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Background Myofibrillar myopathies (MFM) are a subgroup of protein aggregate myopathies (PAM) characterized by a common histological picture of myofibrillar dissolution, Z-disk disintegration, and accumulation of degradation products into inclusions. Mutations in genes encoding components of the Z-disk or Z-disk-associated proteins occur in some patients whereas in most of the cases, the causative gene defect is still unknown. We aimed to search for pathogenic mutations in genes not previously associated with MFM phenotype. Methods We performed whole-exome sequencing in four patients from three unrelated families who were diagnosed with PAM without aberrations in causative genes for MFM. Results In the first patient and her affected daughter, we identified a heterozygous p.(Arg89Cys) missense mutation in LMNA gene which has not been linked with PAM pathology before. In the second patient, a heterozygous p.(Asn4807Phe) mutation in RYR1 not previously described in PAM represents a novel, candidate gene with a possible causative role in the disease. Finally, in the third patient and his symptomatic daughter, we found a previously reported heterozygous p.(Cys30071Arg) mutation in TTN gene that was clinically associated with cardiac involvement. Conclusions Our study identifies a new genetic background in PAM pathology and expands the clinical phenotype of known pathogenic mutations.

Muscular dystrophies in dogs and cats represent a heterogeneous group of inherited, sometimes congenital, but infrequently diagnosed, progressive neuromuscular disorders. A correct identification and characterization of canine and feline muscular dystrophies could increase diagnostic and treatment strategies for veterinary neurologists and could identify useful animal models for the study of human dystrophies. However, in dogs and cats, diagnosis of muscular dystrophies is challenging due to a nonspecific clinical phenotype and pathological lesions, thus is most likely underestimated. We performed immunofluorescence and Western blot techniques using a wide panel of antibodies against proteins involved in human dystrophies (dystrophin mid-rod and carboxyterminal domain, α, β, γ, and δ-sarcoglycan, α-dystroglycan, caveolin-3, emerin, merosin, dysferlin, calpain-3, spectrin epitopes), on 9 canine and 3 feline muscle biopsies characterized by myopathic changes. Dystrophin deficiency was detected in 3 dogs and 2 novel canine muscular dystrophies have been identified, characterized by deficiency of caveolin-3 and calpain-3, respectively. In 2 cats, deficiency of β-SG and carboxyterminal domain of dystrophin in all muscle fibers has been detected. Performing immunofluorescence and Western blot analyses with a wider panel of antibodies allowed a correct identification of muscular dystrophies in dogs and cats and provides a direction for subsequent targeted genetic testing.

Facioscapulohumeral muscular dystrophy (FSHD) is a myopathy with prevalence of 1 in 20,000. Almost all patients affected by FSHD carry deletions of an integral number of tandem 3.3 kilobase repeats, termed D4Z4, located on chromosome 4q35. Assessment of size of D4Z4 alleles is commonly used for FSHD diagnosis. However, the extended molecular testing has expanded the spectrum of clinical phenotypes. In particular, D4Z4 alleles with 9–10 repeat have been found in healthy individuals, in subjects with FSHD or affected by other myopathies. These findings weakened the strict relationship between observed phenotypes and their underlying genotypes, complicating the interpretation of molecular findings for diagnosis and genetic counseling. In light of the wide clinical variability detected in carriers of D4Z4 alleles with 9–10 repeats, we applied a standardized methodology, the Comprehensive Clinical Evaluation Form (CCEF), to describe and characterize the phenotype of 244 individuals carrying D4Z4 alleles with 9–10 repeats (134 index cases and 110 relatives). The study shows that 54.5% of index cases display a classical FSHD phenotype with typical facial and scapular muscle weakness, whereas 20.1% present incomplete phenotype with facial weakness or scapular girdle weakness, 6.7% display minor signs such as winged scapula or hyperCKemia, without functional motor impairment, and 18.7% of index cases show more complex phenotypes with atypical clinical features. Family studies revealed that 70.9% of relatives carrying 9–10 D4Z4 reduced alleles has no motor impairment, whereas a few relatives (10.0%) display a classical FSHD phenotype. Importantly all relatives of index cases with no FSHD phenotype were healthy carriers. These data establish the low penetrance of D4Z4 alleles with 9–10 repeats. We recommend the use of CCEF for the standardized clinical assessment integrated by family studies and further molecular investigation for appropriate diagnosis and genetic counseling. Especially in presence of atypical phenotypes and/or sporadic cases with all healthy relatives is not possible to perform conclusive diagnosis of FSHD, but all these cases need further studies for a proper diagnosis, to search novel causative genetic defects or investigate environmental factors or co-morbidities that may trigger the pathogenic process. These evidences are also fundamental for the stratification of patients eligible for clinical trials. Our work reinforces the value of large genotype–phenotype studies to define criteria for clinical practice and genetic counseling in rare diseases.

Simple Summary Multiple myeloma (MM) is a still uncurable tumor of mainly elderly patients originating from the terminally differentiated B cells. Introduction to the treatment of MM patients of a new class of drugs called proteasome inhibitors (bortezomib followed by carfilzomib and ixazomib) significantly improved disease control. Proteasome inhibitors interfere with the major mechanism of protein degradation in a cell leading to the severe imbalance in the protein turnover that is deadly to MM cells. Currently, these drugs are the mainstream of MM therapy but are also associated with an increased rate of the injuries to multiple organs and tissues. In this review, we summarize the current knowledge on the molecular mechanisms of the first-in-class proteasome inhibitor bortezomib-induced disturbances in the function of peripheral nerves and cardiac and skeletal muscle. Abstract The overall approach to the treatment of multiple myeloma (MM) has undergone several changes during the past decade. and proteasome inhibitors (PIs) including bortezomib, carfilzomib, and ixazomib have considerably improved the outcomes in affected patients. The first-in-class selective PI bortezomib has been initially approved for the refractory forms of the disease but has now become, in combination with other drugs, the backbone of the frontline therapy for newly diagnosed MM patients, as well as in the maintenance therapy and relapsed/refractory setting. Despite being among the most widely used and highly effective agents for MM, bortezomib can induce adverse events that potentially lead to early discontinuation of the therapy with negative effects on the quality of life and outcome of the patients. Although peripheral neuropathy and myelosuppression have been recognized as the most relevant bortezomib-related adverse effects, cardiac and skeletal muscle toxicities are relatively common in MM treated patients, but they have received much less attention. Here we review the neuromuscular and cardiovascular side effects of bortezomib. focusing on the molecular mechanisms underlying its toxicity. We also discuss our preliminary data on the effects of bortezomib on skeletal muscle tissue in mice receiving the drug.

