Myotonic dystrophy (DM) is caused by a (CTG)(n) expansion in the 3'-untranslated region of DMPK gene. Mutant transcripts are retained in nuclear RNA foci, which sequester RNA binding proteins thereby ...misregulating the alternative splicing. Controversy still surrounds the pathogenesis of the DM1 muscle distress, characterized by myotonia, weakness and wasting with distal muscle atrophy. Eight primary human cell lines from adult-onset (DM1) and congenital (cDM1) patients, (CTG)(n) range 90-1800, were successfully differentiated into aneural-immature and contracting-innervated-mature myotubes. Morphological, immunohistochemical, RT-PCR and western blotting analyses of several markers of myogenesis indicated that in vitro differentiation-maturation of DM1 myotubes was comparable to age-matched controls. In all pathological muscle cells, (CTG)(n) expansions were confirmed by long PCR and RNA fluorescence in situ hybridization. Moreover, the DM1 myotubes showed the splicing alteration of insulin receptor and muscleblind-like 1 (MBNL1) genes associated with the DM1 phenotype. Considerable myotube loss and atrophy of 15-day-differentiated DM1 myotubes indicated activated catabolic pathways, as confirmed by the presence of apoptotic (caspase-3 activation, cytochrome c release, chromatin fragmentation) and autophagic (P62/LC3) markers. Z-VAD treatment significantly reduced the decrease in myonuclei number and in average width in 15-day-differentiated DM1 myotubes. We thus propose that the muscle wasting typical in DM1 is due to impairment of muscle mass maintenance-regeneration, through premature apoptotic-autophagic activation, rather than altered myogenesis.
Aims
The peculiar clinical features and the pathogenic mechanism related to calpain‐3 deficiency (impaired sarcomere remodelling) suggest that the ubiquitin‐proteasome degradation pathway may have a ...crucial role in Limb Girdle Muscular Dystrophy 2A (LGMD2A). We therefore investigated muscle atrophy and the role of the ubiquitin‐proteasome and lysosomal‐autophagic degradation pathways.
Methods
We selected 25 adult male LGMD2A patients (and seven controls), classified them using clinical severity score, analysed muscle fibre size by morphometry and protein and/or transcriptional expression levels of the most important atrophy‐ and autophagy‐related genes (MuRF1, atrogin1, LC3, p62, Bnip3).
Results
Muscle fibre size was significantly lower in LGMD2A than in controls and it was significantly correlated with patients' clinical disability score recorded at the time of biopsy, suggesting that functional and structural muscle impairment are dependent. The large majority of atrophic fibres originate from a mechanism different from regeneration, as assessed by neonatal myosin immunolabelling. As compared with controls, LGMD2A muscles have higher MuRF1 (but not atrogin1) protein and MuRF1 gene expression levels, and MuRF1 protein levels significantly correlated with both muscle fibre size and clinical disability score. LGMD2A muscles have slightly increased levels of LC3‐II and p62 proteins and a significant up‐regulation of p62 and Bnip3 gene expression.
Conclusions
In LGMD2A muscles the activation of the atrophy programme appeared to depend mainly upon induction of the ubiquitin‐proteasome system and, to a lesser extent, the autophagic‐lysosomal degradation pathway.
Aims
Pompe disease is an autosomal recessive lysosomal storage disorder resulting from deficiency of acid α‐glucosidase (GAA) enzyme. Histopathological hallmarks in skeletal muscle tissue are fibre ...vacuolization and autophagy. Since 2006, enzyme replacement therapy (ERT) is the only approved treatment with human recombinant GAA alglucosidase alfa. We designed a study to examine ERT‐related skeletal muscle changes in 18 modestly to moderately affected late onset Pompe disease (LOPD) patients along with the relationship between morphological/biochemical changes and clinical outcomes. Treatment duration was short‐to‐long term.
Methods
We examined muscle biopsies from 18 LOPD patients at both histopathological and biochemical level. All patients underwent two muscle biopsies, before and after ERT administration respectively. The study is partially retrospective because the first biopsies were taken before the study was designed, whereas the second biopsy was always performed after at least 6 months of ERT administration.
