Physical activity practiced at a young age is associated with a less severe subsequent clinical presentation in facioscapulohumeral muscular dystrophy.

BACKGROUND: In facioscapulohumeral muscular dystrophy (FSHD), it is not known whether physical activity (PA) practiced at young age is associated with the clinical presentation of disease. To assess this issue, we performed a retrospective cohort study concerning the previous practice of sports and, among them, those with medium-high cardiovascular commitment in clinically categorized carriers of a D4Z4 reduced allele (DRA). METHODS: People aged between 18 and 60 were recruited as being DRA carriers. Subcategory (classical phenotype, A; incomplete phenotype, B; asymptomatic carriers, C; complex phenotype, D) and FSHD score, which measures muscle functional impairment, were assessed for all participants. Information on PAs was retrieved by using an online survey dealing with the practice of sports at a young age. RESULTS: 368 participants were included in the study, average age 36.6 years (SD = 9.4), 47.6% male. The FSHD subcategory A was observed in 157 (42.7%) participants with average (± SD) FSHD score of 5.8 ± 3.0; the incomplete phenotype (category B) in 46 (12.5%) participants (average score 2.2 ± 1.7) and the D phenotype in 61 (16.6%, average score 6.5 ± 3.8). Asymptomatic carriers were 104 (subcategory C, 28.3%, score 0.0 ± 0.2). Time from symptoms onset was higher for patients with A (15.8 ± 11.1 years) and D phenotype (13.3 ± 11.9) than for patients with B phenotype (7.3 ± 9.0). The practice of sports was associated with lower FSHD score (-17%) in participants with A phenotype (MR = 0.83, 95% CI = 0.73-0.95, p = 0.007) and by 33% in participants with D phenotype (MR = 0.67, 95% CI = 0.51-0.89, p = 0.006). Conversely, no improvement was observed in participants with incomplete phenotype with mild severity (B). CONCLUSIONS: PAs at a young age are associated with a lower clinical score in the adult A and D FSHD subcategories. These results corroborate the need to consider PAs at the young age as a fundamental indicator for the correct clinical stratification of the disease and its possible evolution.

A novel in-frame deletion in MYOT causes an early adult onset distal myopathy.

Missense mutations in MYOT encoding the sarcomeric Z-disk protein myotilin cause three main myopathic phenotypes including proximal limb-girdle muscular dystrophy, spheroid body myopathy, and late-onset distal myopathy. We describe a family carrying a heterozygous MYOT deletion (Tyr4_His9del) that clinically was characterized by an early-adult onset distal muscle weakness and pathologically by a myofibrillar myopathy (MFM). Molecular modeling of the full-length myotilin protein revealed that the 4-YERPKH-9 amino acids are involved in local interactions within the N-terminal portion of myotilin. Injection of in vitro synthetized mutated human MYOT RNA or of plasmid carrying its cDNA sequence in zebrafish embryos led to muscle defects characterized by sarcomeric disorganization of muscle fibers and widening of the I-band, and severe motor impairments. We identify MYOT novel Tyr4_His9 deletion as the cause of an early-onset MFM with a distal myopathy phenotype and provide data supporting the importance of the amino acid sequence for the structural role of myotilin in the sarcomeric organization of myofibers.

Whole-exome sequencing in patients with protein aggregate myopathies reveals causative mutations associated with novel atypical phenotypes.

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.

A 5-year clinical follow-up study from the Italian National Registry for FSHD.

BACKGROUND: The natural history of facioscapulohumeral muscular dystrophy (FSHD) is undefined. METHODS: An observational cohort study was conducted in 246 FSHD1 patients. We split the analysis between index cases and carrier relatives and we classified all patients using the Comprehensive Clinical Evaluation Form (CCEF). The disease progression was measured as a variation of the FSHD score performed at baseline and at the end of 5-year follow-up (ΔFSHD score). FINDINGS: Disease worsened in 79.4% (112/141) of index cases versus 38.1% (40/105) of carrier relatives and advanced more rapidly in index cases (ΔFSHD score 2.3 versus 1.2). The 79.1% (38/48) of asymptomatic carriers remained asymptomatic. The highest ΔFSHD score (1.7) was found in subject with facial and scapular weakness at baseline (category A), whereas in subjects with incomplete phenotype (facial or scapular weakness, category B) had lower ΔFSHD score (0.6) p < 0.0001. CONCLUSIONS: The progression of disease is different between index cases and carrier relatives and the assessment of the CCEF categories has strong prognostic effect in FSHD1 patients.

Large genotype-phenotype study in carriers of D4Z4 borderline alleles provides guidance for facioscapulohumeral muscular dystrophy diagnosis.

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.

