[Show abstract][Hide abstract] ABSTRACT: Mutations in the dynamin-2 gene (DNM2) cause autosomal dominant centronuclear myopathy (CNM) and dominant intermediate Charcot-Marie-Tooth (CMT) neuropathy type B (CMTDIB). As the relation between these DNM2-related diseases is poorly understood, we used zebrafish to investigate the effects of two different DNM2 mutations. First we identified a new alternatively spliced zebrafish dynamin-2a mRNA (dnm2a-v2) with greater similarity to human DNM2 than the deposited sequence. Then we knocked-down the zebrafish dnm2a, producing defects in muscle morphology. Finally, we expressed two mutated DNM2 mRNA by injecting zebrafish embryos with human mRNAs carrying the R522H mutation, causing CNM, or the G537C mutation, causing CMT. Defects arose especially in secondary motor neuron formation, with incorrect branching in embryos injected with CNM-mutated mRNA, and total absence of branching in those injected with CMT-mutated mRNA. Muscle morphology in embryos injected with CMT-mutated mRNA appeared less regularly organized than in those injected with CNM-mutated mRNA. Our results showing, a continuum between CNM and CMTDIB phenotypes in zebrafish, similarly to the human conditions, confirm this animal model to be a powerful tool to investigate mutations of DNM2 in vivo.
Full-text · Article · Feb 2016 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Jan 2016 · European Journal of Pediatrics
[Show abstract][Hide abstract] ABSTRACT: Aims:
Tissue fibrosis, characterized by excessive deposition of extracellular matrix proteins, is the end point of diseases affecting 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.
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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Three independent operators analyzed 56 muscle biopsies from 40 patients not treated with steroids, aged 1 to 10 years and 16 individuals treated with steroids, aged 7 to 10 years. We analyzed morphologic measures, normalized every measure for the average number of fibers observed for each year of age, and calculated intraclass correlation coefficients.
The average proportion of connective tissue in patients not treated with steroids was 16.98% from ages 1 to 6 years and 30% from ages 7 to 10 years (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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
No preview · Article · Jul 2015 · Journal of Medical Genetics
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · May 2015 · Journal of Neurology
[Show abstract][Hide abstract] ABSTRACT: 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).
To investigate mitochondrial dysfunction in a large series of muscle biopsy samples from patients with SMA.
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.
We used histochemical, biochemical, and molecular techniques to examine the muscle samples.
Respiratory chain activity and mitochondrial content.
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).
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.
[Show abstract][Hide abstract] ABSTRACT: The EuroBioBank (EBB) network (www.eurobiobank.org) is the first operating network of biobanks in Europe to provide human DNA, cell and tissue samples as a service to the scientific community conducting research on rare diseases (RDs). The EBB was established in 2001 to facilitate access to RD biospecimens and associated data; it obtained funding from the European Commission in 2002 (5th framework programme) and started operation in 2003. The set-up phase, during the EC funding period 2003-2006, established the basis for running the network; the following consolidation phase has seen the growth of the network through the joining of new partners, better network cohesion, improved coordination of activities, and the development of a quality-control system. During this phase the network participated in the EC-funded TREAT-NMD programme and was involved in planning of the European Biobanking and Biomolecular Resources Research Infrastructure. Recently, EBB became a partner of RD-Connect, an FP7 EU programme aimed at linking RD biobanks, registries, and bioinformatics data. Within RD-Connect, EBB contributes expertise, promotes high professional standards, and best practices in RD biobanking, is implementing integration with RD patient registries and 'omics' data, thus challenging the fragmentation of international cooperation on the field.European Journal of Human Genetics advance online publication, 24 December 2014; doi:10.1038/ejhg.2014.272.
Full-text · Article · Dec 2014 · European journal of human genetics: EJHG
[Show abstract][Hide abstract] ABSTRACT: Background Tubular aggregate myopathies (TAMs) are muscle disorders characterised by abnormal accumulations of densely packed single-walled or double-walled membrane tubules in muscle fibres. Recently, STIM1, encoding a major calcium sensor of the endoplasmic reticulum, was identified as a TAM gene.
Methods The present study aims to define the clinical, histological and ultrastructural phenotype of tubular aggregate myopathy and to assess the STIM1 mutation spectrum.
Results We describe six new TAM families harbouring one known and four novel STIM1 mutations. All identified mutations are heterozygous missense mutations affecting highly conserved amino acids in the calcium-binding EF-hand domains, demonstrating the presence of a mutation hot spot for TAM. We show that the mutations induce constitutive STIM1 clustering, strongly suggesting that calcium sensing and consequently calcium homoeostasis is impaired. Histological and ultrastructural analyses define a common picture with tubular aggregates labelled with Gomori trichrome and Nicotinamide adenine dinucleotide (NADH) tetrazolium reductase, substantiating their endoplasmic reticulum origin. The aggregates were observed in both fibre types and were often accompanied by nuclear internalisation and fibre size variability. The phenotypical spectrum ranged from childhood onset progressive muscle weakness and elevated creatine kinase levels to adult-onset myalgia without muscle weakness and normal CK levels.