Results
After ERT, 15 out of 18 patients showed improved 6‐min walking test (6MWT; P = 0.0007) and most of them achieved respiratory stabilization. Pretreatment muscle biopsies disclosed marked histopathological variability, ranging from an almost normal pattern to a severe vacuolar myopathy. After treatment, we detected morphological improvement in 15 patients and worsening in three patients.
Post‐ERT GAA enzymatic activity was mildly increased compared with pretreatment levels in all patients. Protein levels of the mature enzyme increased in 14 of the 18 patients (mean increase = +35%; P < 0.05). Additional studies demonstrated an improved autophagic flux after ERT in some patients.
Conclusions
ERT positively modified skeletal muscle pathology as well as motor and respiratory outcomes in the majority of LOPD patients.
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disease associated with a partial deletion on chromosome 4q35. Few relevant investigations have been reported on its ...epidemiology and were essentially based on clinical diagnosis, having been performed before recognition of the molecular mutation. We report an epidemiological survey on FSHD patients, in which the diagnosis was obtained by combined clinical and molecular evaluation. The survey concerned the north‐east Italian province of Padova, an area of 871,190 inhabitants (1 January 2004). We identified 40 patients affected by FSHD based on clinical diagnosis. In 33 of them, the EcoRI fragment size in the 4q35 region ranged from 14 to 35 kb. Four other patients belonging to the same family harbored a 38‐kb fragment. In these four cases, the relationship between the borderline deletion with the mild FSHD phenotype was corroborated by additional haplotype reconstruction and segregation analysis. Interestingly, the same mild facial‐sparing clinical pattern was apparent only in one other patient with an EcoRI fragment of 32 kb, suggesting that this unusual FSHD phenotype may be due to very small 4q35 deletions. On the whole, estimating a prevalence rate of 44 × 10−6, our survey confirmed FSHD as one of the most frequent neuromuscular disorders in Western populations.
Abstract The aim of this study was to investigate the suitability of the North Star Ambulatory Assessment as a possible outcome measure in multicentric clinical trials. More specifically we wished to ...investigate the level of training needed for achieving a good interobserver reliability in a multicentric setting. The scale was specifically designed for ambulant children with Duchenne Muscular Dystrophy and includes 17 items that are relevant for this cohort. Thirteen Italian centers participated in the study. In the first phase of the study we provided two training videos and an example of the scale performed on a child. After the first session of training, all the 13 examiners were asked to send a video with an assessment performed in their centre and to score all the videos collected. There were no difficulties in performing the items and in obtaining adequate videos with a hand held camera but the results showed a poor interobserver reliability (<.5). After a second training session with review and discussion of the videos previously scored, the same examiners were asked to score three new videos. The results of this session had an excellent interobserver reliability (.995). The level of agreement was maintained even when the same videos were rescored after a month, showing a significant intra-observer reliability (.95). Our results suggest that the NSAA is a test that can be easily performed, completed in 10 min and can be used in a multicentric setting, providing that adequate training is administered.
A multicentre observational study was aimed to assess the prevalence of late-onset Pompe disease (LOPD) in a large high-risk population, using the dried blood spot (DBS) as a main screening tool.
17 ...Italian neuromuscular centres were involved in the late-onset Pompe early diagnosis (LOPED) study. Inclusion criteria were: (1) age ≥5 years, (2) persistent hyperCKaemia and (3) muscle weakness at upper and/or lower limbs (limb-girdle muscle weakness, LGMW). Acid α-glucosidase (GAA) activity was measured separately on DBS by fluorometric as well as tandem mass spectrometry methods. A DBS retest was performed in patients resulted positive at first assay. For the final diagnosis, GAA deficiency was confirmed by a biochemical assay in skeletal muscle, whereas genotype was assessed by GAA molecular analysis.
In a 14-month period, we studied 1051 cases: 30 positive samples (2.9%) were detected by first DBS screening, whereas, after retesting, 21 samples were still positive. Biochemical and molecular genetic studies finally confirmed LOPD diagnosis in 17 cases (1.6%). The median time from the onset of symptoms/signs to diagnosis was 5 years. Among those patients, 35% showed presymptomatic hyperCKaemia and 59% showed hyperCKaemia+LGMW, whereas 6% manifested with LGMW.
