[Show abstract][Hide abstract] ABSTRACT: 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.
[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.
Human Molecular Genetics 06/2014; · 7.69 Impact Factor
[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.
Cell and Tissue Research 04/2014; · 3.68 Impact Factor
[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.
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 10/2013; 32(2):85-90.
[Show abstract][Hide abstract] ABSTRACT: Mutations in the PTRF gene, coding for cavin-1, cause congenital generalized lipodystrophy type 4 (CGL4) associated with myopathy. In CGL4, symptoms are variable comprising, in addition to myopathy, smooth and skeletal muscle hypertrophy, cardiac arrhythmias, and skeletal abnormalities. Secondary features are atlantoaxial instability, acanthosis nigricans, hepatomegaly, umbilical prominence and metabolic abnormalities related to insulin resistance, such as diabetes mellitus, hyperlipidemia and hepatic steatosis.
We describe a 3 year-old child of Moroccan origin with mild muscle phenotype, mainly characterized by mounding, muscle pain, hyperCKemia and mild caveolin 3 reduction on muscle biopsy. No CAV3 gene mutation was detected; instead we found a novel mutation, a homozygous single base pair deletion, in the PTRF gene. Only after detection of this mutation a mild generalized loss of subcutaneous fat, at first underestimated, was noticed and the diagnosis of lipodystrophy inferred.
The PTRF gene should be investigated in patients with hyperCKemia, mild myopathy associated with spontaneous or percussion-induced muscle contractions like rippling or mounding, and no CAV3 mutation. The analysis should be performed even if cardiac or metabolic alterations are absent, particularly in young patients in whom lipodystrophy may be difficult to ascertain.
BMC Medical Genetics 09/2013; 14(1):89. · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Yunis-Varón syndrome (YVS) is an autosomal-recessive disorder with cleidocranial dysplasia, digital anomalies, and severe neurological involvement. Enlarged vacuoles are found in neurons, muscle, and cartilage. By whole-exome sequencing, we identified frameshift and missense mutations of FIG4 in affected individuals from three unrelated families. FIG4 encodes a phosphoinositide phosphatase required for regulation of PI(3,5)P2 levels, and thus endosomal trafficking and autophagy. In a functional assay, both missense substitutions failed to correct the vacuolar phenotype of Fig4-null mouse fibroblasts. Homozygous Fig4-null mice exhibit features of YVS, including neurodegeneration and enlarged vacuoles in neurons. We demonstrate that Fig4-null mice also have small skeletons with reduced trabecular bone volume and cortical thickness and that cultured osteoblasts accumulate large vacuoles. Our findings demonstrate that homozygosity or compound heterozygosity for null mutations of FIG4 is responsible for YVS, the most severe known human phenotype caused by defective phosphoinositide metabolism. In contrast, in Charcot-Marie-Tooth disease type 4J (also caused by FIG4 mutations), one of the FIG4 alleles is hypomorphic and disease is limited to the peripheral nervous system. This genotype-phenotype correlation demonstrates that absence of FIG4 activity leads to central nervous system dysfunction and extensive skeletal anomalies. Our results describe a role for PI(3,5)P2 signaling in skeletal development and maintenance.
The American Journal of Human Genetics 04/2013; · 11.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Severe muscle fibrosis is the endpoint of many chronic myopathies. Identification of factors that regulate fibrosis is important for understanding its pathogenesis and for developing anti-fibrotic treatments that prevent muscle destruction. We have developed an in vitro model for screening potential anti-fibrotic agents. The model consists of three-dimensional clusters (nodules) of fibroblasts derived from Duchenne muscular dystrophy (DMD) muscle. The primary fibroblasts spontaneously and quickly form nodules resembling fibrotic foci (cells plus extracellular matrix) when grown on a solid substrate. We tested the anti-fibrotic action of suramin, decorin, and spironolactone (all with established anti-fibrotic activity) on the model. All three agents significantly reduced nodule number, and spironolactone and suramin significantly reduced nodule diameter. Nodule secretion of soluble collagen was also significantly reduced by decorin and spironolactone treatment, whereas suramin had no significant effect. Collagen I and fibronectin protein expression was significantly reduced in the culture medium of control and DMD fibroblasts by spironolactone treatment, but not by decorin and suramin treatment. Finally, in DMD fibroblast monolayers, collagen deposition was significantly reduced by all three agents. Spironolactone significantly reduced collagen I and fibronectin transcript levels, whereas decorin reduced only fibronectin. Our in vitro model of fibrogenesis has thus revealed differing anti-fibrotic effects in the three anti-fibrotic agents tested. It therefore appears as a useful and sensitive system for the testing of anti-fibrotic drugs and could be adapted for the high-throughput screening of new anti-fibrotic molecules.
