A very late onset AChR and MuSK double positive myasthenia gravis: a case description and literature review.

AChR and MuSK double positive myasthenia gravis has been rarely reported. Generally, it occurs in children and adults after thymectomy or immunotherapy. Furthermore, in a few patients with bulbar or respiratory involvement, MuSK antibodies might be detected after clinical deterioration. We report a man with a very late onset myasthenia gravis (86-year-old) and the coexistence of both antibodies at the time of the diagnosis. Despite the presence of MuSK antibodies, he manifested no bulbar symptoms and had a favorable clinical outcome. However, side effects related to low dose pyridostigmine were evident. Hence, double positivity can also occur in elderly and in more benign forms of myasthenia gravis. Other cases of AChR and MuSK double positive myasthenia gravis could allow a better definition of this condition.

Value of insoluble PABPN1 accumulation in the diagnosis of oculopharyngeal muscular dystrophy.

BACKGROUND AND PURPOSE: The aim was to assess the value of insoluble PABPN1 muscle fibre nuclei accumulation in the diagnosis of atypical cases of oculopharyngeal muscular dystrophy (OPMD). METHODS: Muscle biopsies from a selected cohort of 423 adult patients from several Italian neuromuscular centres were analysed by immunofluorescence: 30 muscle biopsies of genetically proven OPMD, 30 biopsies from patients not affected by neuromuscular disorders, 220 from genetically undiagnosed patients presenting ptosis or swallowing disturbances, progressive lower proximal weakness and/or isolated rimmed vacuoles at muscle biopsy and 143 muscle biopsies of patients affected by other neuromuscular diseases. RESULTS: The detection of insoluble nuclear PABPN1 accumulation is rapid, sensitive (100%) and specific (96%). The revision of our cohort allowed us to discover 23 new OPMD cases out of 220 patients affected with nonspecific muscle diseases. CONCLUSIONS: Oculopharyngeal muscular dystrophy is often misdiagnosed leading to diagnosis delay, causing waste of time and resources. A great number of these cases present symptoms and histological findings frequently overlapping with other muscle diseases, i.e. inclusion body myositis and progressive external ophthalmoplegia. PABPN1 nuclear accumulation is a reliable method for diagnostic purposes and it is safe and useful in helping pathologists and clinicians to direct genetic analysis in the case of suspected OPMD, even when clinical and histological clues are deceptive.

Expanding the histopathological spectrum of CFL2-related myopathies.

Congenital myopathies (CMs) caused by mutation in cofilin-2 gene (CFL2) show phenotypic heterogeneity ranging from early-onset and rapid progressive forms to milder myopathy. Muscle histology is also heterogeneous showing rods and/or myofibrillar changes. Here, we report on three new cases, from two unrelated families, of severe CM related to novel homozygous or compound heterozygous loss-of-function mutations in CFL2. Peculiar histopathological changes showed nemaline bodies and thin filaments accumulations together to myofibrillar changes, which were evocative of the muscle findings observed in Cfl2-/- knockout mouse model.

MicroRNA signatures predict dysregulated vitamin D receptor and calcium pathways status in limb girdle muscle dystrophies (LGMD) 2A/2B.

miRNA expression profile and predicted pathways involved in selected limb-girdle muscular dystrophy (LGMD)2A/2B patients were investigated. A total of 187 miRNAs were dysregulated in all patients, with six miRNAs showing opposite regulation in LGMD2A versus LGMD2B patients. Silico analysis evidence: (1) a cluster of the dysregulated miRNAs resulted primarily involved in inflammation and calcium metabolism, and (2) two genes predicted as controlled by calcium-assigned miRNAs (Vitamin D Receptor gene and Guanine Nucleotide Binding protein beta polypeptide 1gene) showed an evident upregulation in LGMD2B patients, in accordance with miRNA levels. Our data support alterations in calcium pathway status in LGMD 2A/B, suggesting myofibre calcium imbalance as a potential therapeutic target. Copyright © 2016 John Wiley & Sons, Ltd.

MYH7-related myopathies: clinical, histopathological and imaging findings in a cohort of Italian patients.