Mitochondrial diseases (MDs) are a group of heterogeneous disorders due to impaired oxidative phosphorylation causing defective ATP production. The histopathological hallmark is the presence ofragged-red fibers (RRFs), muscle fibers with excessive mitochondrial proliferation. Nitric oxide synthases (NOSs) are enzymes responsible of the synthesis of nitric oxide (NO), a ubiquitous signaling molecule involved in many physio-pathological processes. Three NOS isoenzymes have been identified so far including neuronal NOS (NOS1), inducible NOS (NOS2) and endothelial NOS (NOS3). Despite the expression and the subcellular localization of NOS1 and NOS3 have been previously investigated, a possible involvement of NOS2 in MDs has never been assessed. We evaluated the expression of NOS2 in muscle biopsies from 17 patients with mitochondrial respiratory chain dysfunction. Our data demonstrate that NOS2 is overexpressed in RRFs and the correspondence between NOS2 immunoreactivity and SDH staining suggests that the protein localizes to the mitochondria. Together with previous studies from the literature, these findings indicate a possible role NOSs in the pathogenic events leading to MDs

Background: Reduction of tandemly arrayed D4Z4 repeats is diagnostic for facioscapulohumeral muscular dystrophy (FSHD). However, various clinical phenotypes have been observed in carriers of this molecular lesion. Currently their natural history is obscure. Methods: A prospective observational cohort study is conducted to quantify disease progression over five-year follow-up (2013-2016) in246 carriers of D4Z4 reduced alleles DRA with 1-10 RU from the Italian National Registry for FSHD recruited between January 10th, 2007 and December 20th, 2011. Participants were subdivided in probands (141, males 84) and relatives (105, males 52). Muscle impairment was measured as FSHD score and disease progression as ΔFSHD score. Clinical phenotypes were assessed and categorized on the basis of the Comprehensive Clinical Evaluation Form (CCEF). Findings: The highest ΔFSHD score (1·7) was found in subjects with full FSHD phenotype (category A) or with complex/atypical diseases (category D); subjects with incomplete clinical phenotype (category B) had lower ΔFSHD score (0·6, p

Mitochondrial disorders are heterogeneous multisystemic disorders due to impaired oxidative phosphorylation causing defective mitochondrial energy production. Common histological hallmarks of mitochondrial disorders are RRFs (ragged red fibres), muscle fibres with abnormal focal accumulations of mitochondria. In contrast with the growing understanding of the genetic basis of mitochondrial disorders, the fate of phenotypically affected muscle fibres remains largely unknown. We investigated PCD (programmed cell death) in muscle of 17 patients with mitochondrial respiratory chain dysfunction. We documented that in affected muscle fibres, nuclear chromatin is condensed in lumpy irregular masses and cytochrome c is released into the cytosol to activate, along with Apaf-1 (apoptotic protease-activating factor 1), caspase 9 that, in turn, activates effector caspase 3, caspase 6, and caspase 7, suggesting the execution of the intrinsic apoptotic pathway. Whereas active caspase 3 underwent nuclear translocation, AIF (apoptosis-inducing factor) mainly stayed within mitochondria, into which an up-regulated Bax is relocated. The significant increase in caspase 2, caspase 3 and caspase 6 activity strongly suggest that the cell death programme is caspase-dependent and the activation of caspase 2 together with PUMA (p53 up-regulated modulator of apoptosis) up-regulation point to a role for oxidative stress in triggering the intrinsic pathway. Concurrently, in muscle of patients, the number of satellite cells was significantly increased and myonuclei were detected at different stages of myogenic differentiation, indicating that a reparative programme is ongoing in muscle of patients with mitochondrial disorders. Together, these data suggest that, in patients with mitochondrial disorders, affected muscle fibres are trapped in a mitochondria-regulated caspase-dependent PCD while repairing events take place.

M-line is the narrow transverse band located in the center of the sarcomeric A-band that is mainly responsible for the stabilization of myosin thick filaments. A 27-year-old male patient with a positive medical history for ankylosing spondylitis presented with one month of proximal upper limb muscle weakness associated with pain on both acromioclavicular joints. A biopsy of deltoid muscle documented the disappearance of M-line, the misalignment of myofilaments, and the loss of the distinction between the A and I bands. Complete resolution of muscle weakness occurred after one year of treatment with antiTNFα agent Etanercept. Because of the acute onset of symptoms and the recovery after immunosuppressive treatment we hypothesized that an immune-mediated mechanism was responsible for the muscle disorder. The serum IgG-mediated autoreactivity to skeletal muscle antigens resolved by bidimensional electrophoresis was assessed in the described patient and compared with that of control subjects. The comparative analysis of the immunoreactive spots revealed that ATP synthase subunit α is specifically recognized by patient's serum, suggesting that the protein might represent a putative antigenic target in the disease. This study reports an acute reversible myopathy pathologically characterized by M-line involvement and associated with serological antibodies to the subunit α of ATP synthase.

Brody disease (BD, OMIM #601003) is an inherited skeletal muscle disease clinically characterized by exercise-induced impairment of muscle relaxation and stiffness due to the delay in the Ca2+ re-uptake in the sarcoplasmic reticulum (SR)[1, 2]. The delayed muscle relaxation mainly affects legs, harms, hands and eyelids and usually improves after a few minutes rest [1-3]. Patients frequently report myalgia, painless or mildly painful cramps whereas recurrent rhabdomyolysis have been described in a few cases [2, 3]. A reduction in sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) activity characterizes the skeletal muscle of these patients [2-5]. This article is protected by copyright. All rights reserved.

