Limb girdle muscular dystrophy type 2L presenting as necrotizing myopathy.

Recessive mutations in the ANO5 gene, encoding anoctamin 5, cause proximal limb girdle muscular dystrophy (LGMD2L), Miyoshi-type distal myopathy (MM3) and asymptomatic hyper- CKemia. We report a woman with exertion-induced myalgia and weakness in the hip girdle manifesting at the age of 40. Creatine kinase (CK) was increased 20-fold. Histologically the dominating feature was necrotizing myopathy, but long-term immunosuppressive therapy did not change CK level or myopathic symptoms. Molecular genetic investigation led to the finding of the homozygous ANO5 c.191dupA mutation. This is a report of a muscular dystrophy due to ANO5 mutation presenting histologically as necrotizing myopathy. For this reason our finding extends the histological spectrum of myopathies due to ANO5 mutations as well as the possible differential diagnoses for necrotizing myopathy.

Mitochondrial abnormalities in myofibrillar myopathies.

Histological mitochondrial changes are generally found to be associated with late onset myofibrillar myopathies (MFMs). How these changes contribute to the pathogenesis of MFMs is unknown. Mitochondrial changes, including COX-deficient fibers (n = 8), biochemical activities of respiratory chain complexes (n = 7), and multiple mtDNA deletions by long-range PCR (n = 9) were examined in patients with genetically confirmed MFMs [MYOT (n = 2), DES (n = 1), ZASP (n = 2), FLNC (n = 4)] and compared with age and sex matched normal controls (n = 27) and patients with a mitochondrial disorder with multiple mtDNA deletions due to nuclear genetic defects (n = 8). In 2 MFM patients, micro dissected fibers were analyzed for multiple mtDNA deletions by nested long-range PCR. The COX-deficient fibers only partly corresponded with fibers containing myofibrillar accumulations. In total, there was no difference in the percentage of COX-deficient fibers in MFM patients and normal controls. However, the percentage of COX-deficient fibers was significantly higher in 3 MFM patients. Two MFM patients but none of the controls had multiple mtDNA deletions. Nested long-range PCR detected multiple mtDNA deletions only in COX-deficient fibers. Citrate synthase activities in MFM patients were 1.5-fold increased by compared to those in controls, suggesting initiation of mitochondrial alterations. However, it is unclear whether this is a direct consequence of MFM pathology. *both authors contributed equally to the manuscript.

"Necklace" fibers, a new histological marker of late-onset MTM1-related centronuclear myopathy.

Mutations in the gene encoding the phosphoinositide phosphatase myotubularin 1 protein (MTM1) are usually associated with severe neonatal X-linked myotubular myopathy (XLMTM). However, mutations in MTM1 have also been recognized as the underlying cause of "atypical" forms of XLMTM in newborn boys, female infants, female manifesting carriers and adult men. We reviewed systematically the biopsies of a cohort of patients with an unclassified form of centronuclear myopathy (CNM) and identified four patients presenting a peculiar histological alteration in some muscle fibers that resembled a necklace ("necklace fibers"). We analyzed further the clinical and morphological features and performed a screening of the genes involved in CNM. Muscle biopsies in all four patients demonstrated 4-20% of fibers with internalized nuclei aligned in a basophilic ring (necklace) at 3 microm beneath the sarcolemma. Ultrastructurally, such necklaces consisted of myofibrils of smaller diameter, in oblique orientation, surrounded by mitochondria, sarcoplasmic reticulum and glycogen granules. In the four patients (three women and one man), myopathy developed in early childhood but was slowly progressive. All had mutations in the MTM1 gene. Two mutations have previously been reported (p.E404K and p.R241Q), while two are novel; a c.205_206delinsAACT frameshift change in exon 4 and a c.1234A>G mutation in exon 11 leading to an abnormal splicing and the deletion of nine amino acids in the catalytic domain of MTM1. Necklace fibers were seen neither in DNM2- or BIN1-related CNM nor in males with classical XLMTM. The presence of necklace fibers is useful as a marker to direct genetic analysis to MTM1 in CNM.

Differential involvement of sarcomeric proteins in myofibrillar myopathies: a morphological and immunohistochemical study.