BACKGROUND: Myosin heavy chain 7 (MYH7)-related myopathies are emerging as an important group of muscle diseases of childhood and adulthood, with variable clinical and histopathological expression depending on the type and location of the mutation. Mutations in the head and neck domains are a well-established cause of hypertrophic cardiomyopathy whereas mutation in the distal regions have been associated with a range of skeletal myopathies with or without cardiac involvement, including Laing distal myopathy and Myosin storage myopathy. Recently the spectrum of clinical phenotypes associated with mutations in MYH7 has increased, blurring this scheme and adding further phenotypes to the list. A broader disease spectrum could lead to misdiagnosis of different congenital myopathies, neurogenic atrophy and other neuromuscular conditions. RESULTS: As a result of a multicenter Italian study we collected clinical, histopathological and imaging data from a population of 21 cases from 15 families, carrying reported or novel mutations in MYH7. Patients displayed a variable phenotype including atypical pictures, as dropped head and bent spine, which cannot be classified in previously described groups. Half of the patients showed congenital or early infantile weakness with predominant distal weakness. Conversely, patients with later onset present prevalent proximal weakness. Seven patients were also affected by cardiomyopathy mostly in the form of non-compacted left ventricle. Muscle biopsy was consistent with minicores myopathy in numerous cases. Muscle MRI was meaningful in delineating a shared pattern of selective involvement of tibialis anterior muscles, with relative sparing of quadriceps. CONCLUSION: This work adds to the genotype-phenotype correlation of MYH7-relatedmyopathies confirming the complexity of the disorder.

TRPV4 related scapuloperoneal spinal muscular atrophy: Report of an Italian family and review of the literature.

Scapuloperoneal spinal muscular atrophy (SPSMA) is a rare autosomal dominant disorder caused by heterozygous mutations in the transient receptor potential cation channel (TRPV4) gene, characterized by progressive scapuloperoneal atrophy and weakness. Additional features, such as vocal cord paralysis, scoliosis and/or arthrogryposis, are likely to occur. We report the first Italian family with SPSMA, harboring the c.806G>A mutation in TRPV4 gene (p. R269H). The pattern of expression was variable: the father showed a mild muscular involvement, while the son presented at birth skeletal dysplasia and a progressive course. We reinforce the concept that the disease can be more severe in the following generations. The disorder should be considered in scapuloperoneal syndromes with autosomal dominant inheritance and a neurogenic pattern. The presence of skeletal deformities strongly supports this suspicion. An early diagnosis of SPSMA may be crucial in order to prevent the more severe congenital form.

Clinical and molecular aspects of 30 patients with late-onset Pompe disease (LOPD): unusual features and response to treatment.

Pompe disease is a rare metabolic disorder, due to mutations in the gene encoding acid alpha-glucosidase (GAA), of which infantile and late-onset forms may occur. Aim of the work was to analyze clinical and laboratory data of a cohort of late-onset Pompe disease (LOPD) patients, collected during the last 15 years and to point out unusual phenotypic/genotypic features as well as enzyme replacement therapy (ERT) responses. We diagnosed 30 LOPD patients; at follow-up, they underwent motor, respiratory, cardiac and muscle MRI evaluations. Motor performances were tested by Walton Gardner-Medwin, GSGC and 6MWT tests. Respiratory function was assessed as FVC% in upright/supine position. LOPD presentations were represented by presymptomatic hyperCKemia (37%), proximal/axial muscle weakness (53%) and respiratory impairment (10%). Median diagnostic delay was 8.6 years (± 8.8). Atypical features were observed in 4 patients: marked distal muscle weakness and severe hearing loss at onset, as well as leukoencephalopathy and mesial temporal sclerosis during the disease course. By GAA sequence analysis, two causing mutations were detected in 22/30 patients, only one in the remaining 8 subjects. Overall, 29/30 patients harbored the common c.-32-13T>G mutation (2 were homozygous). Two new DNA variations were discovered (c.2395C>G, c.1771C>T). 14 patients received ERT for up to 60 months. Our study confirms LOPD clinical and genetic heterogeneity: atypical features may contribute to expand the clinical phenotype highlighting its multi-systemic nature. A timely diagnosis could allow early ERT start. An accurate follow-up is recommended to evaluate treatment responses.

Italian recommendations for Lambert-Eaton myasthenic syndrome (LEMS) management.

