 08 27;62(2):266-271. Epub 2020 May 27.

Institute for Neuroscience and Department of Pharmacology and Physiology, George Washington University, Washington, DC.

Background: Biallelic mutations in TBC1-domain containing kinase (TBCK) lead to hypotonia, global developmental delay with severe cognitive and motor deficits, and variable presentation of dysmorphic facial features and brain malformations. It remains unclear whether hypotonia in these individuals is purely neurogenic, or also caused by progressive muscle disease.

Methods: Whole exome sequencing was performed on a family diagnosed with nonspecific myopathic changes by means of histological analysis and immunohistochemistry of muscle biopsy samples.

Results: A novel homozygous truncation in TBCK was found in two sisters diagnosed with muscle disease and severe psychomotor delay. TBCK was completely absent in these patients.

Conclusions: Our findings identify a novel early truncating variant in TBCK associated with a severe presentation and add muscle disease to the variability of phenotypes associated with TBCK mutations. Inconsistent genotype/phenotype correlation could be ascribed to the multiple roles of TBCK in intracellular signaling and endolysosomal function in different tissues.
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http://dx.doi.org/10.1002/mus.26907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7369155PMC
August 2020

The Genetic Landscape of Dystrophin Mutations in Italy: A Nationwide Study.

Front Genet 2020 3;11:131. Epub 2020 Mar 3.

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

Dystrophinopathies are inherited diseases caused by mutations in the dystrophin () gene for which testing is mandatory for genetic diagnosis, reproductive choices and eligibility for personalized trials. We genotyped the gene in our Italian cohort of 1902 patients (BMD n = 740, 39%; DMD n =1162, 61%) within a nationwide study involving 11 diagnostic centers in a 10-year window (2008-2017). In DMD patients, we found deletions in 57%, duplications in 11% and small mutations in 32%. In BMD, we found deletions in 78%, duplications in 9% and small mutations in 13%. In BMD, there are a higher number of deletions, and small mutations are more frequent than duplications. Among small mutations that are generally frequent in both phenotypes, 44% of DMD and 36% of BMD are nonsense, thus, eligible for stop codon read-through therapy; 63% of all out-of-frame deletions are eligible for single exon skipping. Patients were also assigned to Italian regions and showed interesting regional differences in mutation distribution. The full genetic characterization in this large, nationwide cohort has allowed us to draw several correlations between DMD/BMD genotype landscapes and mutation frequency, mutation types, mutation locations along the gene, exon/intron architecture, and relevant protein domain, with effects on population genetic characteristics and new personalized therapies.
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http://dx.doi.org/10.3389/fgene.2020.00131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7063120PMC
March 2020

Improved Criteria for the Classification of Titin Variants in Inherited Skeletal Myopathies.

J Neuromuscul Dis 2020 ;7(2):153-166

Folkhälsan Research Center, Helsinki, Finland.

Background: Extensive genetic screening results in the identification of thousands of rare variants that are difficult to interpret. Because of its sheer size, rare variants in the titin gene (TTN) are detected frequently in any individual. Unambiguous interpretation of molecular findings is almost impossible in many patients with myopathies or cardiomyopathies.

Objective: To refine the current classification framework for TTN-associated skeletal muscle disorders and standardize the interpretation of TTN variants.

Methods: We used the guidelines issued by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) to re-analyze TTN genetic findings from our patient cohort.

Results: We identified in the classification guidelines three rules that are not applicable to titin-related skeletal muscle disorders; six rules that require disease-/gene-specific adjustments and four rules requiring quantitative thresholds for a proper use. In three cases, the rule strength need to be modified.

Conclusions: We suggest adjustments are made to the guidelines. We provide frequency thresholds to facilitate filtering of candidate causative variants and guidance for the use and interpretation of functional data and co-segregation evidence. We expect that the variant classification framework for TTN-related skeletal muscle disorders will be further improved along with a better understanding of these diseases.
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http://dx.doi.org/10.3233/JND-190423DOI Listing
November 2020

Comparative proteomic analyses of Duchenne muscular dystrophy and Becker muscular dystrophy muscles: changes contributing to preserve muscle function in Becker muscular dystrophy patients.

