Toll-like receptors and IL-7 as potential biomarkers for immune-mediated necrotizing myopathies.

We aimed to verify whether the immune system may represent a source of potential biomarkers for the stratification of immune-mediated necrotizing myopathies (IMNMs) subtypes. A group of 22 patients diagnosed with IMNM [7 with autoantibodies against signal recognition particle (SRP) and 15 against 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR)] and 12 controls were included. A significant preponderance of M1 macrophages was observed in both SRP+ and HMGCR+ muscle samples (p < 0.0001 in SRP+ and p = 0.0316 for HMGCR+ ), with higher values for SRP+ (p = 0.01). Despite the significant increase observed in the expression of TLR4 and all endosomal Toll-like receptors (TLRs) at protein level in IMNM muscle tissue, only TLR7 has been shown considerably upregulated compared to controls at transcript level (p = 0.0026), whereas TLR9 was even decreased (p = 0.0223). Within IMNM subgroups, TLR4 (p = 0.0116) mRNA was significantly increased in SRP+ compared to HMGCR+ patients. Within IMNM group, only IL-7 was differentially expressed between SRP+ and HMGCR+ patients, with higher values in SRP+ patients (p = 0.0468). Overall, innate immunity represents a key player in pathological mechanisms of IMNM. TLR4 and the inflammatory cytokine IL-7 represent potential immune biomarkers able to differentiate between SRP+ and HMGCR+ patients.

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.

Sporadic Inclusion Body Myositis at the Crossroads between Muscle Degeneration, Inflammation, and Aging.

Sporadic inclusion body myositis (sIBM) is the most common muscle disease of older people and is clinically characterized by slowly progressive asymmetrical muscle weakness, predominantly affecting the quadriceps, deep finger flexors, and foot extensors. At present, there are no enduring treatments for this relentless disease that eventually leads to severe disability and wheelchair dependency. Although sIBM is considered a rare muscle disorder, its prevalence is certainly higher as the disease is often undiagnosed or misdiagnosed. The histopathological phenotype of sIBM muscle biopsy includes muscle fiber degeneration and endomysial lymphocytic infiltrates that mainly consist of cytotoxic CD8+ T cells surrounding nonnecrotic muscle fibers expressing MHCI. Muscle fiber degeneration is characterized by vacuolization and the accumulation of congophilic misfolded multi-protein aggregates, mainly in their non-vacuolated cytoplasm. Many players have been identified in sIBM pathogenesis, including environmental factors, autoimmunity, abnormalities of protein transcription and processing, the accumulation of several toxic proteins, the impairment of autophagy and the ubiquitin-proteasome system, oxidative and nitrative stress, endoplasmic reticulum stress, myonuclear degeneration, and mitochondrial dysfunction. Aging has also been proposed as a contributor to the disease. However, the interplay between these processes and the primary event that leads to the coexistence of autoimmune and degenerative changes is still under debate. Here, we outline our current understanding of disease pathogenesis, focusing on degenerative mechanisms, and discuss the possible involvement of aging.

Advanced Cellular Models for Rare Disease Study: Exploring Neural, Muscle and Skeletal Organoids.

Organoids are self-organized, three-dimensional structures derived from stem cells that can mimic the structure and physiology of human organs. Patient-specific induced pluripotent stem cells (iPSCs) and 3D organoid model systems allow cells to be analyzed in a controlled environment to simulate the characteristics of a given disease by modeling the underlying pathophysiology. The recent development of 3D cell models has offered the scientific community an exceptionally valuable tool in the study of rare diseases, overcoming the limited availability of biological samples and the limitations of animal models. This review provides an overview of iPSC models and genetic engineering techniques used to develop organoids. In particular, some of the models applied to the study of rare neuronal, muscular and skeletal diseases are described. Furthermore, the limitations and potential of developing new therapeutic approaches are discussed.

The FSHD jigsaw: are we placing the tiles in the right position?

PURPOSE OF REVIEW: Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common myopathies, involving over 870,000 people worldwide and over 20 FSHD national registries. Our purpose was to summarize the main objectives of the scientific community on this topic and the moving trajectories of research from the past to the present. RECENT FINDINGS: To date, research is mainly oriented toward deciphering the molecular and pathogenetic basis of the disease by investigating DUX4-mediated muscle alterations. Accordingly, FSHD drug development has been escalating in the last years in an attempt to silence DUX4 or to block its downstream effectors. Breakthroughs in the field include the awareness that new biomarkers and outcome measures are required for tracking disease progression and patient stratification. The need to develop personalized therapeutic strategies is also crucial according to the phenotypic variability observed in FSHD subjects. SUMMARY: We analysed 121 literature reports published between 2021 and 2023 to assess the most recent advances in FSHD clinical and molecular research.

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.

Human Mutated MYOT and CRYAB Genes Cause a Myopathic Phenotype in Zebrafish.