Aims Myofibrillar myopathies (MFMs) are a group of inherited or sporadic neuromuscular disorders characterized morphologically by foci of myofibril dissolution, disintegration of the Z-disk and insoluble protein aggregates within the muscle fibres. The sequential events leading to muscle fibre damage remains largely unknown. Methods and results We investigated the expression and the cellular localization of RNA polymerase II (RNAPII)-associated proteins (RPAPs) in muscle biopsies from patients with genetically proven and sporadic MFMs. Our data demonstrated that RPAP2, and to a lesser extent GPN1/RPAP4, are accumulated focally in the cytoplasm of MFM muscle fibres in which they co-localize with POLR2A/RPB1, the largest subunit of RNAPII, and correspond to αB-cystallin deposits in distribution and staining intensity. No abnormal staining for RPAP2 has been observed in muscle of patients with central cores, minicores and neurogenic target fibres. Conclusions Together, these findings could provide new insights into the molecular pathogenesis of MFMs and suggest that RPAP2 immunostaining can be a useful diagnostic tool to depict protein aggregates in MFMs.

Here, we report the identification of three novel missense mutations in the calsequestrin-1 (CASQ1) gene in four patients with tubular aggregate myopathy. These CASQ1 mutations affect conserved amino acids in position 44 (p.(Asp44Asn)), 103 (p.(Gly103Asp)), and 385 (p.(Ile385Thr)). Functional studies, based on turbidity and dynamic light scattering measurements at increasing Ca2+ concentrations, showed a reduced Ca2+ -dependent aggregation for the CASQ1 protein containing p.Asp44Asn and p.Gly103Asp mutations and a slight increase in Ca2+ -dependent aggregation for the p.Ile385Thr. Accordingly, limited trypsin proteolysis assay showed that p.Asp44Asn and p.Gly103Asp were more susceptible to trypsin cleavage in the presence of Ca2+ in comparison with WT and p.Ile385Thr. Analysis of single muscle fibers of a patient carrying the p.Gly103Asp mutation showed a significant reduction in response to caffeine stimulation, compared with normal control fibers. Expression of CASQ1 mutations in eukaryotic cells revealed a reduced ability of all these CASQ1 mutants to store Ca2+ and a reduced inhibitory effect of p.Ile385Thr and p.Asp44Asn on store operated Ca2+ entry. These results widen the spectrum of skeletal muscle diseases associated with CASQ1 and indicate that these mutations affect properties critical for correct Ca2+ handling in skeletal muscle fibers.

Multiple myeloma (MM) accounts for ∼13% of all hematologic malignancies. Bortezomib treatment is effective in MM, but can be complicated with neurological side effects. We describe a patient with symptomatic MM who had a reversible metabolic myopathy associated with bortezomib administration and pathologically characterized by excessive storage of lipid droplets together with mitochondrial abnormalities. In a single-center prospective study, 14 out of 24 patients with symptomatic MM were treated with bortezomib and, among these, 7 developed muscular signs and/or symptoms. The myopathy was characterized by a proximal muscle weakness involving lower limbs and was an early complication. Complete resolution of muscle weakness occurred after treatment discontinuation. Conversely, none of the patients who received a treatment without bortezomib developed muscular symptoms. Experimental studies demonstrate that in primary human myoblasts bortezomib at low concentrations leads to excessive storage of lipid droplets together with structural mitochondrial abnormalities, recapitulating the pathologic findings observed in patient's muscle. Our data suggest that patients treated with bortezomib should be monitored for muscular signs and/or symptoms and muscle weakness should alert the clinician to the possibility of myopathy. Bortezomib-induced metabolic myopathy is a potentially reversible entity with important implications for management and treatment of patients with MM.

Introduction Limb girdle muscular dystrophies (LGMDs) are characterized by high molecular heterogeneity, clinical overlap, and a paucity of specific biomarkers. However, their molecular definition is fundamental for prognostic and therapeutic purposes. Methods We created an Italian LGMD registry that included 370 molecularly defined patients. We reviewed detailed retrospective and prospective data and compared each LGMD subtype for differential diagnosis purposes. Results LGMD2A and 2B are the most frequent forms in Italy. The ages at disease onset, clinical progression, and cardiac and respiratory involvement can vary greatly between each LGMD subtype. In a set of extensively studied patients, targeted next-generation sequencing (NGS) identified mutations in 36.5% of cases. Introduction: Limb girdle muscular dystrophies (LGMDs) are characterized by high molecular heterogeneity, clinical overlap, and a paucity of specific biomarkers. Their molecular definition is fundamental for prognostic and therapeutic purposes. Methods: We created an Italian LGMD registry that included 370 molecularly defined patients. We reviewed detailed retrospective and prospective data and compared each LGMD subtype for differential diagnosis purposes. Results: LGMD types 2A and 2B are the most frequent forms in Italy. The ages at disease onset, clinical progression, and cardiac and respiratory involvement can vary greatly between each LGMD subtype. In a set of extensively studied patients, targeted next-generation sequencing (NGS) identified mutations in 36.5% of cases. Conclusion: Detailed clinical characterization combined with muscle tissue analysis is fundamental to guide differential diagnosis and to address molecular tests. NGS is useful for diagnosing forms without specific biomarkers, although, at least in our study cohort, several LGMD disease mechanisms remain to be identified. Muscle Nerve 55: 55–68, 2017.

An adult with Sandhoff disease presented with pure lower motor neuron phenotype. Twenty years later, he showed signs of upper motor neuron involvement. 25 years from the onset, his muscle weakness slightly worsened but he was fully independent in activities of daily living. GM2-gangliosidosis can manifest as a motor neuron disease with a slowly progressive course. The correct knowledge of the natural history can be really important to achieve the diagnosis, design new therapies and evaluate clinical trials.