J Cachexia Sarcopenia Muscle 2020 04 28;11(2):547-563. Epub 2020 Jan 28.

Department of Biomedical Sciences for Health, University of Milan, Milan, Italy.

Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are characterized by muscle wasting leading to loss of ambulation in the first or third decade, respectively. In DMD, the lack of dystrophin hampers connections between intracellular cytoskeleton and cell membrane leading to repeated cycles of necrosis and regeneration associated with inflammation and loss of muscle ordered structure. BMD has a similar muscle phenotype but milder. Here, we address the question whether proteins at variance in BMD compared with DMD contribute to the milder phenotype in BMD, thus identifying a specific signature to be targeted for DMD treatment.

Methods: Proteins extracted from skeletal muscle from DMD/BMD patients and young healthy subjects were either reduced and solubilized prior two-dimensional difference in gel electrophoresis/mass spectrometry differential analysis or tryptic digested prior label-free liquid chromatography with tandem mass spectrometry. Statistical analyses of proteins and peptides were performed by DeCyder and Perseus software and protein validation and verification by immunoblotting.

Results: Proteomic results indicate minor changes in the extracellular matrix (ECM) protein composition in BMD muscles with retention of mechanotransduction signalling, reduced changes in cytoskeletal and contractile proteins. Conversely, in DMD patients, increased levels of several ECM cytoskeletal and contractile proteins were observed whereas some proteins of fast fibres and of Z-disc decreased. Detyrosinated alpha-tubulin was unchanged in BMD and increased in DMD although neuronal nitric oxide synthase was unchanged in BMD and greatly reduced in DMD. Metabolically, the tissue is characterized by a decrement of anaerobic metabolism both in DMD and BMD compared with controls, with increased levels of the glycogen metabolic pathway in BMD. Oxidative metabolism is severely compromised in DMD with impairment of malate shuttle; conversely, it is active in BMD supporting the tricarboxylic acid cycle and respiratory chain. Adipogenesis characterizes DMD, whereas proteins involved in fatty acids beta-oxidation are increased in BMD. Proteins involved in protein/amino acid metabolism, cell development, calcium handling, endoplasmic reticulum/sarcoplasmic reticulum stress response, and inflammation/immune response were increased in DMD. Both disorders are characterized by the impairment of N-linked protein glycosylation in the endoplasmic reticulum. Authophagy was decreased in DMD whereas it was retained in BMD.

Conclusions: The mechanosensing and metabolic disruption are central nodes of DMD/BMD phenotypes. The ECM proteome composition and the metabolic rewiring in BMD lead to preservation of energy levels supporting autophagy and cell renewal, thus promoting the retention of muscle function. Conversely, DMD patients are characterized by extracellular and cytoskeletal protein dysregulation and by metabolic restriction at the level of α-ketoglutarate leading to shortage of glutamate-derived molecules that over time triggers lipogenesis and lipotoxicity.
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http://dx.doi.org/10.1002/jcsm.12527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7113522PMC
April 2020

Glycogen storage in a zebrafish Pompe disease model is reduced by 3-BrPA treatment.

Biochim Biophys Acta Mol Basis Dis 2020 05 7;1866(5):165662. Epub 2020 Jan 7.

Neuromuscular Diseases and Neuroimmunology Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Via Celoria 11, Milan, 20133, Italy. Electronic address:

Pompe disease (PD) is an autosomal recessive muscular disorder caused by deficiency of the glycogen hydrolytic enzyme acid α-glucosidase (GAA). The enzyme replacement therapy, currently the only available therapy for PD patients, is efficacious in improving cardiomyopathy in the infantile form, but not equally effective in the late onset cases with involvement of skeletal muscle. Correction of the skeletal muscle phenotype has indeed been challenging, probably due to concomitant dysfunctional autophagy. The increasing attention to the pathogenic mechanisms of PD and the search of new therapeutic strategies prompted us to generate and characterize a novel transient PD model, using zebrafish. Our model presented increased glycogen content, markedly altered motor behavior and increased lysosome content, in addition to altered expression of the autophagy-related transcripts and proteins Beclin1, p62 and Lc3b. Furthermore, the model was used to assess the beneficial effects of 3-bromopyruvic acid (3-BrPA). Treatment with 3-BrPA induced amelioration of the model phenotypes regarding glycogen storage, motility behavior and autophagy-related transcripts and proteins. Our zebrafish PD model recapitulates most of the defects observed in human patients, proving to be a powerful translational model. Moreover, 3-BrPA unveiled to be a promising compound for treatment of conditions with glycogen accumulation.
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http://dx.doi.org/10.1016/j.bbadis.2020.165662DOI Listing
May 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

Exome sequencing detects compound heterozygous nonsense LAMA2 mutations in two siblings with atypical phenotype and nearly normal brain MRI.

