Variation In Fiber Size Research Articles

G.P.279: Efficient AAV-mediated transfer of FKRP in a new mouse model of Limb Girdle Muscular Dystrophy 2I

Dystroglycanopathies constitute a group of genetic diseases caused by defective glycosylation of alpha-dystroglycan (aDG), a membrane glycoprotein involved in the cell/matrix anchoring of muscle fibers. The aDG glycosylation, a very complex process, requires many proteins whose functions are not fully elucidated. In particular, mutations in the FKRP gene encoding Fukutin related protein, lead to hypoglycosylation of aDG, resulting in different forms of dystroglycanopathies, among which Limb Girdle Muscular Dystrophy type 2I (LGMD2I). We generated a knock-in mouse model of LGMD2I, carrying the most frequent mutation (L276I) encountered in LGMD2I patients. Molecular characterization of this mouse model showed that the introduction of the mutation did not alter the expression of FKRP. However, the protein appears to have altered function since abnormal glycosylation of aDG and reduction of laminin binding was observed. Histologically, the muscles of this model show a dystrophic pattern starting from 6 months of age, consisting both in the presence of central nuclei and in fiber size variability. Interestingly, functional muscle impairment can be observed as early as 2 months of age by a decrease of the muscle resistance to eccentric mechanical stress. To evaluate gene transfer as a therapeutic approach, we cloned the FKRP cDNA in an AAV vector under the transcriptional control of the desmin promoter. The recombinant AAV2/9 vector was injected intramuscularly or intravenously in the mouse model. Expression of the FKRP transgene was obtained, both at RNA and protein levels. The glycosylation of aDG was restored as well as laminin binding. A histological rescue was observed by the decrease of fibers with centrally located nuclei. The AAV vector also improved the muscle function, since it conferred a better resistance to eccentric stress to the injected muscles.

G.P.269: CAP-disease not-related to ACTA1, TPM2 or TPM3 genes

CAP-disease is a rare congenital myopathy characterized by the presence of demarcated areas of subsarcolemmal myofibrillar disorganization that are devoid of myosin ATPase, stained with NADH-TR and strongly immunolabeled for desmin. Mutations in ACTA1, TPM2 or TPM3 have been reported so far. We report on 5 cases: 1 sporadic severe case and four patients from 2 families, respectively. In family 1 mother and daughter were affected; in family 2, two siblings manifested the disease. Mutations in the three documented genes were excluded. Age at onset ranged from birth to childhood. The main clinical phenotype consisted of clear dysmorphic features with elongated face and high-arched palate associated with axial, lower and upper limbs muscle weakness. The most severe cases presented early respiratory distress, and feeding difficulties. One patient had arthrogryposis. Muscle biopsies performed at different ages (from newborn to adult age) showed severe changes with marked variation of fiber size, increased internal nuclei, and type I fiber predominance. Several fibers contained a variable number of CAP in subsarcolemal areas. By electron microscopy, well-delineated CAP structures were constantly encountered. These showed disorganization of myofibrils, loss of thick filaments, and thickened fragments of Z bands without nemaline rod bodies. CAP contained segments of sarcomeres constituted by fragments of Z-lines from where arising thin filaments (”butterfly-like” aspect). Furthermore dense thin filamentous material was found in the subsarcolemmal region and less frequently between the myofibrils. In longitudinal sections, the Z lines appeared occasionally jagged and partially misaligned in some myofibrils. No basal membrane abnormalities were observed. To identify the causative mutation whole exome sequencing is ongoing. In conclusion, we attempt to characterize the clinical and morphological particularities of five cases presenting CAP disease, not-related to known genes.