A 59-year-old male patient complained, from age 36, of insidious appearance in several weeks of a circumscribed painful mass, single or multiple, under the skin with hard consistency on palpation. At first, these masses were localised on sural aspect of the legs, thereafter on arms, thighs and, occasionally, on trunk and face. They had a diameter ranging from 3 cm to 5 cm and the overlying skin tissue was normal. Pain might increase with movement causing functional limitation of the corresponding limb. In the first years, they spontaneously remitted within 2–3 months, successively in 6–7 months. Onset of these lesions was never preceded or accompanied by systemic symptoms such as fever, weakness and fatigue. A muscle biopsy was taken …

Mitochondrial diseases (MDs) are heterogeneous disorders due to impaired respiratory chain function causing defective ATP production. Although the disruption of oxidative phosphorylation is central to the MD pathophysiology, other factors may contribute to these disorders. We investigated the expression and the cellular localization of TNF-α and its receptors, TNFR1 and TNFR2, in muscle biopsies from 15 patients with mitochondrial respiratory chain dysfunction. Our data unambiguously demonstrate that TNF-α is expressed in muscle fibers with abnormal focal accumulations of mitochondria, so-called ragged red fibers, and is delivered to mitochondria where both receptors are localized. Moreover TNF receptors are differentially regulated in patients' muscle in which the expression levels of TNFR1 mRNA are decreased and those of TNFR2 mRNA are increased compared with controls. These findings suggest for the first time that TNF-α could exert a direct effect on mitochondria via its receptors.

Purpose of review To describe the incidence, major risk factors, and the clinical, electrophysiological, and histological features of critical illness myopathy (CIM). Major pathogenetic mechanisms and long-term consequences of CIM are also reviewed. Recent findings CIM is frequently associated with critical illness polyneuropathy (CIP), and may have a relevant impact on patients' outcome. CIM has an earlier onset than CIP, and recovery is faster. Loss of myosin filaments on muscle biopsy is important to diagnose CIM, and has a good prognosis. Critical illness, use of steroids, and immobility concur in causing CIM. Summary A rationale diagnostic approach to CIM using clinical, electrophysiological, and muscle biopsy investigations is important to plan adequate therapy and to predict recovery.

Objectives Facioscapulohumeral muscular dystrophy type 1 (FSHD1) has been genetically linked to reduced numbers (≤8) of D4Z4 repeats at 4q35. Particularly severe FSHD cases, characterised by an infantile onset and presence of additional extra-muscular features, have been associated with the shortest D4Z4 reduced alleles with 1–3 repeats (1–3 DRA). We searched for signs of perinatal onset and evaluated disease outcome through the systematic collection of clinical and anamnestic records of de novo and familial index cases and their relatives, carrying 1–3 DRA. Setting Italy. Participants 66 index cases and 33 relatives carrying 1–3 DRA. Outcomes The clinical examination was performed using the standardised FSHD evaluation form with validated inter-rater reliability. To investigate the earliest signs of disease, we designed the Infantile Anamnestic Questionnaire (IAQ). Comparison of age at onset was performed using the non-parametric Wilcoxon rank-sum or Kruskal-Wallis test. Comparison of the FSHD score was performed using a general linear model and Wald test. Kaplan-Meier survival analysis was used to estimate the age-specific cumulative motor impairment risk. Results No patients had perinatal onset. Among index cases, 36 (54.5%) showed the first signs by 10 years of age. The large majority of patients with early disease onset (26 out of 36, 72.2%) were de novo; whereas the majority of patients with disease onset after 10 years of age were familial (16, 53.3%). Comparison of the disease severity outcome between index cases with age at onset before and over 10 years of age, failed to detect statistical significance (Wald test p value=0.064). Of 61 index cases, only 17 (27.9%) presented extra-muscular conditions. Relatives carrying 1–3 DRA showed a large clinical variability ranging from healthy subjects, to patients with severe motor impairment. Conclusions The size of the D4Z4 allele is not always predictive of severe clinical outcome. The high degree of clinical variability suggests that additional factors contribute to the phenotype complexity.

Contains fulltext : 167645.pdf (Publisher’s version ) (Closed access) Sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) pumps play the major role in lowering cytoplasmic calcium concentration in skeletal muscle by catalyzing the ATP-dependent transport of Ca(2+) from the cytosol to the lumen of the sarcoplasmic reticulum (SR). Although SERCA abnormalities have been hypothesized to contribute to the dysregulation of intracellular Ca(2+) homeostasis and signaling in muscle of patients with myotonic dystrophy (DM) and hypothyroid myopathy, the characterization of SERCA pumps remains elusive and their impairment is still unclear. We assessed the activity of SR Ca(2+)-ATPase, expression levels and fiber distribution of SERCA1 and SERCA2, and oligomerization of SERCA1 protein in muscle of patients with DM type 1 and 2, and with hypothyroid myopathy. Our data provide evidence that SR Ca(2+) ATPase activity, protein levels and muscle fiber distribution of total SERCA1 and SERCA2, and SERCA1 oligomerization pattern are similar in patients with both DM1 and DM2, hypothyroid myopathy and in control subjects. We prove that SERCA1b, the neonatal isoform of SERCA1, is expressed at protein level in muscle of patients with DM2 and, in lower amount, of patients with DM1. Our present study demonstrates that SERCA function is not altered in muscle of patients with DM and with hypothyroid myopathy.