Neuromuscul Disord 2019 05 10;29(5):376-380. Epub 2019 Apr 10.

Muscle Cell Biology Lab, Neuromuscular Diseases and Neuroimmunology Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Via Temolo 4, 20126 Milano, Italy. Electronic address:

LAMA2 mutations cause the most frequent congenital muscular dystrophy subtype MDC1A and a variety of milder phenotypes, characterized by total or partial laminin-α2 deficiency. In both severe and milder cases brain MRI invariably shows abnormal white matter signal intensity. We report clinical, histopathological, imaging and genetic data on two siblings with very subtle, and at first undetected, reduction in laminin-α2 expression, and brain MRI showing minor non-specific abnormalities. Clinical features in the female proband were characterized by muscle weakness involving neck and axial muscles, and pelvic girdle and distal lower limb muscles, reduced tendon reflexes and pes cavus. Clinical features in a younger brother were similar, and remained stable in both siblings during the follow up. Whole exome sequencing (WES) detected two heterozygous truncating LAMA2 mutations. Brain MRI in combination with laminin-α2 immunohistochemistry might not be sufficient and WES might be the only means to reach a diagnosis.
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http://dx.doi.org/10.1016/j.nmd.2019.04.001DOI Listing
May 2019

Collagen XIX Alpha 1 Improves Prognosis in Amyotrophic Lateral Sclerosis.

Aging Dis 2019 Apr 1;10(2):278-292. Epub 2019 Apr 1.

2Neurology Department, ALS Unit, CIBERER U-723, Health Research Institute, October 12th Hospital "IIS I+12", Madrid, Spain.

The identification of more reliable diagnostic or prognostic biomarkers in age-related neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis (ALS), is urgently needed. The objective in this study was to identify more reliable prognostic biomarkers of ALS mirroring neurodegeneration that could be of help in clinical trials. A total of 268 participants from three cohorts were included in this study. The muscle and blood cohorts were analyzed in two cross-sectional studies, while the serial blood cohort was analyzed in a longitudinal study at 6-monthly intervals. Fifteen target genes and fourteen proteins involved in muscle physiology and differentiation, metabolic processes and neuromuscular junction dismantlement were studied in the three cohorts. In the muscle biopsy cohort, the risk for a higher mortality in an ALS patient that showed high Collagen type XIX, alpha 1 (COL19A1) protein levels and a fast progression of the disease was 70.5% ( < 0.05), while in the blood cohort, this risk was 20% ( < 0.01). In the serial blood cohort, the linear mixed model analysis showed a significant association between increasing gene levels along disease progression and a faster progression during the follow-up period of 24 months ( < 0.05). Additionally, higher levels and a faster progression increased 17.9% the mortality risk ( < 0.01). We provide new evidence that can be considered a prognostic biomarker that could help the selection of homogeneous groups of patients for upcoming clinical trial and may be pointed out as a promising therapeutic target in ALS.
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http://dx.doi.org/10.14336/AD.2018.0917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6457048PMC
April 2019

Alteration of mitochondrial membrane inner potential in three Italian patients with megaconial congenital muscular dystrophy carrying new mutations in CHKB gene.

Mitochondrion 2019 07 12;47:24-29. Epub 2019 Apr 12.