Non-Hematologic Toxicity of Bortezomib in Multiple Myeloma: The Neuromuscular and Cardiovascular Adverse Effects.

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.

Phenotypic Variability Among Patients With D4Z4 Reduced Allele Facioscapulohumeral Muscular Dystrophy.

Importance: Facioscapulohumeral muscular dystrophy (FSHD) is considered an autosomal dominant disorder, associated with the deletion of tandemly arrayed D4Z4 repetitive elements. The extensive use of molecular analysis of the D4Z4 locus for FSHD diagnosis has revealed wide clinical variability, suggesting that subgroups of patients exist among carriers of the D4Z4 reduced allele (DRA). Objective: To investigate the clinical expression of FSHD in the genetic subgroup of carriers of a DRA with 7 to 8 repeat units (RUs). Design, Setting, and Participants: This multicenter cross-sectional study included 422 carriers of DRA with 7 to 8 RUs (187 unrelated probands and 235 relatives) from a consecutive sample of 280 probands and 306 relatives from the Italian National Registry for FSHD collected between 2008 and 2016. Participants were evaluated by the Italian Clinical Network for FSHD, and all clinical and molecular data were collected in the Italian National Registry for FSHD database. Data analysis was conducted from January 2017 to June 2018. Main Outcomes and Measures: The phenotypic classification of probands and relatives was obtained by applying the Comprehensive Clinical Evaluation Form which classifies patients in the 4 following categories: (1) participants presenting facial and scapular girdle muscle weakness typical of FSHD (category A, subcategories A1-A3), (2) participants with muscle weakness limited to scapular girdle or facial muscles (category B, subcategories B1 and B2), (3) asymptomatic or healthy participants (category C, subcategories C1 and C2), and (4) participants with myopathic phenotypes presenting clinical features not consistent with FSHD canonical phenotype (category D, subcategories D1 and D2). Results: A total of 187 probands (mean [SD] age at last neurological examination, 53.5 [15.2] years; 103 [55.1%] men) and 235 relatives (mean [SD] age at last neurologic examination, 45.1 [17.0] years; 104 [44.7%] men) with a DRA with 7 to 8 RUs and a molecular diagnosis of FSHD were evaluated. Of 187 probands, 99 (52.9%; 95% CI, 45.7%-60.1%) displayed the classic FSHD phenotype, whereas 86 (47.1%; 95% CI, 39.8%-54.3%) presented incomplete or atypical phenotypes. Of 235 carrier relatives from 106 unrelated families, 124 (52.8%; 95% CI, 46.4%-59.7%) had no motor impairment, whereas a small number (38 [16.2%; 95% CI, 9.8%-23.1%]) displayed the classic FSHD phenotype, and 73 (31.0%; 95% CI, 24.7%-38.0%) presented with incomplete or atypical phenotypes. In 37 of 106 families (34.9%; 95% CI, 25.9%-44.8%), the proband was the only participant presenting with a myopathic phenotype, while only 20 families (18.9%; 95% CI, 11.9%-27.6%) had a member with autosomal dominant FSHD. Conclusions and Relevance: This study found large phenotypic variability associated with individuals carrying a DRA with 7 to 8 RUs, in contrast to the indication that a positive molecular test is the only determining aspect for FSHD diagnosis. These findings suggest that carriers of a DRA with 7 to 8 RUs constitute a genetic subgroup different from classic FSHD. Based on these results, it is recommended that clinicians use the Comprehensive Clinical Evaluation Form for clinical classification and, whenever possible, study the extended family to provide the most adequate clinical management and genetic counseling.

Interpretation of the Epigenetic Signature of Facioscapulohumeral Muscular Dystrophy in Light of Genotype-Phenotype Studies.

Facioscapulohumeral muscular dystrophy (FSHD) is characterized by incomplete penetrance and intra-familial clinical variability. The disease has been associated with the genetic and epigenetic features of the D4Z4 repetitive elements at 4q35. Recently, D4Z4 hypomethylation has been proposed as a reliable marker in the FSHD diagnosis. We exploited the Italian Registry for FSHD, in which FSHD families are classified using the Clinical Comprehensive Evaluation Form (CCEF). A total of 122 index cases showing a classical FSHD phenotype (CCEF, category A) and 110 relatives were selected to test with the receiver operating characteristic (ROC) curve, the diagnostic and predictive value of D4Z4 methylation. Moreover, we performed DNA methylation analysis in selected large families with reduced penetrance characterized by the co-presence of subjects carriers of one D4Z4 reduced allele with no signs of disease or presenting the classic FSHD clinical phenotype. We observed a wide variability in the D4Z4 methylation levels among index cases revealing no association with clinical manifestation or disease severity. By extending the analysis to family members, we revealed the low predictive value of D4Z4 methylation in detecting the affected condition. In view of the variability in D4Z4 methylation profiles observed in our large cohort, we conclude that D4Z4 methylation does not mirror the clinical expression of FSHD. We recommend that measurement of this epigenetic mark must be interpreted with caution in clinical practice.