Based on the 7-year experience of the Italian Clinical Network for FSHD, we revised the FSHD clinical form to describe, in a harmonized manner, the phenotypic spectrum observed in FSHD. The new Comprehensive Clinical Evaluation Form (CCEF) defines various clinical categories by the combination of different features. The inter-rater reproducibility of the CCEF was assessed between two examiners using kappa statistics by evaluating 56 subjects carrying the molecular marker used for FSHD diagnosis. The CCEF classifies: (1) subjects presenting facial and scapular girdle muscle weakness typical of FSHD (category A, subcategories A1–A3), (2) subjects with muscle weakness limited to scapular girdle or facial muscles (category B subcategories B1, B2), (3) asymptomatic/healthy subjects (category C, subcategories C1, C2), (4) subjects with myopathic phenotype presenting clinical features not consistent with FSHD canonical phenotype (D, subcategories D1, D2). The inter-rater reliability study showed an excellent concordance of the final four CCEF categories with a κ equal to 0.90; 95 % CI (0.71; 0.97). Absolute agreement was observed for categories C and D, an excellent agreement for categories A [κ = 0.88; 95 % CI (0.75; 1.00)], and a good agreement for categories B [κ = 0.79; 95 % CI (0.57; 1.00)]. The CCEF supports the harmonized phenotypic classification of patients and families. The categories outlined by the CCEF may assist diagnosis, genetic counseling and natural history studies. Furthermore, the CCEF categories could support selection of patients in randomized clinical trials. This precise categorization might also promote the search of genetic factor(s) contributing to the phenotypic spectrum of disease. Electronic supplementary material The online version of this article (doi:10.1007/s00415-016-8123-2) contains supplementary material, which is available to authorized users.

A 10-year-old neutered female domestic longhaired cat was referred for evaluation of forelimb weakness and lameness. There was hypertrophy and firmness of the musculature with no neurological deficits. Moderate increase of creatine kinase activity was present. Muscle biopsy showed rounded atrophic and hypertrophic fibres, an increased number of centrally located myofibre nuclei, scattered rimmed vacuoles and mild perimysial and endomysial fibrosis. Myofibre necrosis with phagocytosis was present in the gluteal muscle. Immunohistochemistry revealed absence of sarcolemmal caveolin-3 in almost all muscle fibres and sarcoplasmic accumulation of the protein in approximately 30% of myofibres. Normal expression of caveolin-3 was detected by immunoblotting, so protein mislocalization in the sarcoplasm due to aberrant trafficking towards the sarcolemma was suspected. This case represents the first example of muscular dystrophy due to a caveolinopathy in animals.

MDs (mitochondrial diseases) are a clinically heterogeneous group of disorders characterized by impairment of the respiratory chain function with altered oxidative phosphorylation. We tested the hypothesis that the function of vascular endothelium is affected by increased oxidative stress in MDs. A total of 12 patients with MDs and pair-matched controls were studied. Endothelial function was assessed by measuring FMD (flow-mediated vasodilation) of brachial and common femoral arteries. The test was repeated after vitamin C (500 mg, twice a day) and E (400 mg, once a day) supplementation for 30 days and 90 days after vitamin withdrawal. FMD was reduced in patients compared with controls [AUC/τ (time-averaged area under the curve) for the brachial artery, 1.05±0.24 compared with 4.19±0.59% respectively, P

Mitochondria are implicated in several metabolic pathways including cell respiratory processes, apoptosis, and free radical production. Mitochondrial abnormalities have been documented in neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, and amyotrophic lateral sclerosis. Several studies have demonstrated that mitochondrial impairment plays an important role in the pathogenesis of this group of disorders. In this review, we discuss the role of mitochondria in the main neurodegenerative diseases and review the updated knowledge in this field.

To define numerically the clinical severity of facioscapulohumeral muscular dystrophy (FSHD), we developed a protocol that quantifies muscle weakness by combining the functional evaluation of six muscle groups affected in this disease. To validate reproducibility of the protocol, 69 patients were recruited. Each patient was evaluated by at least five neurologists, and an FSHD severity score was given by each examiner. The degree of agreement among clinicians' evaluations was measured by kappa-statistics. Nineteen subjects received a score between 0 and 1, 9 had a score between 2 and 4, 20 received a score between 5 and 10, and 8 had a score between 11 and 15. Of the 13 subjects with D4Z4 alleles within the normal range (ranging from 10 to 150 repeats), 12 obtained a score of 0 and only 1 had a score of 1. Kappa-statistics showed a very high concordance for all muscle groups. We developed a simple, reliable, easily used tool to define the clinical expression of FSHD. Longitudinal studies will assess its sensitivity and utility in measuring changes for widespread use.

Contains fulltext : 88384.pdf (Publisher’s version ) (Closed access) Brody disease is an inherited disorder of skeletal muscle function characterized by increasing impairment of relaxation during exercise. The autosomal recessive form can be caused by mutations in the ATP2A1 gene, which encodes for the sarcoplasmic/endoplasmic reticulum Ca-ATPase 1 (SERCA1) protein. We studied 2 siblings affected by Brody disease. The patients complained of exercise-induced delay of muscle relaxation and stiffness since childhood and had gene analysis of ATP2A1. Morphologic and biochemical studies were performed on a muscle biopsy from 1 patient. The biopsy showed fiber size variation and increased numbers of fibers with internal nuclei. Ultrastructural examination revealed dilatation of lateral cisternae and proliferation of tubular elements of the sarcoplasmic reticulum. By immunohistochemistry, SERCA1 was expressed in a normal pattern, but sarcoplasmic reticulum Ca-ATPase activity was significantly reduced. Immunoblotting after high-resolution 2-dimensional gel electrophoresis showed a significant difference in the amount of SERCA1 protein between the patient and controls. Both patients were found to have 2 previously unreported in-frame deletions in ATP2A1. Because SERCA1 protein has specific biochemical characteristics in our patient, these results underline the importance of a pathologic and biochemical analyses for the diagnosis. In addition, we describe 2 novel mutations in the ATP2A1 gene. 01 maart 2010

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disease associated with a partial deletion on chromosome 4q35. Few relevant investigations have been reported on its epidemiology and were essentially based on clinical diagnosis, having been performed before recognition of the molecular mutation. We report an epidemiological survey on FSHD patients, in which the diagnosis was obtained by combined clinical and molecular evaluation. The survey concerned the north-east Italian province of Padova, an area of 871,190 inhabitants (1 January 2004). We identified 40 patients affected by FSHD based on clinical diagnosis. In 33 of them, the EcoRI fragment size in the 4q35 region ranged from 14 to 35 kb. Four other patients belonging to the same family harbored a 38-kb fragment. In these four cases, the relationship between the borderline deletion with the mild FSHD phenotype was corroborated by additional haplotype reconstruction and segregation analysis. Interestingly, the same mild facial-sparing clinical pattern was apparent only in one other patient with an EcoRI fragment of 32 kb, suggesting that this unusual FSHD phenotype may be due to very small 4q35 deletions. On the whole, estimating a prevalence rate of 44 x 10(-6), our survey confirmed FSHD as one of the most frequent neuromuscular disorders in Western populations.