Medical Genetics Neurogenetics Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy. Electronic address:

Congenital Muscular Dystrophies (CMDs) are a heterogeneous group of autosomal recessive disorders presenting at birth with psychomotor delay, cognitive impairment, muscle weakness and hypotonia. Here we described an alteration of mitochondrial inner membrane potential and mitochondrial network in cells derived from Italian patients carrying three novel mutations in CHKB gene, recently associated with "megaconial CMD". On the bases of our findings, we hypothesize that the mitochondrial membrane potential alteration, presumably as a consequence of the altered biosynthesis of phosphatidylcholine, could be responsible for the peculiar morphological aspect of mitochondria in this disease and might be involved in the disease pathogenesis.
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http://dx.doi.org/10.1016/j.mito.2019.04.002DOI Listing
July 2019

Aging-associated genes and microRNAs: a contribution to myogenic program dysregulation in oculopharyngeal muscular dystrophy.

FASEB J 2019 06 12;33(6):7155-7167. Epub 2019 Mar 12.

Neurology IV-Neuroimmunology and Neuromuscular Diseases Unit, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Istituto Neurologico Carlo Besta, Milan, Italy.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset muscle disease caused by an abnormal (GCN) triplet expansion within the polyadenylate-binding protein nuclear 1 gene and consequent mRNA processing impairment and myogenic defects. Because a reduced cell proliferation potential and the consequent regeneration failure of aging muscle have been shown to be governed by lethal-7 () microRNA-mediated mechanisms, in the present study, we evaluated the role of in the pathogenesis of OPMD. By a multidisciplinary approach, including confocal microscopy, Western blot, and quantitative PCR analyses on muscle biopsies from patients and unaffected individuals, we found a significant increase in expression in OPMD muscles associated with an unusual high percentage of paired box 7-positive satellite cells. Furthermore, IL-6, a cytokine involved in the regulation of satellite cell proliferation and differentiation and a potential target of , was found strongly down-regulated in OPMD compared with control muscles. The decrease in IL-6 transcript levels and protein content was also confirmed during differentiation of patients' and controls' muscle cells. Overall, our data suggest a key role of in the regeneration and degeneration process in OPMD muscle and pointed to IL-6 as a potential target molecule for new therapeutic approaches for this disorder.-Cappelletti, C., Galbardi, B., Bruttini, M., Salerno, F., Canioni, E., Pasanisi, M. B., Rodolico, C., Brizzi, T., Mora, M., Renieri, A., Maggi, L., Bernasconi, P., Mantegazza, R. Aging-associated genes and microRNAs: a contribution to myogenic program dysregulation in oculopharyngeal muscular dystrophy.
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http://dx.doi.org/10.1096/fj.201801577RRDOI Listing
June 2019

Expanding the central nervous system disease spectrum associated with FLNC mutation.

Muscle Nerve 2019 05 20;59(5):E33-E37. Epub 2019 Feb 20.

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

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http://dx.doi.org/10.1002/mus.26443DOI Listing
May 2019

Engineering an Environment for the Study of Fibrosis: A 3D Human Muscle Model with Endothelium Specificity and Endomysium.

Cell Rep 2018 12;25(13):3858-3868.e4

IRCCS Istituto Ortopedico Galeazzi, Cell and Tissue Engineering Laboratory, Milano, Italy; Regenerative Medicine Technologies Lab, Ente Ospedaliero Cantonale, Lugano, Switzerland; Swiss Institute for Regenerative Medicine, Lugano, Switzerland. Electronic address:

The integration of vascular structures into in vitro cultured tissues provides realistic models of complex tissue-vascular interactions. Despite the incidence and impact of muscle-wasting disorders, advanced in vitro systems are still far from recapitulating the environmental complexity of skeletal muscle. Our model comprises differentiated human muscle fibers enveloped by a sheath of human muscle-derived fibroblasts and supported by a vascular network with mural-like cells. Here, we demonstrate the induction of muscle-specific endothelium and the self-organization of endomysial muscle fibroblasts mediated by endothelial cells. We use this model to mimic the fibrotic environment characterizing muscular dystrophies and to highlight key signatures of fibrosis that are neglected or underestimated in traditional 2D monocultures. Overall, this vascularized meso-scale cellular construct finely recapitulates the human skeletal muscle environment and provides an advanced solution for in vitro studies of muscle physiology and pathology.
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http://dx.doi.org/10.1016/j.celrep.2018.11.092DOI Listing
December 2018

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

Copy Number Variants Account for a Tiny Fraction of Undiagnosed Myopathic Patients.

Genes (Basel) 2018 Oct 26;9(11). Epub 2018 Oct 26.