Cell and Tissue Research 04/2013; · 3.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutations in dynamin 2 (DNM2) gene cause autosomal dominant centronuclear myopathy and occur in around 50% of patients with centronuclear myopathy. We report clinical, morphological, muscle imaging and genetic data of 10 unrelated Italian patients with centronuclear myopathy related to DNM2 mutations. Our results confirm the clinical heterogeneity of this disease, underlining some peculiar clinical features, such as severe pulmonary impairment and jaw contracture that should be considered in the clinical follow-up of these patients. Muscle MRI showed a distinct pattern of involvement, with predominant involvement of soleus and tibialis anterior in the lower leg muscles, followed by hamstring muscles and adductor magnus at thigh level and gluteus maximus. The detection of three novel DNM2 mutations and the first case of somatic mosaicism further expand the genetic spectrum of the disease.
[Show abstract][Hide abstract] ABSTRACT: Whole-exome sequencing (WES), which analyzes the coding sequence of most annotated genes in the human genome, is an ideal approach to studying fully penetrant autosomal-recessive diseases, and it has been very powerful in identifying disease-causing mutations even when enrollment of affected individuals is limited by reduced survival. In this study, we combined WES with homozygosity analysis of consanguineous pedigrees, which are informative even when a single affected individual is available, to identify genetic mutations responsible for Walker-Warburg syndrome (WWS), a genetically heterogeneous autosomal-recessive disorder that severely affects the development of the brain, eyes, and muscle. Mutations in seven genes are known to cause WWS and explain 50%-60% of cases, but multiple additional genes are expected to be mutated because unexplained cases show suggestive linkage to diverse loci. Using WES in consanguineous WWS-affected families, we found multiple deleterious mutations in GTDC2 (also known as AGO61). GTDC2's predicted role as an uncharacterized glycosyltransferase is consistent with the function of other genes that are known to be mutated in WWS and that are involved in the glycosylation of the transmembrane receptor dystroglycan. Therefore, to explore the role of GTDC2 loss of function during development, we used morpholino-mediated knockdown of its zebrafish ortholog, gtdc2. We found that gtdc2 knockdown in zebrafish replicates all WWS features (hydrocephalus, ocular defects, and muscular dystrophy), strongly suggesting that GTDC2 mutations cause WWS.
The American Journal of Human Genetics 09/2012; 91(3):541-7. · 11.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Episodic ataxia type 1 (EA1) is an autosomal dominant neurological disorder characterized by myokymia and attacks of ataxic gait often precipitated by stress. Several genetic mutations have been identified in the Shaker-like K(+) channel Kv1.1 (KCNA1) of EA1 individuals, including V408A, which result in remarkable channel dysfunction. By inserting the heterozygous V408A, mutation in one Kv1.1 allele, a mouse model of EA1 has been generated (Kv1.1(V408A/+)). Here, we investigated the neuromuscular transmission of Kv1.1(V408A/+) ataxic mice and their susceptibility to physiologically relevant stressors. By using in vivo preparations of lateral gastrocnemius (LG) nerve-muscle from Kv1.1(+/+) and Kv1.1(V408A/+) mice, we show that the mutant animals exhibit spontaneous myokymic discharges consisting of repeated singlets, duplets or multiplets, despite motor nerve axotomy. Two-photon laser scanning microscopy from the motor nerve, ex vivo, revealed spontaneous Ca(2+) signals that occurred abnormally only in preparations dissected from Kv1.1(V408A/+) mice. Spontaneous bursting activity, as well as that evoked by sciatic nerve stimulation, was exacerbated by muscle fatigue, ischemia and low temperatures. These stressors also increased the amplitude of compound muscle action potential. Such abnormal neuromuscular transmission did not alter fiber type composition, neuromuscular junction and vascularization of LG muscle, analyzed by light and electron microscopy. Taken together these findings provide direct evidence that identifies the motor nerve as an important generator of myokymic activity, that dysfunction of Kv1.1 channels alters Ca(2+) homeostasis in motor axons, and also strongly suggest that muscle fatigue contributes more than PNS fatigue to exacerbate the myokymia/neuromyotonia phenotype. More broadly, this study points out that juxtaparanodal K(+) channels composed of Kv1.1 subunits exert an important role in dampening the excitability of motor nerve axons during fatigue or ischemic insult.
