A fourth case of POMT2-related limb girdle muscle dystrophy with mild reduction of α-dystroglycan glycosylation.

BACKGROUND: POMT2 mutations have been identified in Walker-Warburg syndrome or muscle-eye-brain-like, but rarely in limb girdle muscular dystrophy (LGMD). RESULTS: Two POMT2 mutations, one null and one missense, were found in a patient with LGMD and mild mental impairment, no brain or ocular involvement, minor histopathological features, and slight reduction of α-dystroglycan (α-DG) glycosylation and α-DG laminin binding. CONCLUSIONS: Our case, the fourth LGMD POMT2-mutated reported to date, provides further evidence of correlation between level of α-DG glycosylation and phenotype severity.

The neurotrophin receptor p75NTR is induced on mature myofibres in inflammatory myopathies and promotes myotube survival to inflammatory stress.

AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Altered extracellular matrix transcript expression and protein modulation in primary Duchenne muscular dystrophy myotubes.

Extent of muscle fibrosis contributes to disease severity in muscular dystrophies. To investigate whether extracellular matrix (ECM) components contribute to the severe fibrosis observed in Duchenne muscular dystrophy (DMD) skeletal muscle, we quantitated several ECM components (transcripts and proteins) in primary DMD and control myotube cultures. We evaluated the fibrogenic transforming growth factor- beta1 (TGF-beta1); the small pleiotropic proteoglycan decorin, involved in collagen fibrillogenesis and TGF-beta1 modulation; metalloproteinases MMP-2 and MMP-9; tissue inhibitors of metalloproteinase (TIMP) 1, 2 and 3; collagens I and VI; and the tissue factor myostatin that inhibits muscle growth. Dystrophic myotube cultures had significantly lower levels of decorin mRNA, as also observed in DMD muscle biopsies, and significantly higher levels of TGF-beta1, myostatin, and collagens I and VI. MMP-2, TIMP-1 and TIMP-2 transcript levels were also significantly increased in DMD, but MMP-9 and TIMP-3 transcripts were unchanged. By zymography, MMP-2 activity was significantly higher in DMD than control. Protein levels were similar in DMD and controls but myostatin protein was significantly increased in DMD. We have found that transcript expression and protein modulation of several ECM components is altered in DMD muscle cells in vitro, indicating that these cells contribute fundamentally to the pathological process, since the inflammation and degeneration characterizing DMD muscle in vivo are presumably absent in culture. Our findings that myostatin-potent inhibitor of satellite cell activation and muscle renewal--is increased, and that decorin-binder and downregulator of TGFbeta1 and myostatin--is decreased, may have implications for DMD therapy to reduce muscle fibrosis.

Possible function of two insect phospholipid-hydroperoxide glutathione peroxidases.

We compared the functional properties of two insect members of the phospholipid hydroperoxide glutathione peroxidases (PHGPx) family, VLP1, a major component of virus-like particles from the hymenopteran endoparasitoid Venturia canescens and its closest Drosophila relative, one of the putative PHGPx-proteins predicted from the Berkeley Drosophila genome sequence project. Recombinant Drosophila PHGPx shows enzymatic activity towards a number of PHGPx substrates, while the recombinant PHGPx-like domain of VLP1 lacks a functionally relevant cysteine and enzyme activity. A possible function of a non-enzymatic extracellular PHGPx-like protein is discussed.

Development of muscle pathology in canine X-linked muscular dystrophy. II. Quantitative characterization of histopathological progression during postnatal skeletal muscle development.

We have characterized the time course of muscle pathology development during the postnatal maturation of quadriceps and tibialis anterior muscle in dystrophic golden retriever dogs. We determined the percentages of degenerating, regenerating, calcium-positive, hypercontracted, albumin-positive, and C3 complement fraction-positive muscle fibers and the extent of connective tissue proliferation in animals from neonate to adult. Necrotic fibers increased from days 2 to 30, decreased at 60 days (to 0.8%) and increased in older animals to a stable level of around 2%. Hypercontracted fibers peaked at 15 days (19.1%) and declined to 3.7% in adults. Regenerating fibers were numerous at 15 and 30 days (10%), declined at 60 days to 4.7% and declined further in adults. Calcium- and albumin-positive fibers peaked at 30 days (6.5% and 13.8%, respectively) and then declined to around 3% and 5%, respectively, in older dogs. In dystrophic dogs, the extent of fibrosis was significantly greater on 15 days than in controls, but did not then increase with age. In carriers, calcium- and albumin-positive fibers always expressed dystrophin abnormally. Muscle damage occurs before completion of muscle maturation in dystrophic dogs. While necrosis and hypercontraction remain stable in adults, fiber regeneration declines to very low levels. In contrast to Duchenne muscular dystrophy, muscle fibrosis in the muscle studied does not increase with age.

Unusual expression of emerin in a patient with X-linked Emery-Dreifuss muscular dystrophy.

We report on a patient with the typical clinical findings of Emery-Dreifuss muscular dystrophy due to a mutation in the emerin gene that should have produced a higher molecular weight protein. Immunohistochemical analysis showed emerin localized only in the cytoplasm of muscle fibres and lymphoblastoid cells. The emerin molecule contained the nucleoplasmic domain and the transmembrane domain responsible for nuclear membrane targeting, so its incorrect localization and lack of function could be due to abnormal folding resulting in rapid degradation or inability to bind other nuclear proteins.

Partial laminin alpha2 chain deficiency in a patient with myopathy resembling inclusion body myositis.