Fondazione Hospital S.Camillo IRCCS, 30126 Venezia, Italy.

Next-generation sequencing (NGS) technologies have led to an increase in the diagnosis of heterogeneous genetic conditions. However, over 50% of patients with a genetically inherited disease are still without a diagnosis. In these cases, different hypotheses are usually postulated, including variants in novel genes or elusive mutations. Although the impact of copy number variants (CNVs) in neuromuscular disorders has been largely ignored to date, missed CNVs are predicted to have a major role in disease causation as some very large genes, such as the dystrophin gene, have prone-to-deletion regions. Since muscle tissues express several large disease genes, the presence of elusive CNVs needs to be comprehensively assessed following an accurate and systematic approach. In this multicenter cohort study, we analyzed 234 undiagnosed myopathy patients using a custom array comparative genomic hybridization (CGH) that covers all muscle disease genes at high resolution. Twenty-two patients (9.4%) showed non-polymorphic CNVs. In 12 patients (5.1%), the identified CNVs were considered responsible for the observed phenotype. An additional ten patients (4.3%) presented candidate CNVs not yet proven to be causative. Our study indicates that deletions and duplications may account for 5⁻9% of genetically unsolved patients. This strongly suggests that other mechanisms of disease are yet to be discovered.
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http://dx.doi.org/10.3390/genes9110524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6267442PMC
October 2018

Broad phenotypic spectrum and genotype-phenotype correlations in GMPPB-related dystroglycanopathies: an Italian cross-sectional study.

Orphanet J Rare Dis 2018 09 26;13(1):170. Epub 2018 Sep 26.

Department of Developmental Neuroscience and Molecular Medicine Neuromuscular Unit and Child Neurology, IRCCS Fondazione Stella Maris, Via dei Giacinti 2, 56018, Pisa, Italy.

Background: Dystroglycanopathy (α-DG) is a relatively common, clinically and genetically heterogeneous category of congenital forms of muscular dystrophy (CMD) and limb-girdle muscular dystrophy (LGMD) associated with hypoglycosylated α-dystroglycan. To date, mutations in at least 19 genes have been associated with α-DG. One of them, GMPPB, encoding the guanosine-diphosphate-mannose (GDP-mannose) pyrophosphorylase B protein, has recently been associated with a wide clinical spectrum ranging from severe Walker-Warburg syndrome to pseudo-metabolic myopathy and even congenital myasthenic syndromes. We re-sequenced the full set of known disease genes in 73 Italian patients with evidence of either reduced or nearly absent α-dystroglycan to assess genotype-phenotype correlations in this cohort. We used innovative bioinformatic tools to calculate the effects of all described GMPPB mutations on protein function and attempted to correlate them with phenotypic expressions.

Results: We identified 13 additional cases from 12 families and defined seven novel mutations. Patients displayed variable phenotypes including less typical pictures, ranging from asymptomatic hyperCKemia, to arthrogryposis and congenital clubfoot at birth, and also showed neurodevelopmental comorbidities, such as seizures and ataxic gait, as well as autism-spectrum disorder, which is seldom described in clinical reports of dystroglycanopathies. We also demonstrated that few mutations recur in the Italian GMPPB-mutated population and that alterations of protein stability are the main effects of GMPPB missense variants.

Conclusion: This work adds to the data on genotype-phenotype correlations in α-DG and offers new bionformatic tools to provide the conceptual framework needed to understand the complexity of these disorders.
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http://dx.doi.org/10.1186/s13023-018-0863-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158856PMC
September 2018

HDAC8 regulates canonical Wnt pathway to promote differentiation in skeletal muscles.

J Cell Physiol 2019 05 24;234(5):6067-6076. Epub 2018 Sep 24.

Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Università degli Studi di Milano, Milano, Italy.