Lambert-Eaton myasthenic syndrome (LEMS) is a pre-synaptic disorder of the neuromuscular and autonomic transmission mediated by antibodies to voltage-gated calcium channels at the motor nerve terminal. LEMS is a quite rare and probably under-diagnosed disease: the onset may be slow and clinical signs are typically fluctuating, thus adding to the delay in diagnosis. LEMS weakness typically involves lower and upper limbs and the proximal muscles are predominantly affected. A significant proportion of patients also have dysfunction of the autonomic nervous system that may include dry mouth, constipation, blurred vision, impaired sweating, and orthostatic hypotension. LEMS recognition is based on clinical, electrophysiological and immunological criteria. Nearly 50-60% of patients with LEMS have an underlying tumour that, in almost all cases, is a small-cell lung cancer; the onset of neurological symptoms generally precedes tumour detection. A careful screening for the early detection of the possible associated cancer is a crucial step for optimal disease management. The Italian Working Group on Myasthenic Syndromes developed diagnostic and therapeutic algorithms that could serve in routine clinical practice as tools for a patient-tailored approach.

Long-term follow-up in infantile-onset lambert-eaton myasthenic syndrome.

Lambert-Eaton myasthenic syndrome is a neuromuscular junction disorder characterized by proximal limb muscle weakness, fatigability, decreased deep-tendon reflexes, and autonomic symptoms. There are 2 forms of Lambert-Eaton myasthenic syndrome: one most frequently associated with small-cell lung cancer (P-Lambert-Eaton myasthenic syndrome) and the other that is a pure autoimmune form (NP-Lambert-Eaton myasthenic syndrome). Lambert-Eaton myasthenic syndrome is a very rare disorder in children younger than age 12 years. Herein, we report a 25-year-old man with NP-Lambert-Eaton myasthenic syndrome, which onset was at the age of 10 years. To date, this is the most long-term follow-up of NP-Lambert-Eaton myasthenic syndrome in childhood. In our patient, the only symptomatic treatment with 3,4-diaminopyridine phosphate has been sufficient to guarantee him a good quality of life. Our data remind physicians to keep in mind the diagnosis of Lambert-Eaton myasthenic syndrome in children with a proximal myopathic pattern and they confirm the specificity of compound muscle action potential incremental pattern after brief maximal effort in Lambert-Eaton myasthenic syndrome.

ANT1 is reduced in sporadic inclusion body myositis.

To investigate ANT1 and NF-κB expression in inclusion body myositis (IBM) muscle and to verify their possible roles in the pathogenesis of the disease, we collected muscle samples from five patients with IBM, polimyositis (PM) and controls. p65 form of NF-κB was analyzed using immunocytochemistry, Western blot and EMSA. Western blot of ANT1 was performed and confirmed by gene expression study. Mann-Whitney test was used for groups comparisons. NF-κB (p65) was found over-expressed both with western blot and EMSA, either in IBM or PM patients versus controls (p < 0.01). Expression of ANT1 were lower in IBM samples versus both PM and controls (p < 0.01). ANT1 reduction and NF-κB over-expression in IBM muscle could explain the lack of apoptosis in such disease. Normal ANT1 expression in PM could be related to the scarcity of mitochondrial abnormalities in the disease, but it could also suggest that these two conditions diverge in activating different anti-apoptotic pathways.

Activation of NF-kappaB pathway in Duchenne muscular dystrophy: relation to age.

Muscle degeneration in Duchenne muscular dystrophy (DMD) is exacerbated by increased oxidative stress and the endogenous inflammatory response, with a key role played by nuclear factor kappa-B (NF-kappaB) and other related factors such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-6. However the time course of expression of these molecules and the relation with the amount of necrosis and regeneration have never been investigated. The expression of NF-kappaB, the cytokines TNF-alpha and IL-6 and the antioxidant enzyme glutathione peroxidase (GPx) was studied in muscle samples from 14 patients with DMD aged between 2 and 9 years. Moreover a quantitative morphological evaluation was performed to evaluate necrotic and regenerative areas. The highest percentage of necrosis was revealed within 4 years of age, with a significant negative correlation with age (p < 0.003), which paralleled to a significant decrement of regenerating area (p < 0.0004). We reported the novel observation that the number of NF-kappaB positive fibers and the NF-kappaB DNA-binding activity, revealed by EMSA, are high at two years of life and significantly decline with age (p < 0.0005 and p < 0.0001). The expression of TNF-alpha, IL-6 and GPx was upregulated in DMD muscles compared to controls and significantly increased with age on real-time PCR analysis (p < 0.0002; p < 0.0005; p < 0.03 respectively) and ELISA (p < 0.002; p < 0.02; p < 0.0001 respectively). Since anti-inflammatory and anti-oxidant drugs are nowadays being translated to clinical studies in DMD, the reported insights on these therapeutic targets appear relevant. Further studies on the interactions among these pathways in different DMD phases and on the response of these cascades to treatments currently under investigation are needed to better elucidate their relevance as therapeutic targets.