We describe the case of a 59 year old man with systemic sclerosis and superficial siderosis of the central nervous system characterized by progressive worsening of walking with signs of pyramidal liberation associated with cerebellar ataxia and bilateral hypoacusia. Ferritin and oligoclonal bands were found on lumbar puncture and Magnetic Resonance Imaging of the brain and spinal cord revealed a rim of hypointensity in T2-weighted images enveloping the surface of brain and spinal cord, typical of hemosiderosis. Neurological complications, both central and peripheral, are thought to be rare and coincidental in systemic sclerosis; however over the past decade, some authors have reported a high incidence (up to 40%) of neurological involvement in the disease. In this report we consider the hypothesis that superficial siderosis may be a direct complication of systemic sclerosis. To our knowledge, this case represents the first description of superficial siderosis in a patient with systemic sclerosis.

The ataxic form of chronic inflammatory demyelinating polyradiculoneuropathy (ataxic-CIDP) has been recently described as a subtype of chronic ataxic neuropathy, distinguished by steroid responsiveness and relative preservation of myelinated fibres at sural nerve biopsy. We report on a case of progressive, predominantly sensory, steroid-responsive neuropathy with clinical, laboratory, electrophysiological and pathological features of this uncommon form of CIDP. Moreover, the present case displays peculiar hyperpyrexia-triggered relapses leading to transitory severe tetraparesis, bilateral facial drooping, dysphonia, dysphagia and dyspnoea, which leave clinicians with some unresolved questions.

We describe four patients, from four different families, affected by a mild myopathy or asymptomatic elevated serum creatine kinase levels, in whom toluidine blue-stained semithin sections of muscle specimens revealed inclusions of different size and shape. The inclusions did not stain by routine histochemical studies. The sarcoplasmic or endoplasmic reticulum calcium 1 (SERCA1) ATPase and/or calsequestrin reactivity of inclusions, by immunohistochemistry, and the SERCA1- and calsequestrin-increased expression, by immunoblot, suggested that inclusions were constituted by an excess of proteins normally present in the terminal cisternae of sarcoplasmic reticulum. Our cases, both sporadic and familial, represent a new type of surplus protein myopathy.

M. Filosto, P. Tonin, G. Vattemi, C. Savio, N. Rizzuto and G. Tomelleri (2003) Neuropathology and Applied Neurobiology 29, 52–59 Transcription factors c-Jun/activator protein-1 and nuclear factor-kappa B in oxidative stress response in mitochondrial diseases Mitochondrial dysfunction leads to oxygen free radical (ROS) generation with consequent oxidative stress and cellular damage. Recently, activation of the cellular antioxidant system and apoptosis were demonstrated in skeletal muscle fibres from patients with mitochondrial diseases, but the underlying mechanisms remain unknown. Hydrogen peroxide, a by-product of ROS generation, is a chemical inducer of gene expression able to activate apoptosis and to promote the antioxidant response through the activation of nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1) transcription factor. Using immunohistochemistry and confocal microscopy, we evaluated the expression of NF-κB and AP-1 in muscle biopsies from patients with mitochondrial disease. In addition, we examined the expression of factors involved in their activation, such as NF-κB inducing kinase (NIK) and phosphorylated Jun-N-terminal kinase (p-JNK). Most fibres with respiratory chain dysfunction displayed nuclear staining for activated c-Jun/AP-1, but not for NF-κB. The same fibres reacted for p-JNK. Only some ragged red fibres immunoreacted for NIK. These data suggest that AP-1 is involved in the oxidative stress response in muscle fibres from patients with mitochondrial disease.

The gold standard to characterize idiopathic inflammatory myopathies is the morphological, immunohistochemical and immunopathological analysis of muscle biopsy. Mononuclear cell infiltrates and muscle fiber necrosis are commonly shared histopathological features. Inflammatory cells that surround, invade and destroy healthy muscle fibers expressing MHC class I antigen are the typical pathological finding of polymyositis. Perifascicular atrophy and microangiopathy strongly support a diagnosis of dermatomyositis. Randomly distributed necrotic muscle fibers without mononuclear cell infiltrates represent the histopathological hallmark of immune-mediated necrotizing myopathy; meanwhile, endomysial inflammation and muscle fiber degeneration are the two main pathological features in sporadic inclusion body myositis. A correct differential diagnosis requires immunopathological analysis of the muscle biopsy and has important clinical implications for therapeutic approach. In particular, unnecessary, potentially harmful, immune-suppressive therapy should be avoided alike in dystrophic myopathies with secondary inflammation.

A missense mutation in the calsequestrin-1 gene (CASQ1) was found in a group of patients with a my- opathy characterized by weakness, fatigue, and the pres- ence of large vacuoles containing characteristic inclusions resulting from the aggregation of sarcoplasmic reticulum (SR) proteins. The mutation affects a conserved aspar- tic acid in position 244 (p.Asp244Gly) located in one of the high-affinity Ca 2+ -binding sites of CASQ1 and alters the kinetics of Ca 2+ release in muscle fibers. Expression of the mutated CASQ1 protein in COS-7 cells showed a markedly reduced ability in forming elongated polymers, whereas both in cultured myotubes and in in vivo mouse fibers induced the formation of electron-dense SR vac- uoles containing aggregates of the mutant CASQ1 protein that resemble those observed in muscle biopsies of pa- tients. Altogether, these results support the view that a single missense mutation in the CASQ1 gene causes the formation of abnormal SR vacuoles containing aggregates of CASQ1, and other SR proteins, results in altered Ca 2+ release in skeletal muscle fibers, and, hence, is responsible for the clinical phenotype observed in these patients.