Myofibrillar myopathies (MFMs) are rare inherited or sporadic progressive neuromuscular disorders with considerable clinical and genetic heterogeneity. In the current study, we have analyzed histopathological and immunohistochemical characteristics in genetically identified MFMs. We performed a morphological and morphometrical study in a cohort of 24 genetically identified MFM patients (12 desmin, 6 alphaB-crystallin, 4 ZASP, 2 myotilin), and an extensive immunohistochemical study in 15 of these patients, using both well-known and novel antibodies directed against distinct compartments of the muscle fibers, including Z-disc and M-band proteins. Our morphological data revealed some significant differences between the distinct MFM subgroups: the consistent presence of 'rubbed-out' fibers in desminopathies and alphaB-crystallinopathies, an elevated frequency of vacuoles in ZASPopathies and myotilinopathies, and the presence of a few necrotic fibers in the two myotilinopathy patients. Immunohistochemistry showed that in MFM only a subset of Z-disc proteins, such as filamin C and its ligands myotilin and Xin, exhibited significant alterations in their localization, whereas other Z-disc proteins like alpha-actinin, myopodin and tritopodin, did not. In contrast, M-band proteins revealed no abnormalities in MFM. We conclude that the presence of 'rubbed-out' fibers are a suggestive feature for desminopathy or alphaB-crystallinopathy, and that MFM is not a general disease of the myofibril, but primarily affects a subgroup of stress-responsive Z-disc proteins.

Functional relevance of mitochondrial abnormalities in sporadic inclusion body myositis.

Cytochrome c oxidase (COX)-deficient fibers and multiple mitochondrial DNA (mtDNA) deletions are frequent findings in sporadic inclusion body myositis (s-IBM). However, the functional impact of these defects is not known. We investigated oxygen desaturation during exercise using the forearm exercise test, accumulation of lactate during exercise using a cycle ergometry test and mitochondrial changes (COX-deficient fibers, biochemical activities of respiratory chain complexes, multiple mtDNA deletions by long-range polymerase chain reaction) in 10 patients with s-IBM and compared the findings with age and sex-matched normal and diseased controls (without mitochondrial disorders) as well as patients with mitochondrial disorder due to nuclear gene defects resulting in multiple mtDNA deletions (MITO group). The mean age of the s-IBM patients was 68.2 ± 5.7 years (range: 56-75). Patients with s-IBM had statistically significantly reduced oxygen desaturation (ΔsO2) during the handgrip exercise (p<0.05) and elevated peak serum lactate levels during cycle ergometry compared to normal controls (p<0.05). The percentage of COX-deficient fibers in s-IBM and MITO patients was significantly increased compared to normal controls (p<0.01). Five out of nine s-IBM patients had multiple mtDNA deletions. Thirty-three percent of s-IBM patients showed an increased citrate synthase content and decreased activities of complex IV (COX). The biochemical pattern of respiratory chain complexes in patients with s-IBM and MITO was similar. Histopathological analysis showed similar changes in s-IBM and MITO due to nuclear gene defects. Functional tests reflecting mitochondrial impairment suggest a contribution of mitochondrial defects to disease-related symptoms such as fatigue and exertion-induced symptoms.

Sialylation and muscle performance: sialic acid is a marker of muscle ageing.

Sialic acids (Sia) are widely expressed as terminal monosaccharides on eukaryotic glycoconjugates. They are involved in many cellular functions, such as cell-cell interaction and signal recognition. The key enzyme of sialic acid biosynthesis is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE), which catalyses the first two steps of Sia biosynthesis in the cytosol. In this study we analysed sialylation of muscles in wild type (C57Bl/6 GNE (+/+)) and heterozygous GNE-deficient (C57Bl/6 GNE (+/-)) mice. We measured a significantly lower performance in the initial weeks of a treadmill exercise in C57Bl/6 GNE (+/-) mice compared to wild type C57Bl/6 GNE (+/+) animals. Membrane bound Sia of C57Bl/6 GNE (+/-) mice were reduced by 33-53% at week 24 and by 12-15% at week 80 in comparison to C57Bl/6 GNE (+/+) mice. Interestingly, membrane bound Sia concentration increased with age of the mice by 16-46% in C57Bl/6 GNE (+/+), but by 87-207% in C57Bl/6 GNE (+/-). Furthermore we could identify specific morphological changes in aged muscles. Here we propose that increased Sia concentrations in muscles are a characteristic feature of ageing and could be used as a marker for age-related changes in muscle.

Myopathy with hexagonally cross-linked crystalloid inclusions: delineation of a clinico-pathological entity.