Histone deacetylase 8 (HDAC8) is a class 1 histone deacetylase and a member of the cohesin complex. HDAC8 is expressed in smooth muscles, but its expression in skeletal muscle has not been described. We have shown for the first time that HDAC8 is expressed in human and zebrafish skeletal muscles. Using RD/12 and RD/18 rhabdomyosarcoma cells with low and high differentiation potency, respectively, we highlighted a specific correlation with HDAC8 expression and an advanced stage of muscle differentiation. We inhibited HDAC8 activity through a specific PCI-34051 inhibitor in murine C2C12 myoblasts and zebrafish embryos, and we observed skeletal muscles differentiation impairment. We also found a positive regulation of the canonical Wnt signaling by HDAC8 that might explain muscle differentiation defects. These findings suggest a novel mechanism through which HDAC8 expression, in a specific time window of skeletal muscle development, positively regulates canonical Wnt pathway that is necessary for muscle differentiation.
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http://dx.doi.org/10.1002/jcp.27341DOI Listing
May 2019

Exosomes and exosomal miRNAs from muscle-derived fibroblasts promote skeletal muscle fibrosis.

Matrix Biol 2018 12 5;74:77-100. Epub 2018 Jul 5.

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

Exosomes, natural carriers of mRNAs, non-coding RNAs and proteins between donor and recipient cells, actively contribute to cell-cell communication. We investigated the potential pro-fibrotic role of exosomes released by muscle-derived fibroblasts of Duchenne muscular dystrophy (DMD) patients, and of miRNAs carried by exosomes. By fibrosis focused array analysis we found that exosomes from DMD fibroblasts, had significantly higher levels of miR-199a-5p, a miRNA up-regulated in fibrotic conditions, compared to control exosomes, while levels in myoblast-derived exosomes were not increased. In control fibroblasts, exposure to DMD fibroblast-derived exosomes induced a myofibroblastic phenotype with increase in α-smooth actin, collagen and fibronectin transcript and protein expression, soluble collagen production and deposition, cell proliferation, and activation of Akt and ERK signaling, while exposure to control exosomes did not. Transfecting control fibroblasts or loading control exosomes with miR-199a-5p mimic or inhibitor induced opposing effects on fibrosis-related mRNAs and proteins, on collagen production and Akt and ERK pathways. Finally, injection of DMD fibroblast-derived exosomes into mouse tibialis anterior muscle after cardiotoxin-induced necrosis, produced greater fibrosis than control exosomes. Our findings indicate that exosomes produced by local fibroblasts in the DMD muscle are able to induce phenotypic conversion of normal fibroblasts to myofibroblasts thereby increasing the fibrotic response. This conversion is related to transfer of high levels of miR-199a-5p and to reduction of its target caveolin-1; both, therefore, are potential therapeutic targets in muscle fibrosis.
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http://dx.doi.org/10.1016/j.matbio.2018.07.003DOI Listing
December 2018

Up-regulation of Toll-like receptors 7 and 9 and its potential implications in the pathogenic mechanisms of LMNA-related myopathies.

Nucleus 2018 ;9(1):398-409

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

Laminopathies are a heterogeneous group of diseases, caused by mutations in lamin A/C proteins. The most common laminopathy (LMNA-related myopathies, LMNA-RM) affects skeletal and cardiac muscles; muscle histopathology is variable, ranging from mild unspecific changes to dystrophic features, sometimes with inflammatory evidence. Whether the genetic defect might activate innate immune components, leading to chronic inflammation, myofiber necrosis and fibrosis, is still unknown. By qPCR, a significant up-regulation of Toll-like receptor (TLR) 7 and 9 transcripts was found in LMNA-RM compared to other myopathic and non-myopathic muscles. A marked TLR7/9 staining was observed on LMNA-RM blood vessels and muscle fibers and, when present, on infiltrating cells, mainly macrophages, scattered in the tissue or localized close to degenerated muscle fibers and connective tissue. Our results recognize innate immunity as a player in LMNA-RM pathogenesis. Modulation of TLR7/9 signaling pathways and decrease of macrophage-mediated inflammation might be potential therapeutic strategies in LMNA-RM management.

Abbreviations: DMD, Duchenne muscular dystrophy; EDMD2, Emery-Dreifuss muscular dystrophy type 2; FSHD, facio-scapulo-humeral muscular dystrophy; LGMD1B, limb-girdle muscular dystrophy type 1B; LMNA-CMD, LMNA-related congenital muscular dystrophy; LMNA-RM, LMNA-related myopathies; sIBM, sporadic inclusion body myositis; TLR, Toll-like receptor.
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http://dx.doi.org/10.1080/19491034.2018.1471947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7000140PMC
September 2019

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

Zc3h10 is a novel mitochondrial regulator.