G.P.74: Anti-Signal Recognition Particle associated myopathy in a multiethnic Malaysian population

Anti-Signal Recognition Particle (SRP) myopathy is a complement dependent antibody-mediated necrotising myopathy which is often clinically more severe and require longer and more aggressive immunosuppressive treatment. We describe a series of Malaysian patients with anti-SRP myopathy. Of 30 patients with idiopathic inflammatory myopathy, seen prospectively at the University of Malaya Medical Centre, Kuala Lumpur between 2012 and 2014, eight (26.7%) had positive anti-SRP antibodies. All patients were female, of whom four were ethnic Malays and four, ethnic Chinese. Their mean age of disease onset was 37years (range 24–60years). All presented with progressive severe proximal muscle weakness. Mean serum creatine kinase at presentation was 4994u/L (range 385–14,000iu/L). Muscle histopathology showed muscle fibre necrosis with variable regenerative features. Inflammation ranged from minimal to severe infiltration of lymphocytes in seven patients and in one patient, there was marked variation of fibre size with interstitial fibrosis, morphological features similar to that of muscular dystrophy. All patients responded only partially to corticosteroids and other immunosuppressive agents including methotrexate, mycophenolate mofetil, azathioprine and IVIG. Rituximab was given to one patient but with minimal improvement. Other associated autoantibodies found included anti Ro-52 in 50% and anti PM-Scl75 and anti PL-12 in 25% respectively. None had associated interstitial lung disease nor underlying malignancy. In prospective series inflammatory myopathy patients seen at a tertiary Malaysian hospital, anti-SRP myopathy was seen a quarter of the patients. The clinical picture was similar to anti-SRP myopathy reported from other populations.

G.P.181: Early onset and severe X-linked dilated cardiomyopathy with epilepsy, behavioral abnormalities and elevated CK due to DMD gene intron 1 splice site mutation

DMD gene mutations are responsible for the dystrophinopathies including Duchenne and Becker muscular dystrophies (DMD/BMD). Although usually associated with DMD and BMD, an almost exclusive cardiac involvement was found in rare patients carrying DMD gene mutations. We here report on a patient who developed dilated cardiomyopathy since 11years old and required heart transplantation at 14years. An older brother was also affected and transplanted. Moreover, the patient was treated by Risperidone and Lamotrigine for behavioral abnormalities and generalized epilepsy. First neuromuscular assessment at 24years did not show any muscle weakness, atrophy or hypertrophy, neither exercise intolerance nor rhabdomyolysis. CPK levels were always high (from 300 to 14000IU/L). Electromyographic examination showed myopathic pattern with normal nerve conditions. Muscle CT scan imaging was normal. As LMNA, MYH7 and LAMP2 genes analyses did not reveal any mutation, deltoid muscle biopsy was performed and revealed fiber size variation, few centralized nuclei and regenerating fibers without overt muscle fibrosis. Dystrophin protein immunohistochemical studies showed irregular staining with DYS1, MANDYS8, MANDRA1 antibodies while it was absent with DYS3 antibody. Moreover, muscle western blot studies showed highly reduced amounts of dystrophin with normal molecular weight. Subsequent DMD gene analysis did not found any genomic deletion or duplication. However, dystrophin mRNA analysis showed no amplification for the exon 1 to exon 6 fragment suggesting skipping of these exons. Finally, DMD gene sequencing using next generation technologies lead to the identification of an intron 1 splice donor site variation (c.31+4A>G). This new case further illustrates the clinical heterogeneity of dystrophinopathies. Additional molecular analyses are required to decipher the molecular basis of the predominantly cardiac and central nervous system involvement in this patient.