Acute Sarcomeric M-Line Disease Associated With ATP Synthase Subunit α Autoantibodies in Ankylosing Spondylitis.

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.

Identification and characterization of three novel mutations in the CASQ1 gene in four patients with tubular aggregate myopathy.

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.

Bortezomib-Induced Muscle Toxicity in Multiple Myeloma.

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.

The italian limb girdle muscular dystrophy registry: Relative frequency, clinical features, and differential diagnosis.

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.

A novel clinical tool to classify facioscapulohumeral muscular dystrophy phenotypes.

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.

Clinical expression of facioscapulohumeral muscular dystrophy in carriers of 1-3 D4Z4 reduced alleles: experience of the FSHD Italian National Registry.

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.

Evidence for caspase-dependent programmed cell death along with repair processes in affected skeletal muscle fibres in patients with mitochondrial disorders.

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.

Immunoblot as a potential diagnostic tool for myofibrillar myopathies.

Myofibrillar myopathies (MFMs) are a group of inherited or sporadic neuromuscular disorders morphologically characterized by foci of myofibril dissolution, disintegration of the Z-disk, and insoluble protein aggregates within the muscle fibers. The diagnosis is based on muscle biopsy. Light and electron microscopy has a central role in the diagnostic work up, and immunohistochemistry shows abnormal deposition of several proteins including αB-crystallin, desmin, and myotilin. In contrast, immunoblotting does not have any diagnostic value because it does not highlight differences in the amount of involved proteins. We investigated the pattern and level expression of desmin, αB-crystallin, myotilin, and ZASP (Z-band alternatively spliced PDZ motif-containing protein) in muscle of seven patients with MFMs by immunoblotting after SDS-PAGE and 2D-PAGE using two different solubilizing solutions, one radioimmunoprecipitation assay (RIPA) buffer, and the other urea-containing buffer. Our data demonstrated that urea-containing buffer improves the solubilization and recovery of desmin, αB-crystallin, myotilin, and ZASP as compared with RIPA buffer and that the total content of these proteins is increased in muscles of patients. The present results provide evidence that immunoblotting is an additional tool for confirming diagnosis of MFMs.

Abnormal expression of RNA polymerase II-associated proteins in muscle of patients with myofibrillar myopathies.

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.

Autophagy, inflammation and innate immunity in inflammatory myopathies.

Autophagy has a large range of physiological functions and its dysregulation contributes to several human disorders, including autoinflammatory/autoimmune diseases such as inflammatory myopathies (IIMs). In order to better understand the pathogenetic mechanisms of these muscular disorders, we sought to define the role of autophagic processes and their relation with the innate immune system in the three main subtypes of IIM, specifically sporadic inclusion body myositis (sIBM), polymyositis (PM), dermatomyositis (DM) and juvenile dermatomyositis (JDM). We found that although the mRNA transcript levels of the autophagy-related genes BECN1, ATG5 and FBXO32 were similar in IIM and controls, autophagy activation in all IIM subgroups was suggested by immunoblotting results and confirmed by immunofluorescence. TLR4 and TLR3, two potent inducers of autophagy, were highly increased in IIM, with TLR4 transcripts significantly more expressed in PM and DM than in JDM, sIBM and controls, and TLR3 transcripts highly up-regulated in all IIM subgroups compared to controls. Co-localization between autophagic marker, LC3, and TLR4 and TLR3 was observed not only in sIBM but also in PM, DM and JDM muscle tissues. Furthermore, a highly association with the autophagic processes was observed in all IIM subgroups also for some TLR4 ligands, endogenous and bacterial HSP60, other than the high-mobility group box 1 (HMGB1). These findings indicate that autophagic processes are active not only in sIBM but also in PM, DM and JDM, probably in response to an exogenous or endogenous 'danger signal'. However, autophagic activation and regulation, and also interaction with the innate immune system, differ in each type of IIM. Better understanding of these differences may lead to new therapies for the different IIM types.

A mutation in the CASQ1 gene causes a vacuolar myopathy with accumulation of sarcoplasmic reticulum protein aggregates.

A missense mutation in the calsequestrin-1 gene (CASQ1) was found in a group of patients with a myopathy characterized by weakness, fatigue, and the presence of large vacuoles containing characteristic inclusions resulting from the aggregation of sarcoplasmic reticulum (SR) proteins. The mutation affects a conserved aspartic 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 vacuoles containing aggregates of the mutant CASQ1 protein that resemble those observed in muscle biopsies of patients. 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.