EMBO Rep 2018 04 5;19(4). Epub 2018 Mar 5.

DiSFeB, Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, Italy

Mitochondria are the energy-generating hubs of the cell. In spite of considerable advances, our understanding of the factors that regulate the molecular circuits that govern mitochondrial function remains incomplete. Using a genome-wide functional screen, we identify the poorly characterized protein Zinc finger CCCH-type containing 10 (Zc3h10) as regulator of mitochondrial physiology. We show that Zc3h10 is upregulated during physiological mitochondriogenesis as it occurs during the differentiation of myoblasts into myotubes. Zc3h10 overexpression boosts mitochondrial function and promotes myoblast differentiation, while the depletion of Zc3h10 results in impaired myoblast differentiation, mitochondrial dysfunction, reduced expression of electron transport chain (ETC) subunits, and blunted TCA cycle flux. Notably, we have identified a loss-of-function mutation of Zc3h10 in humans (Tyr105 to Cys105) that is associated with increased body mass index, fat mass, fasting glucose, and triglycerides. Isolated peripheral blood mononuclear cells from individuals homozygotic for Cys105 display reduced oxygen consumption rate, diminished expression of some ETC subunits, and decreased levels of some TCA cycle metabolites, which all together derive in mitochondrial dysfunction. Taken together, our study identifies Zc3h10 as a novel mitochondrial regulator.
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http://dx.doi.org/10.15252/embr.201745531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5891430PMC
April 2018

Interpreting Genetic Variants in Titin in Patients With Muscle Disorders.

JAMA Neurol 2018 05;75(5):557-565

Fondazione Hospital S.Camillo Institute for Research and Health Care, Venezia, Italy.

Importance: Mutations in the titin gene (TTN) cause a wide spectrum of genetic diseases. The interpretation of the numerous rare variants identified in TTN is a difficult challenge given its large size.

Objective: To identify genetic variants in titin in a cohort of patients with muscle disorders.

Design, Setting, And Participants: In this case series, 9 patients with titinopathy and 4 other patients with possibly disease-causing variants in TTN were identified. Titin mutations were detected through targeted resequencing performed on DNA from 504 patients with muscular dystrophy, congenital myopathy, or other skeletal muscle disorders. Patients were enrolled from 10 clinical centers in April 2012 to December 2013. All of them had not received a diagnosis after undergoing an extensive investigation, including Sanger sequencing of candidate genes. The data analysis was performed between September 2013 and January 2017. Sequencing data were analyzed using an internal custom bioinformatics pipeline.

Main Outcomes And Measures: The identification of novel mutations in the TTN gene and novel patients with titinopathy. We performed an evaluation of putative causative variants in the TTN gene, combining genetic, clinical, and imaging data with messenger RNA and/or protein studies.

Results: Of the 9 novel patients with titinopathy, 5 (55.5%) were men and the mean (SD) age at onset was 25 (15.8) years (range, 0-46 years). Of the 4 other patients (3 men and 1 woman) with possibly disease-causing TTN variants, 2 (50%) had a congenital myopathy and 2 (50%) had a slowly progressive distal myopathy with onset in the second decade. Most of the identified mutations were previously unreported. However, all the variants, even the already described mutations, require careful clinical and molecular evaluation of probands and relatives. Heterozygous truncating variants or unique missense changes are not sufficient to make a diagnosis of titinopathy.

Conclusions And Relevance: The interpretation of TTN variants often requires further analyses, including a comprehensive evaluation of the clinical phenotype (deep phenotyping) as well as messenger RNA and protein studies. We propose a specific workflow for the clinical interpretation of genetic findings in titin.
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http://dx.doi.org/10.1001/jamaneurol.2017.4899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5885217PMC
May 2018

The RD-Connect Registry & Biobank Finder: a tool for sharing aggregated data and metadata among rare disease researchers.

Eur J Hum Genet 2018 05 2;26(5):631-643. Epub 2018 Feb 2.

National Center for Rare Diseases, Istituto Superiore di Sanità, Rome, Italy.