G.P.59: Muscle pathology as a diagnostic clue to Allgrove syndrome

Allgrove or triple A syndrome is an autosomal-recessive disorder caused by mutations in the AAAS gene, coding for the nucleoporin ALADIN. While the eponymous combination of Achalasia, Addisonianism and Alacrimia seems hard to miss, these cases are a diagnostic challenge when this trias is incomplete and other neurological signs from a broad range of associated symptoms predominate. This was the case with a 15year old boy from a consanguineous family suffering from progressive distal limb muscle weakness and atrophy as well as mild learning disability, whose muscle and sural nerve biopsy samples we received. No abnormalities of the sural nerve were found, but the gastrocnemius muscle sample showed considerable fatty replacement, increased endo- and perimysial connective tissue, rounded fibre profiles, increased fibre size variability, increased internalised myonuclei and nuclear clumps, numerous degenerating and some regenerating fibres, massive fibre splitting and many whirled fibres as well as some rimmed vacuoles, few target fibres, but more frequent targetoid/ core-like lesions. Conspicuous nuclear inclusions in electron microscopy and particularly reduced immunoreactivity with an antibody against lamin A/C led us to suspect a nuclear pathology. When he developed dysphagia due to achalasia, sequencing of the AAAS gene was initiated, revealing homozygous c.762delC mutations in exon 8 leading to a premature stop of translation due to frameshift (p.S255V fs ∗ 35). Adrenal deficiency and autonomic dysfunction can result in life threatening situations for these patients, but can be addressed by symptomatic medication. Thus making the diagnosis in time is important. Triple A syndrome with its variable, multi-system symptoms including optic atrophy, cerebellar ataxia, upper and lower motoneuron signs and various neuropathic presentations is a differential diagnosis for mixed pathologies with signs of nuclear changes that myopathologists need to be aware of.

Silencing of drpr Leads to Muscle and Brain Degeneration in Adult Drosophila

Mutations in the gene encoding the single transmembrane receptor multiple epidermal growth factor-like domain 10 (MEGF10) cause an autosomal recessive congenital muscle disease in humans. Although mammalian MEGF10 is expressed in the central nervous system as well as in skeletal muscle, patients carrying mutations in MEGF10 do not show symptoms of central nervous system dysfunction. drpr is the sole Drosophila homolog of the human genes MEGF10, MEGF11, and MEGF12 (JEDI, PEAR). The functional domains of MEGF10 and drpr bear striking similarities, and residues affected by MEGF10 mutations in humans are conserved in drpr. Our analysis of drpr mutant flies revealed muscle degeneration with fiber size variability and vacuolization, as well as reduced motor performance, features that have been observed in human MEGF10 myopathy. Vacuolization was also seen in the brain. Tissue-specific RNAi experiments demonstrated that drpr deficiency in muscle, but not in the brain, leads to locomotor defects. The histological and behavioral abnormalities seen in the affected flies set the stage for further studies examining the signaling pathway modulated by MEGF10/Drpr in muscle, as well as assessing the effects of genetic and/or pharmacological manipulations on the observed muscle defects. In addition, the absence of functional redundancy for Drpr in Drosophila may help elucidate whether paralogs of MEGF10 in humans (eg, MEGF11) contribute to maintaining wild-type function in the human brain.

RNA Transcription and Maturation in Skeletal Muscle Cells are Similarly Impaired in Myotonic Dystrophy and Sarcopenia: The Ultrastructural Evidence.

OPINION article Front. Aging Neurosci., 30 July 2014Sec. Neuroinflammation and Neuropathy Volume 6 - 2014 | https://doi.org/10.3389/fnagi.2014.00196

Controlling Fiber Morphology and Scaffold Design for Treatment of Dry Age-Related Macular Degeneration

Polystyrene (PS), poly(ethylene terephthalate) (PET), and polyurethane (PU) were electrospun in various solvents, to ascertain the ideal conditions for reproducible scaffold production, in order to develop a synthetic Bruch's membrane. Effects of different environmental factors under laboratory conditions and controlled conditions, with and without 1% NaCl, were investigated. For PS, environmental conditions were more important than NaCl addition in fiber diameter reduction; however, NaCl addition showed reduced fiber size variation. For PET, reduction in fiber diameter on addition of NaCl compared to controlled environmental conditions was observed. Fiber size variation for PET was unaffected by NaCl addition or controlled conditions.

