C. Angelini

Ultrastructural changes in dysferlinopathy support defective membrane repair mechanism

Background: The dysferlin gene has recently been shown to be involved in limb girdle muscular dystrophy type 2B and its allelic disease, Miyoshi myopathy, both of which are characterised by an active muscle degeneration and regeneration process. Dysferlin is known to play an essential role in skeletal muscle fibre repair, but the process underlying the pathogenetic mechanism of dysferlinopathy is not completely understood. Aims: To define both specific alterations of muscle fibres and a possible sequential mechanism of myopathy development. Methods: A histological, immunohistochemical, and ultrastructural analysis of 10 muscle biopsies from patients with molecularly diagnosed dysferlinopathy. Results: An inflammatory response was seen in most of the muscle biopsies. The immunohistochemical pattern demonstrated active regeneration and inflammation. Non-necrotic fibres showed alterations at different submicroscopic levels, namely: the sarcolemma and basal lamina, subsarcolemmal region, and sarcoplasmic compartment. In the subsarcolemmal region there were prominent aggregations of small vesicles, probably derived from the Golgi apparatus, which consisted of empty, swollen cisternae. In the sarcolemma there were many gaps and microvilli-like projections, whereas the basal lamina was multilayered. Conclusions: The histopathological, immunohistochemical, and ultrastructural data show that dysferlinopathy is characterised by a very active inflammatory/degenerative process, possibly associated with an inefficient repair and regenerative system. The presence of many crowded vesicles just beneath the sarcolemma provides submicroscopical proof of a defective resealing mechanism, which fails to repair the sarcolemma.

LOPED study: looking for an early diagnosis in a late-onset Pompe disease high-risk population

Objective 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. Design/methods 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. Results 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. Conclusions 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 clinical course of calpainopathy (LGMD2A) and dysferlinopathy (LGMD2B).

Abstract Objective: Autosomal recessive limb girdle muscular dystrophies (LGMD type 2) are a clinically and genetically heterogeneous group of disorders, characterized by progressive involvement and wasting of limb girdle muscles. In order to describe the peculiar clinical features of LGMD2A (calpainopathy) and LGMD2B (dysferlinopathy), the most frequent forms of LGMD in European countries, we analysed and compared the phenotype and the clinical course in two relatively large groups of these patients. Methods: We selected 22 patients with a molecular diagnosis of LGMD2A and 21 patients with LGMD2B and reported their clinical data collected from both clinical history and during periodical neuromuscular examinations: age and distribution of muscle involvement at onset, clinical functional score by the use of ten-point modified scale of Gardner–Medwin and Walton at onset and at last clinical examination, and the rate of disease progression. Results: LGMD2A group included patients with different ages at onset (early-onset or late-onset), different phenotypes (upper girdle in Erb LGMD or lower girdle in Leyden–Moebius LGMD) and different disease progressions (rapid or slow course). LGMD2B patients differed for pattern of muscle involvement at onset (distal in Miyoshi dystrophy or proximal in Leyden–Moebius LGMD) but they had a rather homogeneous age at onset (in the second/third decade) and rate of disease progression. Discussion: Our data show that besides the clinical differences within each group of patients, the two forms of LGMD present distinctive clinical features. The various phenotypes and courses can be attributed to specific pathogenetic mechanisms and might suggest differential therapeutic strategies.

European consensus for starting and stopping enzyme replacement therapy in adult patients with Pompe disease: a 10-year experience.

Pompe disease is a rare inheritable muscle disorder for which enzyme replacement therapy (ERT) has been available since 2006. Uniform criteria for starting and stopping ERT in adult patients were developed and reported here.

Muscle atrophy in Limb Girdle Muscular Dystrophy 2A: A morphometric and molecular study

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.

T.P.18

Glycogenosis type II (OMIM 23230) is an autosomal recessive lysosomal storage disorder resulting from a deficiency in the glucosidase alpha acid (GAA) enzyme. The disease is characterized by progressive accumulation of lysosomal glycogen in various tissues, primarily heart and skeletal muscle, and it is clinically classified into three forms: infantile, juvenile, and late-onset. The histopathological hallmarks in muscle tissue are fiber vacuolization and autophagy. Recombinant human GAA is the only approved enzyme replacement therapy (ERT) available for disease treatment. It is effective in most infantile patients, whereas the improvement is quite variable in adults. We analyzed muscle biopsies from 14 late-onset patients at molecular, biochemical, and histopathological level before and after ERT. We evaluated the following morphological parameters: CSA, number of vacuolated fibers, degree of glycogen accumulation, percentage of vacuolization in type I and II fibers. Pre-treatment muscle biopsies showed marked histopathological variability ranging from almost normal morphology to severe vacuolar myopathy. Post-treatment muscle biopsies morphologically improved in seven patients, worsened in two patients and were unchanged in all the other subjects. Immunohistochemical analysis of the autophagic/lysosomal markers EEA1 (early endosome antigen 1), LC3 (microtubule-associated protein 1 light chain 3), and LAMP2 (lysosome associated membrane protein 2) showed a variable binding in both the first and the second biopsies. GAA enzymatic activity, tested by a fluorimetric assay in both lymphocytes and muscle tissue from 5 patients after ERT, was mildly increased in skeletal muscle compared with pre-treatment levels. Also, GAA expression assessed by immunoblotting slightly increased in a few patients. All patients clinically improved or remained stable after ERT.

G.O.7

Limb-girdle muscular dystrophies (LGMDs) are a highly heterogeneous group of muscle disorders affecting the pelvic and the shoulder girdle musculature. Until now, over thirty disease genes are known. Even if the age of onset and other clinical features could address the diagnosis towards the specific LGMD form, the phenotypic heterogeneity within each form hampers an easy and rapid molecular identification of causative mutations. We developed a unique and exhaustive platform, based on Next Generation Sequencing on Illumina HiSeq, to analyze 89 muscular disease genes at very high coverage. This regains 20–30% of missing sequences when compared with whole exome sequencing of the same DNA samples. Using this protocol, we have sequenced 312 patients with a LGMD phenotype. Most patients have been previously studied using a gene-by-gene approach without success. Thus, these cases are somewhat enriched for unknown and elusive mutations. Nevertheless, in about 20% of patients we found typical causative mutations in predicted genes, missed by Sanger sequencing or generally poorly studied. An additional 30% of patients carry other novel pathogenic variations. As easily predictable, the diagnostic rate sensibly increases in naive samples not previously screened. Our extensive procedure allowed us to obtain a full comprehensive view of all sequence variants in these samples and to refine the genotype-phenotype correlation. More interestingly, we found in each patient at least five rare mutations (