In rare disease (RD) research, there is a huge need to systematically collect biomaterials, phenotypic, and genomic data in a standardized way and to make them findable, accessible, interoperable and reusable (FAIR). RD-Connect is a 6 years global infrastructure project initiated in November 2012 that links genomic data with patient registries, biobanks, and clinical bioinformatics tools to create a central research resource for RDs. Here, we present RD-Connect Registry & Biobank Finder, a tool that helps RD researchers to find RD biobanks and registries and provide information on the availability and accessibility of content in each database. The finder concentrates information that is currently sparse on different repositories (inventories, websites, scientific journals, technical reports, etc.), including aggregated data and metadata from participating databases. Aggregated data provided by the finder, if appropriately checked, can be used by researchers who are trying to estimate the prevalence of a RD, to organize a clinical trial on a RD, or to estimate the volume of patients seen by different clinical centers. The finder is also a portal to other RD-Connect tools, providing a link to the RD-Connect Sample Catalogue, a large inventory of RD biological samples available in participating biobanks for RD research. There are several kinds of users and potential uses for the RD-Connect Registry & Biobank Finder, including researchers collaborating with academia and the industry, dealing with the questions of basic, translational, and/or clinical research. As of November 2017, the finder is populated with aggregated data for 222 registries and 21 biobanks.
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http://dx.doi.org/10.1038/s41431-017-0085-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5945774PMC
May 2018

Rare Disease Biospecimens and Patient Registries: Interoperability for Research Promotion, a European Example: EuroBioBank and SpainRDR-BioNER.

Adv Exp Med Biol 2017 ;1031:141-147

Neuromuscular and Neuroimmunology Unit, C. Besta Neurological Institute, Milan, Italy.

Well-annotated and properly preserved specimens are crucial both for diagnostic purposes and for use in basic and pre-clinical research, and are especially important for rare disease (RD) studies. Several consortia have been established in the recent years in order to facilitate research and to maximise access to rare biological samples and data stored in rare disease biobanks and registries, among them the EuroBioBank network and the Spain National Rare Disease Registry (RDR) and Biobank (BioNER).EuroBioBank, established in 2001, was the first network of RD biobanks to operate in Europe as a service distributing human DNA, cells, and tissue to the scientific community conducting research on rare diseases.The Spanish RDR and BioNER were created for facilitating rare disease research and health-related matters. The coordination of these two bodies represents an example of great scientific value as biological samples donated by patients at BioNER are linked to clinical information collected in the RDR.Rare disease biobanks and registries will need for the future to increase their effort to improve interconnection so to enable investigators to better locate samples and associated data, while protecting security of the data and privacy of the participants and adhering to international ethical and legal requirements.
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http://dx.doi.org/10.1007/978-3-319-67144-4_7DOI Listing
June 2018

Test of Antifibrotic Drugs in a Cellular Model of Fibrosis Based on Muscle-Derived Fibroblasts from Duchenne Muscular Dystrophy Patients.

Methods Mol Biol 2018 ;1687:205-217

Muscle Cell Biology Lab, Neuromuscular Diseases and Neuroimmunology Unit, Fondazione IRCCS Istituto Neurologico "C. Besta", Via Temolo 4, 20126, Milan, Italy.

An in vitro model of muscle fibrosis, based on the use of primary human fibroblasts isolated from muscle biopsies of patients affected by Duchenne muscular dystrophies (DMD) and cultivated in monolayer and 3D conditions, is used to test the potential antifibrotic activity of pirfenidone (PFD). This in vitro model may be usefully also to evaluate the toxicity and efficacy of other candidate molecules for the treatment of fibrosis. The drug toxicity is evaluated using a colorimetric assay based on the conversion of tetrazolium salt (MTT) to insoluble formazan, while the effect of the drug on cell proliferation is measured with the bromodeoxyuridine incorporation assay. The efficacy of the drug is evaluated in fibroblast monolayers by quantitating synthesis and deposition of intracellular collagen with a spectrophotometric picrosirius red-based assay, and by quantitating cell migration using a "scratch" assay. The efficacy of PFD as antifibrotic drug is also evaluated in a 3D fibroblast model by measuring diameters and number of nodules.
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http://dx.doi.org/10.1007/978-1-4939-7374-3_15DOI Listing
June 2018