A case of adult-onset reducing body myopathy presenting a novel clinical feature, asymmetrical involvement of the sternocleidomastoid and trapezius muscles

We herein report a 32-year-old woman with adult-onset reducing body myopathy (RBM) who had a mutation in the four-and-a-half LIM domain 1 gene (FHL1) and showed a marked asymmetrical involvement of sternocleidomastoid and trapezius muscles. At 30years of age she noticed bilateral foot drop, and over the next two years developed difficulty raising her right arm. At 32years of age she was admitted to our hospital for a diagnostic evaluation. Neurological examination showed moderate weakness and atrophy of her right sternocleidomastoid muscle, right trapezius muscle, and bilateral upper proximal muscles. There were severe weakness and atrophy of her bilateral tibialis anterior muscles. Her deep tendon reflexes were hypoactive in her upper extremities. Her serum creatine kinase level was mildly increased. Muscle biopsy specimens from the left tibialis anterior muscle revealed marked variation in fiber size, some necrotic or regenerating fibers, and reducing bodies. Gene analysis of FHL1 demonstrated a mutation: a heterozygous missense mutation of c.377G>A (p. C126T) in FHL1. Compared with previous adult-onset RBM cases harboring mutations in FHL1, our case was characterized by asymmetrical atrophy of the sternocleidomastoid and trapezius muscles.

Muscle Disease

On the basis of strong research evidence, Duchenne muscular dystrophy (DMD), the most common severe childhood form of muscular dystrophy, is an X-linked recessive disorder caused by out-of-frame mutations of the dystrophin gene. Thus, it is classified asa dystrophinopathy. The disease onset is before age 5 years. Patients with DMD present with progressive symmetrical limb-girdle muscle weakness and become wheelchair dependent after age 12 years. (2)(3). On the basis of some research evidence,cardiomyopathy and congestive heart failure are usually seen in the late teens in patients with DMD. Progressive scoliosis and respiratory in sufficiency often develop once wheelchair dependency occurs. Respiratory failure and cardiomyopathy are common causes of death, and few survive beyond the third decade of life. (2)(3)(4)(5)(6)(7). On the basis of some research evidence, prednisone at 0.75 mg/kg daily (maximum dose, 40 mg/d) or deflazacort at 0.9 mg/kg daily (maximum dose, 39 mg/d), a derivative of prednisolone (not available in the United States), as a single morning dose is recommended for DMD patients older than 5 years, which may prolong independent walking from a few months to 2 years. (2)(3)(16)(17). Based on some research evidence, treatment with angiotensin-converting enzyme inhibitors, b-blockers, and diuretics has been reported to be beneficial in DMD patients with cardiac abnormalities. (2)(3)(5)(18). Based on expert opinion, children with muscle weakness and increased serum creatine kinase levels may be associated with either genetic or acquired muscle disorders (Tables 1 and 3). (14)(15)

Letter to the Editor Complex-I defect with minimal manifestations

Letter to the Editor Complex-I defect with minimal manifestations

SMASH - semi-automatic muscle analysis using segmentation of histology: a MATLAB application.

BackgroundHistological assessment of skeletal muscle tissue is commonly applied to many areas of skeletal muscle physiological research. Histological parameters including fiber distribution, fiber type, centrally nucleated fibers, and capillary density are all frequently quantified measures of skeletal muscle. These parameters reflect functional properties of muscle and undergo adaptation in many muscle diseases and injuries. While standard operating procedures have been developed to guide analysis of many of these parameters, the software to freely, efficiently, and consistently analyze them is not readily available. In order to provide this service to the muscle research community we developed an open source MATLAB script to analyze immunofluorescent muscle sections incorporating user controls for muscle histological analysis.ResultsThe software consists of multiple functions designed to provide tools for the analysis selected. Initial segmentation and fiber filter functions segment the image and remove non-fiber elements based on user-defined parameters to create a fiber mask. Establishing parameters set by the user, the software outputs data on fiber size and type, centrally nucleated fibers, and other structures. These functions were evaluated on stained soleus muscle sections from 1-year-old wild-type and mdx mice, a model of Duchenne muscular dystrophy. In accordance with previously published data, fiber size was not different between groups, but mdx muscles had much higher fiber size variability. The mdx muscle had a significantly greater proportion of type I fibers, but type I fibers did not change in size relative to type II fibers. Centrally nucleated fibers were highly prevalent in mdx muscle and were significantly larger than peripherally nucleated fibers.ConclusionsThe MATLAB code described and provided along with this manuscript is designed for image processing of skeletal muscle immunofluorescent histological sections. The program allows for semi-automated fiber detection along with user correction. The output of the code provides data in accordance with established standards of practice. The results of the program have been validated using a small set of wild-type and mdx muscle sections. This program is the first freely available and open source image processing program designed to automate analysis of skeletal muscle histological sections.