A novel myopathy characterized by hexagonally cross-linked tubular arrays has been reported in five patients. We studied the clinical and histopathological features of five additional unrelated patients with this myopathy. Patients experienced exercise intolerance with exercise-induced myalgia and weakness, without rhabdomyolysis. One patient additionally presented mild permanent pelvic girdle muscle weakness. Age at onset varied between 13 and 56 years. The inclusions were eosinophilic on H and E, bright red with modified Gomori's trichrome stains, present in type 2 fibers, and revealed immunoreactivity selectively for a caveolin-3-antibody. Ultrastructurally, the inclusions showed a highly organized, hexagonally cross-linked crystalloid structure. Mutations in the caveolin-3 encoding gene were excluded. Biochemical assessment of glycogenolysis in muscle was normal. Inherited or sporadic myopathy with hexagonally cross-linked tubular arrays is associated with a homogeneous clinical and histopathological phenotype. This myopathy should be included in the differential diagnosis of patients with exercise intolerance and myalgia.

Cross-linking of scleral collagen in the rabbit using riboflavin and UVA.

PURPOSE: Scleral biomechanical weakness and thinning is known to be one of the main factors in the pathogenesis of progressive myopia. We tried to strengthen rabbit sclera by cross-linking scleral collagen using ultraviolet A (UVA) and the photosensitizer riboflavin. METHODS: Circumscribed 10 x10 mm sectors of the posterior--equatorial sclera of six chinchilla rabbit eyes were treated in vivo using a UVA double diode with 4.2 mW/cm(2) UVA at 370 nm and applying 0.1% riboflavin-5-phosphate drops as photosensitizer for 30 min. 1 day postoperatively biomechanical stress--strain measurements of three treated scleral strips were performed using a microcomputer-controlled biomaterial testing device and compared to non-treated contralateral control sclera. In addition, three treated eyes were examined histologically by light microscopy, TUNEL staining and electron microscopy to evaluate side-effects. RESULTS: Following the cross-linking treatment, the ultimate stress was 11.87+/-1.8 MPa versus 3.63+/-0.40 in the controls (increase of 227.9%, p=0.014), Young's modulus 27.67+/-4.16 MPa versus 4.9+/-.15 MPa in the controls (increase of 464.7%, p=0.021) and ultimate strain 92.2+/-7.43% versus 165.63+/-19.09% in the controls (decrease of 54.52%, p=0.012). Histologically, serious side-effects were found in the entire posterior globe with almost complete loss of the photoreceptors, the outer nuclear layer and the retinal pigment epithelium (RPE). CONCLUSIONS: Our new method of scleral collagen cross-linking proved very effective in increasing the scleral mechanical strength; the new treatment may represent an option for strengthening scleral tissue in progressive myopia. However, serious side-effects were observed in the outer retina. In future studies these side-effects could be avoided by reducing the irradiation dose below the cytotoxic level of the retina. Before its clinical application, the new method should be tested in a myopia animal model.

Hemosiderin deposits in chronic graft-vs.-host disease related myopathy.

Chronic graft-vs.-host disease (cGVHD) occurs in 20-50% of patients who survive for at least 100 d after allogeneic stem cell transplantation (SCT). cGVHD includes scleroderma-like skin changes, chronic cholangitis, obstructive lung disease and general wasting syndrome. Polymyositis or myopathy are rare manifestations of cGVHD with approximately 40 reported cases. Polymyositis accompanied by hemosiderin deposits in cGVHD has been reported only once, and there are no reports on lipofuscin deposits in skeletal muscle cells in cGVHD. We report here on a 56-yr-old male who underwent allogeneic SCT in 1999 for osteomyelofibrosis and progressive hematopoietic insufficiency. In February 2004, the patient was hospitalized for progressive muscular weakness with loss of the ability to walk. Laboratory tests demonstrated normal values for serum creatine kinase, aldolase and lactic dehydrogenase; the ferritin level was highly elevated. The femoral muscle biopsy showed mostly perifascicular atrophy as well as numerous subsarcolemmal hemosiderin and lipofuscin deposits. Intravenous administration of the chelating agent deferoxamine was ineffective. Three weeks later the patient died of aspiration pneumonia. Interestingly, autopsy disclosed moderate hemosiderin deposits in the liver, the organ usually involved in hemosiderosis.

Arthrogryposis multiplex with deafness, inguinal hernias, and early death: a family report of a probably autosomal recessive trait.

We report on three male newborn infants of a highly inbred Lebanese family presenting with a characteristic phenotype: arthrogryposis multiplex, deafness, large inguinal hernia, hiccup-like diaphragmatic contractions, and inability to suck, requiring nasogastric gavage feeding. All three boys died from respiratory failure during the first 3 months of life. Intra vitam or post mortem examinations revealed myopathic changes and elevated glycogen content of muscle tissue. This new syndrome is probably transmitted in an autosomal recessive mode, although X-linked inheritance cannot be excluded.