Analysis of NCAM helps identify unusual phenotypes of hereditary inclusion-body myopathy.

BACKGROUND: Hereditary inclusion-body myopathy or distal myopathy with rimmed vacuoles (h-IBM/DMRV) is due to mutations of the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene, which codes for an enzyme of the sialic acid biosynthetic pathway. By Western blot (WB) analysis, we have previously shown that in h-IBM/DMRV muscle, the neural cell adhesion molecule (NCAM) has increased electrophoretic mobility that reflects reduced sialylation of the protein. OBJECTIVE: To identify patients with h-IBM/DMRV with atypical clinical or pathologic phenotype using NCAM analysis and the possible cellular mechanism associated with the overall abnormal sialylation of NCAM observed in this disorder. METHODS: WB analysis of NCAM was performed on muscle biopsies of 84 patients with an uncharacterized muscle disorder who were divided in the following 2 groups: 1) 46 patients with a proximal muscle weakness in whom the main limb-girdle muscular dystrophy syndromes had been ruled out; and 2) 38 patients with a distal distribution of weakness in whom a neurogenic affection had been excluded. Patients in whom a reduced sialylation of NCAM was suspected were studied for the presence of GNE mutations. RESULTS: In 3 patients, we found that NCAM had increased electrophoretic mobility, thus suggesting an abnormal sialylation of the protein. The genetic study demonstrated that they all carried pathogenic GNE mutations. Further studies demonstrated that hyposialylated NCAM, showing increased electrophoretic mobility on WB, is expressed by nonregenerating fibers in h-IBM/DMRV muscle. CONCLUSIONS: WB analysis of NCAM may be instrumental in the identification of h-IBM/DMRV with atypical clinical or pathologic features.

Wound botulism in drug users: a still underestimated diagnosis.

Wound botulism is a rare infectious disease that is becoming a frequent complication of parental drug use. Diagnosis is often difficult and based on clinical suspicion. We report the first Italian case of wound botulism due to intramuscular heroin injection in a 48-year-old man with an acute onset of slurred speech and dysphagia. The most considerable finding of electrophysiological study was the reduction in amplitude of compound muscle action potential which should be considered a useful initial electrodiagnostic sign in the clinical context of botulism. Alerting clinicians to botulism is crucial for a rapid diagnosis and appropriate treatment and thus decreasing mortality and complications.

Charcot-Marie-Tooth disease type 1B: marked phenotypic variation of the Ser78Leu mutation in five Italian families.

OBJECTIVES: To describe clinical, electrophysiological and genetic data of five unrelated Sicilian pedigrees harbouring a heterozygous Ser78Leu mutation in the myelin protein zero (MPZ) extracellular domain. MATERIALS AND METHODS: Clinical, electrophysiological and genetic findings of 16 patients were reported. Polymorphic markers flanking the coding sequence of MPZ gene were also analysed. RESULTS: A wide range of age at onset was observed in families 1 and 3, with a clinical heterogeneity, in terms of severity of the disease, within the same family (families 1 and 3), and among families. A markedly unsteady gait was a distinctive feature of many members of family 1. All patients in family 2 complained of severe cramps and painful paresthesia. Molecular genetic analysis showed that all affected subjects shared a common haplotype at three microsatellite loci D1S2858, D1S2624 and D1S484. CONCLUSIONS: Our study provides further evidence that phenotypic features of MPZ mutations can vary within and among different families. High frequency of Ser78Leu mutation in Sicily as well as the results of haplotype analyses suggest that the mutation may have been inherited from a common ancestor.

Expression of transglutaminase 2 does not differentiate focal myositis from generalized inflammatory myopathies.