Neurobiology of Disease 05/2012; 47(3):310-21. · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The neurotrophin receptor p75NTR is a marker for human differentiation-prone muscle satellite cells and regulates myogenesis. Here, we wondered whether inflammation could modify p75NTR expression on muscle precursor cells in vitro and in vivo. In vitro experiments on human primary skeletal myoblasts demonstrated that exposure to IFN-γ or IL-1α decreased transcript and protein levels of p75NTR. Furthermore, histological investigations showed a reduction in the pool of p75NTR expressing satellite cells in inflammatory myopathy biopsies. These data suggest a link between muscle inflammation and reduction of p75NTR expression on precursor cells.
Journal of neuroimmunology 12/2011; 243(1-2):100-2. · 2.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Congenital muscular dystrophy type 1A is caused by mutations in the LAMA2 gene that encodes the laminin α2 chain, a component of the skeletal muscle extracellular matrix protein laminin-211. The clinical spectrum of the disease is more heterogeneous than previously thought, particularly in terms of motor achievement and disease progression. We investigated clinical findings and performed molecular genetic analysis in 3 families from Saudi Arabia and 1 from Sudan in whom congenital muscular dystrophy 1A was suspected based on homozygosity mapping and laminin α2 chain deficiency.
We investigated 9 affected individuals from 1 Sudanese and 3 Saudi families in whom MDC1A was suggested by clinical, neuroimaging and/or pathological findings and by homozygosity mapping at the LAMA2 locus. Morphological and immunohistochemical analysis were performed in 3 patients from the 3 Saudi families. SSCP analysis, DNA sequencing and microsatellite analysis were carried out in the 4 index cases.
A previously described mutation in the LAMA2 gene, a homozygous T > C substitution at position +2 of the consensus donor splice site of exon 26, was found in the 4 index patients. Clinical evaluation of 9 patients from the 4 families revealed variable disease severity particularly as regards motor achievement and disease progression. Microsatellite analysis showed an identical mutation-associated haplotype in the 4 index cases indicating a founder effect of the mutation in all 4 families.
Our data provide further evidence that the clinical spectrum of MDC1A due to a single mutation is heterogeneous, particularly in terms of motor achievement and disease progression, making it difficult to give a reliable prognosis even in patients with identical LAMA2-associated haplotype. The c.3924 + 2 T > C mutation to date has been found only in patients originating from the Middle East or Sudan; therefore laminin 2 chain deficiency in patients from those regions should initially prompt a search for this mutation.
[Show abstract][Hide abstract] ABSTRACT: To increase our understanding of profibrotic mechanisms in dystrophic muscle.
Extracellular matrix, fibrosis-related molecules and histopathology were assessed in skeletal muscle of patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and congenital muscular dystrophy type 1A (MDC1A).Osteopontin expression was much higher in DMD and MDC1A than in BMD and control muscle. Osteopontin was expressed in mononuclear cell infiltrates, on some muscle fibre surfaces, in regenerating fibres, and in calcified fibres. In all pathological muscles, matrix metalloproteinase (MMP)-1 was increased around groups of fibres that were also characterized by absence of collagen 1. The amounts of MMP-2, MMP-9 and tissue inhibitor of MMP -1 transcripts were also increased, whereas their proteins were variably expressed in muscle fibres (surface or cytoplasm) and at foci of necrosis and regeneration. Inflammatory cells, fibroblasts and myofibroblasts were more numerous in DMD and MDC1A than in BMD muscle.
Several fibrosis-related factors are greatly altered in severely dystrophic skeletal muscle. Osteopontin was the most conspicuously upregulated, both as transcript and as protein, in muscle fibres and infiltrating cells, indicating an intimate involvement in fibrosis, and also in inflammation and muscle regeneration, although its precise roles in these processes remain to be elucidated.
[Show abstract][Hide abstract] ABSTRACT: Recent studies propose the neurotrophin receptor p75NTR as a marker for muscle satellite cells and a key regulator of regenerative processes after injury. Here, we investigated the contribution of cellular compartments other than satellite cells and regenerating myofibres to p75NTR signal in diseased skeletal muscle.
We checked regulation of p75NTR expression in muscle biopsies from patients with inflammatory myopathies (polymyositis, dermatomyositis and inclusion body myositis), or Becker muscular dystrophy, and in nonmyopathic tissues. Quantitative PCR, immunohistochemistry, immunofluorescence or electron microscopy were used. RNA interference approaches were applied to myotubes to explore p75NTR function.
We found p75NTR transcript and protein upregulation in all inflammatory myopathies but not in dystrophic muscle, suggesting a role for inflammatory mediators in induction of p75NTR expression. In inflamed muscle p75NTR was localized on distinct cell types, including immune cells and mature myofibres. In vitro assays on human myotubes confirmed that inflammatory factors such as IL-1 could induce p75NTR. Finally, RNA interference experiments in differentiated cells showed that, in the absence of p75NTR, myotubes were more susceptible to apoptosis when exposed to inflammatory stimuli.