Conclusions The present study expands the phenotypical spectrum of STIM1-related tubular aggregate myopathy. STIM1 should therefore be considered for patients with tubular aggregate myopathies involving either muscle weakness or myalgia as the first and predominant clinical sign.
Full-text · Article · Oct 2014 · Journal of Medical Genetics
[Show abstract][Hide abstract] ABSTRACT: Dystroglycan is a transmembrane glycoprotein whose interactions with the extracellular matrix (ECM) are necessary for normal muscle and brain development, and disruptions of its function lead to dystroglycanopathies, a group of congenital muscular dystrophies showing extreme genetic and clinical heterogeneity. Specific glycans bound to the extracellular portion dystroglycan, α-dystroglycan, mediate ECM interactions and most known dystroglycanopathy genes encode glycosyltransferases involved in glycan synthesis. POMK, which was found mutated in one severe dystroglycanopathy case, is instead involved in a glycan phosphorylation reaction critical for ECM binding, but little is known about the clinical presentation of POMK mutations or of the function of this protein in the muscle. Here we describe two families carrying different truncating alleles, both removing the kinase domain in POMK, with different clinical manifestations ranging from Walker Warburg Syndrome, the most severe form of dystroglycanopathy, to limb-girdle muscular dystrophy with cognitive defects. We explored POMK expression in fetal and adult human muscle and identified widespread expression primarily during fetal development in myocytes and interstitial cells suggesting a role for this protein during early muscle differentiation. Analysis loss of function in the zebrafish embryo and larva showed that pomk function is necessary for normal muscle development, leading to locomotor dysfuction in the embryo and signs of muscular dystrophy in the larva. In summary, we defined diverse clinical presentations following POMK mutations and showed that this gene is necessary for early muscle development.
Full-text · Article · Jun 2014 · Human Molecular Genetics
[Show abstract][Hide abstract] ABSTRACT: The Sgcb-null mouse, with knocked-down β-sarcoglycan, develops severe muscular dystrophy as in type 2E human limb girdle muscular dystrophy. The mdx mouse, lacking dystrophin, is the most used model for Duchenne muscular dystrophy (DMD). Unlike DMD, the mdx mouse has mild clinical features and shows little fibrosis in limb muscles. To characterize ECM protein deposition and the progression of muscle fibrosis, we evaluated protein and transcript levels of collagens I, III and VI, decorin, and TGF-β1, in quadriceps and diaphragm, at 2, 4, 8, 12, 26, and 52 weeks in Sgcb-null mice, and protein levels at 12, 26, and 52 weeks in mdx mice. In Sgcb-null mice, severe morphological disruption was present from 4 weeks in both quadriceps and diaphragm, and included conspicuous deposition of extracellular matrix components. Histopathological features of Sgcb-null mouse muscles were similar to those of age-matched mdx muscles at all ages examined, but, in the Sgcb-null mouse, the extent of connective tissue deposition was generally greater than mdx. Furthermore, in the Sgcb-null mouse, the amount of all three collagen isoforms increased steadily, while, in the mdx, they remained stable. We also found that, at 12 weeks, macrophages were significantly more numerous in mildly inflamed areas of Sgcb-null quadriceps compared to mdx quadriceps (but not in highly inflamed regions), while, in the diaphragm, macrophages did not differ significantly between the two models, in either region. Osteopontin mRNA was also significantly greater at 12 weeks in laser-dissected highly inflamed areas of the Sgcb-null quadriceps compared to the mdx quadriceps. TGF-β1 was present in areas of degeneration-regeneration, but levels were highly variable and in general did not differ significantly between the two models and controls. The roles of the various subtypes of macrophages in muscle repair and fibrosis in the two models require further study. The Sgcb-null mouse, which develops early fibrosis in limb muscles, appears more promising than the mdx mouse for probing pathogenetic mechanisms of muscle fibrosis and for developing anti-fibrotic treatments. Highlights • The Sgcb-null mouse develops severe muscular dystrophy, the mdx mouse does not.• Fibrosis developed earlier in Sgcb-null quadriceps and diaphragm than mdx.• Macrophages were commoner in mildly inflamed parts of Sgcb-null quadriceps than mdx.• The Sgcb-null model appears more useful than mdx for studying fibrotic mechanisms.• The Sgcb-null model also appears more useful for developing anti-fibrotic treatments.
No preview · Article · Apr 2014 · Cell and Tissue Research
[Show abstract][Hide abstract] ABSTRACT: The adult-onset form of Pompe disease had a wide clinical spectrum, ranging from asymptomatic patients with increased CK to muscle cramps and pain syndrome or rigid-spine syndrome. In addition clinical severity and disease progression are greatly variable. We report on a family with 3 siblings characterized by an unusual adult-onset Pompe disease including dysphagia and weakness of tongue, axial and limb-girdle muscles, in association with atypical globular inclusions in muscle fibres. Our study confirms the great clinical and histological variability of adult-onset Pompe disease and further supports the need of careful evaluation of bulbar function in patients affected by this pathology.
Full-text · Article · Oct 2013 · Acta myologica: myopathies and cardiomyopathies: official journal of the Mediterranean Society of Myology / edited by the Gaetano Conte Academy for the study of striated muscle diseases