Tacrolimus, also known as FK506, is an immunosuppressive agent widely used for the prevention of acute allograft rejection in organ transplantation and for the treatment of immunological diseases. This study reports two male patients who underwent solid organ transplantation (liver and kidney). After transplant, the patients received continuous immunosuppressive therapy with oral tacrolimus and later presented clinical manifestations and laboratory signs of myopathy. Muscle biopsies of both patients clearly documented an inflammatory myopathy with the histological features of polymyositis including CD8 + T cells which invaded healthy muscle fibers and expressed granzyme B and perforin, many CD68 + macrophages and MHC class I antigen upregulation on the surface of most fibers. Because of the temporal association while receiving tacrolimus and since other possible causes for myopathy were excluded, the most likely cause of polymyositis in our patients was tacrolimus toxicity. We suggest that patients on tacrolimus should be carefully monitored for serum CK levels and clinical signs of muscle disease.

Facioscapulohumeral muscular dystrophy (FSHD), a common hereditary myopathy, is characterized by atrophy and weakness of selective muscle groups. FSHD is considered an autosomal dominant disease with incomplete penetrance and unpredictable variability of clinical expression within families. Mice overexpressing FRG1 (FSHD region gene 1), a candidate gene for this disease, develop a progressive myopathy with features of the human disorder. Here, we show that in FRG1-overexpressing mice, fast muscles, which are the most affected by the dystrophic process, display anomalous fast skeletal troponin T (fTnT) isoform, resulting from the aberrant splicing of the Tnnt3 mRNA that precedes the appearance of dystrophic signs. We determine that muscles of FRG1 mice develop less strength due to impaired contractile properties of fast-twitch fibers associated with an anomalous MyHC-actin ratio and a reduced sensitivity to Ca2+. We demonstrate that the decrease of Ca2+ sensitivity of fast-twitch fibers depends on the anomalous troponin complex and can be rescued by the substitution with the wild-type proteins. Finally, we find that the presence of aberrant splicing isoforms of TNNT3 characterizes dystrophic muscles in FSHD patients. Collectively, our results suggest that anomalous TNNT3 profile correlates with the muscle impairment in both humans and mice. On the basis of these results, we propose that aberrant fTnT represents a biological marker of muscle phenotype severity and disease progression.

We describe a new mutation in the tRNA(Ala) gene, a T-->C transition at nucleotide position 5628, in a 62-year-old woman with late onset chronic progressive external ophthalmoplegia, dysphagia and mild proximal myopathy. The mutation is heteroplasmic and disrupts a highly conserved A-U base pair within the anticodon stem of the tRNA(Ala). Cytochrome c oxidase-negative fibers harbor a significantly higher level of mutated mtDNA than cytochrome c oxidase-positive fibers. This is the first mutation in the tRNA(Ala) gene which satisfies accepted criteria for pathogenicity.

Phosphoglycerate mutase (PGAM) deficiency causes a rare metabolic myopathy characterized by exercise-related myalgia and myoglobinuria. This disorder was described in 13 patients and five different mutations in the PGAM-M gene were identified. We report on a new patient with an unusual clinical presentation. As a youth, he participated in different sports without complaining of muscular symptoms, but at 44 years of age, after a brief, intense effort, he experienced lightheadedness without fainting. Serum CK was elevated and the ischemic exercise test showed a pathological lactate response. Muscle biopsy showed only mild abnormalities, but biochemical study revealed a defect of PGAM and genetic analysis showed two different mutations in the PGAM-M gene. Our case expands the clinical spectrum of PGAM deficiency and suggests that the frequency of this metabolic myopathy may be underestimated.

Item does not contain fulltext Brody disease is an inherited myopathy associated with a defective function of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 1 (SERCA1) protein. Mutations in the ATP2A1 gene have been reported only in some patients. Therefore it has been proposed to distinguish patients with ATP2A1 mutations, Brody disease (BD), from patients without mutations, Brody syndrome (BS). We performed a detailed study of SERCA1 protein expression in muscle of patients with BD and BS, and evaluated the alternative splicing of SERCA1 in primary cultures of normal human muscle and in infant muscle. SERCA1 reactivity was observed in type 2 muscle fibers of patients with and without ATP2A1 mutations and staining intensity was similar in patients and controls. Immunoblot analysis showed a significant reduction of SERCA1 band in muscle of BD patients. In addition we demonstrated that the wild type and mutated protein exhibits similar solubility properties and that RIPA buffer improves the recovery of the wild type and mutated SERCA1 protein. We found that SERCA1b, the SERCA1 neonatal form, is the main protein isoform expressed in cultured human muscle fibers and infant muscle. Finally, we identified two novel heterozygous mutations within exon 3 of the ATP2A1 gene from a previously described patient with BD.