It is becoming evident that clinical phenotypes associated with partial laminin alpha2 chain deficiency are variable. We recently observed a 29-year-old man with leukoencephalopathy and vacuolar myopathy resembling inclusion body myositis. Laminin alpha2 immunohistochemical analysis showed reduction of the protein on muscle fiber surfaces. Molecular analysis revealed two novel compound heterozygous mutations in the LAMA2 gene. This is the first report linking a mutation in the LaMA2 gene with leukoencephalopathy and inclusion body-like myositis.

Dystrophin-associated protein abnormalities in dystrophin-deficient muscle fibers from symptomatic and asymptomatic Duchenne/Becker muscular dystrophy carriers.

The absence of dystrophin in muscle fibers is associated with a major reduction in dystrophin-associated proteins (DAPs) and disruption of the linkage between the subsarcolemmal cytoskeleton and the extracellular matrix. We investigated the expression of the DAPs beta-dystroglycan, alpha-sarcoglycan, gamma-sarcoglycan and syntrophin as well as utrophin in the muscles of 13 Duchenne muscular dystrophy (DMD) carriers (with variable percentages of dystrophin-deficient fibers and with a range of clinical symptoms), 2 Becker muscular dystrophy (BMD) carriers (expressing a highly truncated protein in some fibers), 2 girls with a DMD-like phenotype, and 11 BMD carriers with almost normal dystrophin expression (reduced or patchy distribution in a few fibers only and rare dystrophin-deficient fibers). DAPs were highly reduced in all fibers lacking dystrophin in the DMD carriers, but were almost normal in the dystrophin-deficient fibers of the 2 BMD carriers with highly truncated dystrophin. In the 11 BMD carriers with nearly normal dystrophin, the few fibers with reduced or patchy dystrophin immunostaining also showed reduced DAP expression in correlation with dystrophin expression. Immunoblot for beta-dystroglycan and alpha-sarcoglycan confirmed the immunohistochemical findings. Utrophin expression was slightly increased in a proportion of fibers in the DMD and BMD carriers with dystrophin mosaicism. We found no correlation between utrophin expression and DAP expression. We conclude that absence or reduction of dystrophin in muscle fibers of DMD and BMD carriers causes a reduction of DAPs in the same fibers, as observed in DMD and BMD patients, while utrophin does not seem to play a role in DAP expression in adult muscle.

Generalized lysosomal storage in Yunis Varón syndrome.

Muscle biopsy in a neonate with features of Yunis Varón syndrome revealed a vacuolar myopathy with evidence of lysosomal storage disease. Similar vacuoles were also present in heart, cartilage, central nervous system and cultured fibroblasts. Although the histologic findings in the central nervous system resembled those of infantile acid maltase deficiency, the essayed lysosomal enzymes were normal. Chromatography of urine revealed abnormal bands of unidentified oligosaccharides. This is the first report of generalized storage disease in Yunis Varón syndrome. The biochemical defect is unknown.

Fetus-like dystrophin expression and other cytoskeletal protein abnormalities in centronuclear myopathies.

We have investigated supposed maturational arrest of muscle in centronuclear myopathies (CNMs) by characterizing the expression of dystrophin, other cytoskeletal proteins, and fetal myosin in the muscle fibers of 9 CNM patients (4 sporadic, 3 familial, 2 adult sporadic). Dystrophin and beta-spectrin localized intracytoplasmically in centrally nucleated fibers. Talin and vinculin were normally expressed. Desmin was radially organized in several fibers in all patients. Scattered vimentinpositive fibers were found in 3 cases. Six myotonic dystrophy cases and 4 inflammatory myopathy cases with regenerating fibers were also studied: dystrophin and the membrane cytoskeletal proteins were normally expressed in the former; and dystrophin, spectrin, and vinculin were reduced in the latter. Intracytoplasmic dystrophin is further evidence of maturational arrest in CNMs. Spectrin and dystrophin codistribute in these pathological conditions as in normal muscle. We conclude that the altered cytoskeletal network found in CNMs likely plays a pathogenetic role in these conditions.

Dystrophin abnormalities in Duchenne and Becker dystrophy carriers: correlation with cytoskeletal proteins and myosins.

Characterization with a panel of six antibodies revealed abnormal dystrophin expression in 6 of 20 Duchenne muscular dystrophy (DMD) carriers examined, and in 5 of 12 Becker muscular dystrophy (BMD) carriers examined. The immunocytochemistry of muscle fibres was normal with five of the antibodies in two BMD carriers, but some muscle fibres were negative to the antibody directed against a portion of the dystrophin rod domain. Mosaicism was detected with all six antibodies in the other three BMD (but in only a small number of fibres) and in all DMD carriers muscles. Spectrin, vinculin and talin were immunolocalized in the same muscle specimens in order to assess membrane cytoskeletal integrity and to correlate their expression with that of dystrophin. These proteins, including vinculin, which was previously reported to be reduced in DMD patient muscles, were normally present on the surface of all dystrophin-deficient fibres. Muscle fibre types were characterized using monoclonal antibodies against fetal myosin and adult fast and adult slow myosin heavy chains. In both the DMD and BMD carriers, a significant reduction in type 2B fibres, as well as an increase in type 2C and fetal myosin-containing fibres was found - as has also been reported in DMD patients. Altered dystrophin expression was observed more frequently in type 2 than type 1 fibres. Dystrophin deficiency was found in a high percentage of type 2C fibres as well as in all fibres expressing fetal myosin; this suggests that dystrophin-deficient fibres are more susceptible to degeneration, leading to regeneration.

Needle biopsy for muscle diagnosis and research: an Italian experience.

We report our experience of needle biopsy over the past two and a half, viz. 395 cases. We find that the technique meets the needs of diagnosis and research very satisfactorily. We describe its advantages over open biopsy and recommend it in preference to the latter, provided that the necessary technical skill for processing the sample is available.