Characterization of intercostal muscle pathology in canine degenerative myelopathy: A disease model for amyotrophic lateral sclerosis

Dogs homozygous for missense mutations in the SOD1 gene develop a late-onset neuromuscular disorder called degenerative myelopathy (DM) that has many similarities to amyotrophic lateral sclerosis (ALS). Both disorders are characterized by widespread progressive declines in motor functions, accompanied by atrophic changes in the descending spinal cord tracts. Some forms of ALS are also associated with SOD1 mutations. In end-stage ALS, death usually occurs as a result of respiratory failure from severe functional impairment of respiratory muscles. The mechanisms that lead to this loss of function are not known. Dogs with DM are euthanized at all stages of disease progression, providing an opportunity to characterize the onset and progression of any pathological changes in the respiratory muscles that may precede respiratory failure. To characterize such potential disease-related pathology, we evaluated intercostal muscles from Boxer and Pembroke Welsh Corgi dogs that were euthanized at various stages of DM disease progression. DM was found to result in intercostal muscle atrophy, fibrosis, increased variability in muscle fiber size and shape, and alteration in muscle fiber type composition. This pathology was not accompanied by retraction of the motor neuron terminals from the muscle acetylcholine receptor complexes, suggesting that the muscle atrophy did not result from physical denervation. These findings provide a better understanding of the mechanisms that likely lead to respiratory failure in at least some forms of ALS and will be useful in the development and evaluation of potential therapeutic interventions using the DM model.

P.4.14 A novel mutation in RYR1 gene for central core disease in a Chinese family

Central core disease (CCD) is a mild, non-progressive or slowly progressive congenital myopathy with proximal symmetrical muscle weakness. CCD is transmitted as an autosomal dominant or recessive trait and at least one gene responsible is the ryanodine receptor gene at chromosome 19q13.1. Here we report the clinical and myopathological changes in a Chinese family with central core disease in an autosomal dominant pattern. The proband showed early onset congenital myopathy with severe contractures, delayed motor milestones, both proximal and distal muscle weakness, normal serum creatine kinase (CK), a non-progressive course, with muscle biopsy findings of central cores and in addition, marked proliferation of connective and adipose tissue, and variation in fibre size. The mother and the younger sister of the proband had similar but mild symptoms with the proband. Molecular genetic analysis showed a heterozygous sequence change in exon 102 (c.14692G > A; p.Gly4897Asp) in RYR1 gene not previously reported. The uncommon clinical distribution of weakness and the severe morphological abnormalities on muscle histology, which had some features of a muscular dystrophy had never been reported in an autosomal dominant CCD family.

P.18.12 Histological and magnetic resonance imaging findings of Vastus Lateralis in Myotonic Dystrophies: Do they match up?