OBJECTIVES: Idiopathic inflammatory myopathies (IIM), including dermatomyositis (DM), polymyositis (PM), sporadic inclusion-body myositis (s-IBM) and focal myositis (FM) are a heterogeneous group of autoimmune disorders of skeletal muscle. An increased transglutaminase 2 (TG2) expression has been found in DM, PM and s-IBM. The aim of our study was to investigate TG2 expression in FM in comparison with other IIM. MATERIALS AND METHODS: We re-examined tissue material we have gathered in the course of our previous studies on IIM, investigating muscle expression of TG2 in patients with FM in comparison with DM, PM and s-IBM using immunocytochemistry and real-time RT-PCR. RESULTS: Immunocytochemistry revealed an increased TG2 signal in endomysial vessels, in atrophic and degenerating/regenerating muscle fibres in PM, DM, s-IBM and FM; in s-IBM, some vacuoles were immunostained too. Real-time RT-PCR study confirmed a significantly increased expression of TG2 in all IIM muscles examined. CONCLUSIONS: Our study demonstrates the presence of TG2 in FM muscles. The study suggests that TG2 expression does not represent a distinctive marker to differentiate FM from generalized IIM. TG2 over-expression in inflamed skeletal muscle does not seem have a pathogenetic role in such a disease, but it could represent a way to contain the inflammatory process.

Novel SHOX gene mutation in a short boy with Becker muscular dystrophy: double trouble in two adjacent genes.

Short stature is a well-recognized feature of Duchenne muscular dystrophy, whilst it has been reported rarely in Becker muscular dystrophy (BMD). Here we report two brothers with BMD, who exhibited a very different growth pattern. Whereas in the short brother (-2.2 SDS) molecular investigation revealed a G367A mutation in the short stature homeobox containing (SHOX) gene located in the Xp22.3 region, no abnormality was found in the brother with normal height (-0.1 SDS). Our data suggest that abnormal growth observed in a boy with BMD may be related to an additional genetic alteration, already known as correlated with short stature.

Phenotypic clustering of lamin A/C mutations in neuromuscular patients.

BACKGROUND: Mutations in the LMNA gene, encoding human lamin A/C, have been associated with an increasing number of disorders often involving skeletal and cardiac muscle, but no clear genotype/phenotype correlation could be established to date. METHODS: We analyzed the LMNA gene in a large cohort of patients mainly affected by neuromuscular or cardiac disease and clustered mutated patients in two groups to unravel possible correlations. RESULTS: We identified 28 variants, 9 of which reported for the first time. The two groups of patients were characterized by clinical and genetic differences: 1) patients with childhood onset displayed skeletal muscle involvement with predominant scapuloperoneal and facial weakness associated with missense mutations; 2) patients with adult onset mainly showed cardiac disorders or myopathy with limb girdle distribution, often associated with frameshift mutations presumably leading to a truncated protein. CONCLUSIONS: Our findings, supported by meta-analysis of previous literature, suggest the presence of two different pathogenetic mechanisms: late onset phenotypes may arise through loss of function secondary to haploinsufficiency, while dominant negative or toxic gain of function mechanisms may explain the severity of early phenotypes. This model of patient stratification may help patient management and facilitate future studies aimed at deciphering lamin A/C pathogenesis.

RAGE-NF-kappaB pathway activation in response to oxidative stress in facioscapulohumeral muscular dystrophy.

OBJECTIVES: An increased expression of adenine nucleotide translocator (ANT1), found in facioscapulohumeral muscular dystrophy (FSHD), is known to lead to a decrease in nuclear factor-kappaB (NF-kappaB) DNA binding and to sensitize muscle cells to oxidative stress and apoptosis. Receptor for advanced glycation end products (RAGE) mediated by NF-kappaB activation is involved in proinflammatory pathomechanism and in muscle fiber regeneration in inflammatory myopathies and in limb girdle muscular dystrophy. Oxidative stress can stimulate RAGE- NF-kappaB pathway. Our purpose was to verify if oxidative stress may induce RAGE- NF-kappaB pathway activation in FSHD, contributing to the pathogenesis of such a disease. MATERIALS AND METHODS: On muscle samples of eight patients with FSHD, eight patients with Duchenne muscular dystrophy and eight normal controls the following studies were carried out: immunocytochemistry for activated NF-kappaB; electrophoretic mobility shift assay of NF-kappaB DNA binding activity; Western blot studies of RAGE and ANT1; hydrogen peroxide (HP), peroxidase and glutathione peroxidase (GPx) assays. RESULTS: An increased RAGE and ANT1 expression in FSHD with moderate increase of NF-kappaB DNA binding activity was found together with an increased production of HP and a reduced activity of peroxidase and GPx. CONCLUSIONS: Our data confirm that response to oxidative stress and ANT1 increased activity are early events in FSHD muscle. The study also reveals that the RAGE- NF-kappaB pathway, induced by oxidative stress, is activated independently of the presence of a clear histochemical evidence of muscle damage in FSHD.