LOPED study suggests that GAA activity should be accurately screened by DBS in all patients referring for isolated hyperCKaemia and/or LGMW. A timely diagnosis was performed in five patients with presymptomatic hyperCKaemia, but two had already manifested with relevant changes on muscle morphology and MRI. Consequently, enzyme replacement therapy was started in 14/17 patients, including the 2 patients still clinically presymptomatic but with a laboratory evidence of disease progression.
The objective of this study was to describe a large Italian cohort of patients with late-onset glycogen storage disease type 2 (GSDII) at various stages of disease progression and to evaluate the ...clinical effectiveness of alglucosidase alpha enzyme replacement therapy (ERT). Previous studies showed in late-onset patients ERT efficacy against placebo and variable response in uncontrolled studies. Seventy-four juvenile or adult GSDII patients were treated with ERT in a multicenter open label, non-randomized study, from 12 months up to 54 months. Recombinant human alpha glucosidase (rh-GAA) was injected by intravenous route at 20 mg/kg every second week. Patients were divided into three groups according to ERT duration: Group A received treatment for 12–23 months (
n
= 16), Group B for 24–35 months (
n
= 14), and Group C for more than 36 months (
n
= 44). Clinical assessment included a 6-min walk test (6MWT), forced vital capacity (FVC), the Walton and Gardner-Medwin score, the number of hours of ventilation, body mass index, echocardiography and blood creatine kinase (CK). Included in our cohort were 33 males and 41 females (M:F = 0.8:1), with a mean age at first symptoms of 28.3 years (range 2–55 years) and a mean age of 43 years at study entry (range 7–72 years). Seven wheelchair bound patients, as well as 27 patients requiring ventilation support, were included. After treatment we could observe an increase in distance walked on the 6MWT in the large majority of patients (48/58; 83%), with an overall mean increase of 63 m (from 320 ± 161 to 383 ± 178 m). After treatment in the majority of patients FVC was improved or unchanged (45/69; 65%). In ventilated patients we observed an improvement in average number of hours off the ventilator (from 15.6 to 12.1 h). Six patients stopped mechanical ventilation and two others started it. The effect of therapy was not related to ERT duration. Nine of 64 patients (13%) that underwent to echocardiography showed a variable degree of cardiac hypertrophy (left ventriculum or septum), and a positive effect was observed after 36 months of ERT in one adult case. Discontinuation of treatment occurred in four patients: one drop-off case, one patient died for a sepsis after 34 months of treatment and two patients stopped ERT for worsening of general clinical condition. Mild adverse effects were observed in four cases (5%). This study represents the largest cohort of late-onset GSDII patients treated with ERT, and confirm a positive effect of treatment. These results, obtained in a large case series on therapy, indicate a favourable effect of ERT therapy, even in more advanced stage of the disease.
Muscle pathology in dysferlin deficiency Fanin, M.; Angelini, C.
Neuropathology and applied neurobiology,
December 2002, Letnik:
28, Številka:
6
Journal Article
Recenzirano
Dysferlin deficiency is being increasingly recognized in limb‐girdle dystrophy and distal myopathy but its role in the development of muscle pathology is still poorly understood. For this purpose, 26 ...muscle biopsies from 25 dysferlinopathy patients were analysed by routine histochemistry and by immunohistochemistry with eight different antibodies, and scored for inflammatory response and type of cell infiltrate, fibre degeneration and regeneration, fibre type composition and severity of histopathological changes. In cases with an advanced‐stage dystrophic pattern we observed type 1 fibre predominance exceeding 80%, suggesting a selective loss of type 2 fibres or a conversion process.
The extent of muscle fibre regeneration and degeneration in dysferlinopathy was intermediate between sarcoglycanopathy and Duchenne dystrophy or myositis, suggesting a rather aggressive course of the disease. An increased inflammatory response was observed in the majority of our patients (16/26), who also showed an active dystrophic pattern. Type and localization of cellular infiltrates suggest that inflammatory reaction is secondary to necrosis. Major histocompatibility complex (MHC) class I molecules were overexpressed in dysferlinopathy, mainly in association with fibre phagocytosis and regeneration; their occasional expression in non‐necrotic fibres might represent a marker of ongoing necrosis. Muscle inflammation might be triggered by the structurally altered membrane consequent to dysferlin defect.
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