In myotonic dystrophy type 1 (DM1), MRI shows a characteristic pattern of skeletal muscle involvement, and data are usually in line with the clinical presentation. In contrast, myotonic dystrophy type 2 (DM2) patients often show discrepancies between their clinical phenotype and MRI. Systematic data on MRI abnormalities and their histological correlates are not available to date. We analyzed Vastus Lateralis biopsies, corresponding 3T MRI, and clinical data in 1 DM1 (f, 150–200 CTGs; MIRS IV; ages at biopsy/MRI: 49/50 y) and 6 DM2 patients (f/m:3/3). In DM2, mean ages at biopsy and subsequent MRI examinations were 53.7+/−12.2 y and 57.7+/−13.3 y, respectively. 3 female DM2 patients had pathological MRI results indicative of fatty degeneration: 2 patients showed moderate fatty changes, 1 patient had a complete fibroadipose tissue replacement. The corresponding histological findings included increased fiber size variation, type I fiber predominance, increased numbers of internal nuclei, numerous pyknotic nuclear clumps, acid phosphatase-positive sarcoplasmic granules in normal appearing and/or degenerative fibers, and widespread fatty tissue replacement in all 3 patients, and results correlated well with the extent of MRI changes. Further 3 DM2 patients had normal MRI, however also showed histological abnormalities which were milder and included type I fiber predominance, type II fiber atrophy, an increase of internal nuclei, and numerous pyknotic nuclear clumps but no fatty degeneration. In DM1, MRI of Vastus Lateralis was normal. The histological findings were in line and showed only mild variations in fiber size and few nuclear clumps. In DM, histological and imaging findings correlated well with the clinical data on muscle strength of knee extensors. We conclude that MRI most reliably reflected fatty tissue replacement in vivo. In contrast, changes in fiber type distribution, abnormal position of myonuclei, and numerous nuclear clumps did not show MRI correlates.

P.10.16 The presence of developmental and fetal/neonatal myosin in neonatal human muscle

Myosin heavy chains are developmentally regulated but the spectrum of the down-regulation of the developmental and fetal/neonatal isoforms in human muscle is not known. We have reviewed 20 paediatric muscle biopsies, aged 15 days to 22 months with minimal pathology, in particular with minimal variation in fibre size, no excess internal nuclei, no fibrosis, no necrosis, no inflammation and no structural abnormalities, to ascertain the presence of extrafusal fibres with developmental and fetal myosin using the antibodies MHCd and MHCn (Leica). Although pathology was minimal, the biopsies were all taken for a clinical reason, and phenotype often severe, thus a chromosomal, neurometabolic or neuromuscular disorder could not be excluded, in particular a metabolic or myasthenic condition. In all cases very few fibres, often none, showed developmental myosin. Fetal myosin was present in many fibres before 3 months of age but after 6 months it was not present, or in only a few fibres of normal size. We conclude (1) the presence of developmental myosin is a useful indicator of fibre regeneration (based on separate studies of dystrophic muscle), and its presence in a large number of fibres after birth is likely to reflect an abnormality; (2) the widespread presence of fetal myosin after the age of 3–6 months is likely to reflect a delay in maturation and/or an abnormality although this cannot always be defined. This study is limited by the lack of knowledge of the effects of abnormal movement, contractures, hypotonia, in utero and postnatally, on muscle structure and maturation, in addition to those of a neuromuscular disorder that has minimal effect on muscle pathology.

P.1.9 Repeated cardiotoxin treatment progresses muscle phenotype in Collagen VI deficient mice

Collagen VI-related myopathies are inherited muscle disorders characterized clinically by muscle weakness and pathologically by fiber size variation and enhanced fibrosis. These disorders show broad spectrum of clinical presentation from severe Ullrich congenital muscular dystrophy (UCMD) to milder Bethlem myopathy (BM), both of which and both are caused by mutations in one of Col6A1–3 genes, In mice, collagen VI deficiency has been reported to reveal remarkable phenotypes in some organs such as bones, cartilages and adipose tissues, however their skeletal muscle presents a milder phenotype as that in BM. We have generated collagen VI-deficient mice and observed their muscle pathology chronologically. Along aging, distal muscles showed fiber size variation remarkably but only milder moderate fibrosis. Since collagen VI is produced in interstitial mesenchymal cells, we hypothesized if activation of mesenchymal cells contributes the muscle pathological phenotypes in collagen VI-deficient mice. We injected cardiotoxin (CTX) to TA muscles of collagen VI-deficient mice repeatedly to induce muscle necrosis and regeneration-mediated activation of mesenchymal cells artificially. After CTX injections, myofibers were well regenerated even with multiple CTX treatment, and along the number of CTX injections, muscle weakness, myofiber atrophy and interstitial fibrosis were progressed as observed in UCMD. The finding suggests that dysregulation of interstitial mesenchymal cells may be associated with the pathogenesis of collagen VI-related myopathies.