Facioscapulohumeral muscular dystrophy has been genetically linked to reduced numbers (≤ 8) of D4Z4 repeats at 4q35 combined with 4A(159/161/168) DUX4 polyadenylation signal haplotype. However, we have recently reported that 1.3% of healthy individuals carry this molecular signature and 19% of subjects affected by facioscapulohumeral muscular dystrophy do not carry alleles with eight or fewer D4Z4 repeats. Therefore, prognosis for subjects carrying or at risk of carrying D4Z4 reduced alleles has become more complicated. To test for additional prognostic factors, we measured the degree of motor impairment in a large group of patients affected by facioscapulohumeral muscular dystrophy and their relatives who are carrying D4Z4 reduced alleles. The clinical expression of motor impairment was assessed in 530 subjects, 163 probands and 367 relatives, from 176 unrelated families according to a standardized clinical score. The associations between clinical severity and size of D4Z4 allele, degree of kinship, gender, age and 4q haplotype were evaluated. Overall, 32.2% of relatives did not display any muscle functional impairment. This phenotype was influenced by the degree of relation with proband, because 47.1% of second- through fifth-degree relatives were unaffected, whereas only 27.5% of first-degree family members did not show motor impairment. The estimated risk of developing motor impairment by age 50 for relatives carrying a D4Z4 reduced allele with 1-3 repeats or 4-8 repeats was 88.7% and 55%, respectively. Male relatives had a mean score significantly higher than females (5.4 versus 4.0, P = 0.003). No 4q haplotype was exclusively associated with the presence of disease. In 13% of families in which D4Z4 alleles with 4-8 repeats segregate, the diagnosis of facioscapulohumeral muscular dystrophy was reported only in one generation. In conclusion, this large-scale analysis provides further information that should be taken into account when counselling families in which a reduced allele with 4-8 D4Z4 repeats segregates. In addition, the reduced expression of disease observed in distant relatives suggests that a family's genetic background plays a role in the occurrence of facioscapulohumeral muscular dystrophy. These results indicate that the identification of new susceptibility factors for this disease will require an accurate classification of families.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in the gene encoding thymidine phosphorylase (TP). Allogeneic hematopoietic stem cell transplantation (HSCT) has been proposed as a treatment for patients with MNGIE and a standardized approach to HSCT in this condition has recently been developed. We report on the transplant course, management and short-term follow-up in two MNGIE patients who underwent HSCT. The source of stem cells was bone marrow taken from an HLA 9/10 allele-matched unrelated donor in the first patient and from an HLA 10/10 allele-matched sibling donor in the second. Both patients achieved full donor chimerism, and we observed restoration of buffy coat TP activity and lowered urine nucleoside concentrations in both of them. The post-transplant clinical follow-up showed improvement in gastrointestinal dysmotility, abdominal cramps and diarrhea. Neurological assessment remained unchanged. However, the first patient died 15 months after HSCT due to gastrointestinal obstruction and shock; the second patient died 8 months after the procedure due to respiratory distress following septic shock. Although HSCT corrects biochemical abnormalities and improves gastrointestinal symptoms, the procedure can be risky in subjects already in poor medical condition as are many MNGIE patients. Since transplant-related morbidity and mortality increases with progression of the disease and number of comorbidities, MNGIE patients should be submitted to HSCT when they are still relatively healthy, in order to minimize the complications of the procedure. Anyway, there is still incomplete knowledge on the natural history of the disease in many affected patients and it is not yet clear when the best time to do a transplant is. Further clues to the therapeutic potential of HSCT could result from a prolonged observation in a greater number of non-transplanted and transplanted patients, which would allow us to answer the questions of if, how and when MNGIE patients require HSCT treatment.

Background Facioscapulohumeral muscular dystrophy (FSHD) is considered an autosomal dominant disease with a prevalence of 1 in 20 000. Almost all patients with FSHD carry deletions of integral copies of tandem 3.3 kb repeats (D4Z4) located on chromosome 4q35. However, FSHD families have been reported in which individuals carrying a D4Z4-reduced allele remain asymptomatic. Recently, it has been proposed that the D4Z4-reduced allele is pathogenic only in association with the permissive haplotype, 4APAS. Methods and results Through the Italian National Registry for FSHD (INRF), genotype-phenotype correlations were extensively studied in 11 non-consanguineous families in which two D4Z4-reduced alleles segregate. Overall, 68 subjects carrying D4Z4-reduced alleles were examined, including 15 compound heterozygotes. It was found that in four families the only FSHD-affected subject was the compound heterozygote for the D4Z4-reduced allele, and 52.6% of subjects carrying a single D4Z4-reduced 4A161PAS haplotype were non-penetrant carriers; moreover, the population frequency of the 4A161PAS haplotype associated with a D4Z4-reduced allele was found to be as high as 1.2%. Conclusions This study reveals a high frequency of compound heterozygotes in the Italian population and the presence of D4Z4-reduced alleles with the 4A161PAS pathogenic haplotype in the majority of non-penetrant subjects in FSHD families with compound heterozygosity. These data suggest that carriers of FSHD-sized alleles with 4A161PAS haplotype are more common in the general population than expected on the basis of FSHD prevalence. These findings challenge the notion that FSHD is a fully penetrant autosomal dominant disorder uniquely associated with the 4A161PAS haplotype, with relevant repercussions for genetic counselling and prenatal diagnosis.

INTRODUCTION Calpain 3 deficiency causes limb girdle muscular dystrophy type 2A, which is one of the most common forms of limb girdle muscular dystrophy. Nevertheless, calpainopathy is not always associated with mutations in the specific gene and secondary reduction in protein expression has been described. CASE REPORT We report a case of a 43-year-old man who complained of thigh muscle stiffness and had muscle hypertrophy of both vastus medialis with prolonged myotonic contraction by percussion. A muscle biopsy showed dystrophic features and calpain 3 deficiency was shown by immunoblot analysis although mutations in the specific gene were not found. Known cases of secondary calpain 3 protein deficiency were ruled out and mutations in MD1 and MD2 genes were excluded. CONCLUSIONS This patient represents the first case of calpain 3 deficiency with selective pseudohypertrophy of vastus medialis muscles.

Hearing impairment is common in patients with mitochondrial disorders, affecting over half of all cases at some time in the course of the disease. In some patients, deafness is only part of a multisystem disorder. By contrast, there are also a number of “pure” mitochondrial deafness disorders, the most common probably being maternally inherited. We retrospectively analyzed the last 60 genetically confirmed mitochondrial disorders diagnosed in our Department: 28 had bilateral sensorineural hearing loss, whereas 32 didn't present ear's abnormalities, without difference about sex and age of onset between each single group of diseases. We reported also a case of MELAS patient with sensorineural hearing loss, in which cochlear implantation greatly contributed to the patient's quality of life. Our study suggests that sensorineural hearing loss is an important feature in mitochondrial disorders and indicated that cochlear implantation can be recommended for patients with MELAS syndrome and others mitochondrial disorders.