Our observations that p75NTR is upregulated on skeletal myofibres in inflammatory myopathies in vivo and promotes resistance to inflammatory mediators in vitro suggest that neurotrophin signalling through p75NTR may mediate a tissue-protective response to inflammation in skeletal myofibres.
Neuropathology and Applied Neurobiology 08/2011; 38(4):367-78. · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sporadic inclusion-body myositis (s-IBM) is a chronic progressive inflammatory myopathy leading to severe disability. It has been suggested that statins may benefit s-IBM patients based on their pleiotropic effects on autoimmunity and possible adverse influence of increased cholesterol on muscle pathological changes. We carried out a pilot, open-label trial to evaluate safety and tolerability of oral simvastatin in s-IBM patients. Fourteen patients were treated with 40 mg of simvastatin over 12 months. Primary outcome measures included the assessment tools proposed by International Myositis Outcome Assessment Collaborative Study group and the IBM-Functional Rating Scale. As additional data, we report the results obtained from muscle MRI, biopsy and oropharyngeal scintigraphy. Ten patients completed the trial and the treatment appeared safe and well tolerated. None of the patients showed a significant clinical improvement. Outcome measures used in this study proved to be valuable tools for global assessment of s-IBM patients. At present, we cannot recommend simvastatin as a treatment for s-IBM though our data may warrant a placebo-controlled study.
[Show abstract][Hide abstract] ABSTRACT: Satellite cells are resident stem cells of adult skeletal muscle that have roles in tissue repair. Although several efforts have led to the functional characterization of distinct myogenic populations in animal models, the translation of these findings to humans has been limited. Here, we analyzed the expression and function of the neurotrophin receptor p75NTR in human skeletal muscle precursor cells. We combined histological investigations of muscle biopsies with molecular and cellular analyses of primary muscle precursor cells. p75NTR is expressed by most satellite cells in vivo and is a marker for regenerating fibers in inflamed and dystrophic muscle. p75NTR mRNA and protein are also detectable in primary myoblasts, and these levels increase transiently when cell differentiation is triggered. Transcriptome analyses of p75NTR high versus p75NTR low muscle cells showed that p75NTR is the prototype marker for a precursor cell population that has a broad transcriptional repertoire associated with muscle development and maturation. Several in vitro experiments, including receptor blockade and gene silencing in myoblasts, proved that p75NTR specifically regulates myogenesis and dystrophin expression. Taken together, the results indicate that p75NTR is a novel marker of human differentiation-prone muscle precursor cells that is involved in myogenesis in vivo and in vitro.
Journal of Neuropathology and Experimental Neurology 02/2011; 70(2):133-42. · 4.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background / Purpose:
We investigated two male infant patients born from monozygotic twin sisters and unrelated fathers, suggesting an X-linked trait.
Fibroblasts from both patients showed reduction of cIII and cIV but not of cI. We SNP-screened the X chromosome, and found a disease-segregating haploidentical region containing a mutation in the Apoptosis Inducing Factor 1 (AIF1) gene that eliminates amino acid R201. AIF1 is bound to the inner mitochondrial membrane (AIFmit) as an NADH oxidase flavoprotein of unknown function; under apoptogenic stimuli, a soluble form (AIFsol) is released by proteolytic cleavage of AIFmit, and migrates to the nucleus, where it induces parthanatos, i.e. caspase-independent fragmentation of DNA.We found that in vitro the R201del mutation increases the DNA binding affinity of AIFsol and decreases the stability of both forms. Staurosporine-induced parthanatos, and galactose-induced mitochondrial fragmentation were markedly increased, whereas re-expression of AIF1wt induced recovery of cIII and cIV activities, in AIFR201del fibroblast cells.This is the first report of an AIF1 mutation in a human mitochondrial encephalomyopathy. Unlike the Harlequin mouse, a spontaneous AIF1 knockdown model, which displays partial cI deficiency, the human disease showed multiple respiratory enzyme defects associated with reduced mtDNA amount in skeletal muscle.
[Show abstract][Hide abstract] ABSTRACT: We report on a patient with muscle pain not associated with muscle weakness. Microscopic examination of the muscle biopsy revealed rod-like cytoplasmic bodies in many fast fibres. By electron microscopy these had a crystalloid structure identical to the hexagonally cross-linked caveolin 3-positive tubular arrays, previously described in patients with similarly benign myopathy. We found that these inclusions were positive for calsequestrin and for the calsequestrin-binding protein junctin, as well as for caveolin 3. However, the genes coding for these proteins were not mutated. For diagnostic purposes calsequestrin and caveolin 3 positivity should be checked when rods are encountered in muscle biopsy for mild myopathy.