P.11.1 Peptide-PMO induced exon-skipping restores muscle physiology in the mdx mouse

Antisense directed exon-skipping has been shown to be a powerful method to induce expression of dystrophin in dystrophic muscle for a range of species including man. Dystrophin expression can be induced in the mdx mouse by oligonucleotides targeting exon 23 which removes the premature stop mutation in the murine dystrophin gene. We had previously shown improved resistance to eccentric exercise (10% length change of the active muscle) following intramuscular delivery of a phosphorodiamidate morpholino oligomer (PMO) targeting exon 23 in the mdx mouse (Sharp et al., 2011). Little improvement was seen in specific force which is commonly only 60% of wild-type in the mdx mouse. We now report similar physiology results following a single intravenous treatment with a peptide-PMO (PPMO) using the Pip6a peptide. Importantly, repeated intravenous PPMO treatment every two weeks starting at 12 weeks old and continuing for 10 doses, completely prevented the force drop associated with eccentric exercise and significantly improved the specific force, albeit not quite to wild-type levels. Both the acute and chronic dosing results correlated with the increased expression of dystrophin in muscle fibres and decreased levels of TIMP-1 in the serum. The chronic dosing study also showed a reduction in inflammation, restoration of a more normal fibre type pattern and reduction in fibre size variability. These studies show the potential of the PPMO formulation to significantly improve the results of exon-skipping in clinical trials.

P.12.2 Dominant distal myopathy due to slow channelopathy

Slow channel syndrome, first recognized by Engel in 1982, has distinct phenotypic features, dominant inheritance, selective weakness of cervical scapular and finger extensor weakness, ophtalmoparesis. Histochemical findings show type 1 fibers preponderance, small group of atrophic fibers of either fiber type, increased fiber size variability, tubular aggregates. We examined 3 biopsies in 3 patients (2 males – 55 years and 66 years, 1 female – 61 years) presenting a dominant distal myopathy with variable age at onset (from second to third decade). The patients had ophtalmoparesis, upper limb extensor, forearm and finger weakness and distal myopathy with a progressive slow course. The woman had also some difficulty in anterior tibial flexor muscle but also atrophy of forearms muscles. Electrophysiology on single stimulus of ulnar nerve showed repetitive CMAP. The genomic wide linkage analysis identified 3 regions co-segregating with the disease in chromosome 1, 13, 17. Next generation sequencing showed in all patients a known mutation in epsilon-subunit of AchR receptor and a mutation in phospholipase. Muscle showed mild changes consisting in atrophic fibers, mostly of type 2, some fiber type grouping, central nuclei and ring fibers. Such fibers are consistent with abnormal regeneration.We suggest that the occurrence of mutations involving both AchR receptor and phospholipase might concur in determining a new distal phenotype with permanent weakness. Our family was previously diagnosed as myotonic dystrophy or distal myopathy. The presence of the histochemical findings of both myopathy and neuropathy were present. We suggest the possibility of calcium overload due to prolonged open time of the AChR channel with a presynaptic component might determine a distal neuro-myopathy with slow evolution and muscle rearrangement due to a muscle fiber degeneration and functional denervation. The slow channel mutation should be searched in families with permanent distal weakness.

Sarcoplasmic Masses in the Skeletal Muscle of a Stranded Pigmy Sperm Whale (Kogia breviceps)

We measured the abundance of sarcoplasmic masses within skeletal muscle myocytes of an adult female stranded pigmy sperm whale (Kogia breviceps). The presence of these masses in other species has been reported in association with myopathies, including myotonic dystrophy, the most frequently related pathology. Other histopathologic muscle changes included a high number of internal nuclei, variations in fiber size and shape, and the predominance of type I fibers.