Myofibrillar myopathies (MFMs) are a group of hereditary neuromuscular disorders sharing common histological features, such as myofibrillar derangement, Z-disk disintegration, and the accumulation of degradation products into protein aggregates. They are caused by mutations in several genes that encode either structural proteins or molecular chaperones. Nevertheless, the mechanisms by which mutated genes result in protein aggregation are still unknown. To unveil the role of myotilin and αB-crystallin in the pathogenesis of MFM, we injected zebrafish fertilized eggs at the one-cell stage with expression plasmids harboring cDNA sequences of human wildtype or mutated MYOT (p.Ser95Ile) and human wildtype or mutated CRYAB (p.Gly154Ser). We evaluated the effects on fish survival, motor behavior, muscle structure and development. We found that transgenic zebrafish showed morphological defects that were more severe in those overexpressing mutant genes. which developed a myopathic phenotype consistent with that of human myofibrillar myopathy, including the formation of protein aggregates. Results indicate that pathogenic mutations in myotilin and αB-crystallin genes associated with MFM cause a structural and functional impairment of the skeletal muscle in zebrafish, thereby making this non-mammalian organism a powerful model to dissect disease pathogenesis and find possible druggable targets.

Ryanodine receptor 1 (RYR1) mutations in two patients with tubular aggregate myopathy.

Two likely causative mutations in the RYR1 gene were identified in two patients with myopathy with tubular aggregates, but no evidence of cores or core-like pathology on muscle biopsy. These patients were clinically evaluated and underwent routine laboratory investigations, electrophysiologic tests, muscle biopsy and muscle magnetic resonance imaging (MRI). They reported stiffness of the muscles following sustained activity or cold exposure and had serum creatine kinase elevation. The identified RYR1 mutations (p.Thr2206Met or p.Gly2434Arg, in patient 1 and patient 2, respectively) were previously identified in individuals with malignant hyperthermia susceptibility and are reported as causative according to the European Malignant Hyperthermia Group rules. To our knowledge, these data represent the first identification of causative mutations in the RYR1 gene in patients with tubular aggregate myopathy and extend the spectrum of histological alterations caused by mutation in the RYR1 gene.

Expanding the clinical and genetic spectrum of pathogenic variants in STIM1.

INTRODUCTION/AIMS: Stromal interaction molecule 1 (STIM1) is a reticular Ca2+ sensor composed of a luminal and a cytosolic domain. Autosomal dominant mutations in STIM1 cause tubular aggregate myopathy and Stormorken syndrome or its variant York platelet syndrome. In this study we aimed to expand the features related to new variants in STIM1. METHODS: We performed a cross-sectional study of individuals harboring monoallelic STIM1 variants recruited at five tertiary centers involved in a study of inherited myopathies analyzed with a multigene-targeted panel. RESULTS: We identified seven individuals (age range, 26-57 years) harboring variants in STIM1, including five novel changes: three located in the EF-hand domain, one in the sterile α motif (SAM) domain, and one in the cytoplasmatic region of the protein. Functional evaluation of the pathogenic variants using a heterologous expression system and measuring store-operated calcium entry demonstrated their causative role and suggested a link of new variants with the clinical phenotype. Muscle contractures, found in three individuals, showed variability in body distribution and in the number of joints involved. Three patients showed cardiac and respiratory involvement. Short stature, hyposplenism, sensorineural hearing loss, hypothyroidism, and Gilbert syndrome were variably observed among the patients. Laboratory tests revealed hyperCKemia in six patients, thrombocytopenia in two patients, and hypocalcemia in one patient. Muscle biopsy showed the presence of tubular aggregates in three patients, type I fiber atrophy in one patient, and nonspecific myopathic changes in two patients. DISCUSSION: Our clinical, histological, and molecular data expand the genetic and clinical spectrum of STIM1-related diseases.

Clinical, morphological and genetic characterization of Brody disease: an international study of 40 patients.

Brody disease is an autosomal recessive myopathy characterized by exercise-induced muscle stiffness due to mutations in the ATP2A1 gene. Almost 50 years after the initial case presentation, only 18 patients have been reported and many questions regarding the clinical phenotype and results of ancillary investigations remain unanswered, likely leading to incomplete recognition and consequently under-diagnosis. Additionally, little is known about the natural history of the disorder, genotype-phenotype correlations, and the effects of symptomatic treatment. We studied the largest cohort of Brody disease patients to date (n = 40), consisting of 22 new patients (19 novel mutations) and all 18 previously published patients. This observational study shows that the main feature of Brody disease is an exercise-induced muscle stiffness of the limbs, and often of the eyelids. Onset begins in childhood and there was no or only mild progression of symptoms over time. Four patients had episodes resembling malignant hyperthermia. The key finding at physical examination was delayed relaxation after repetitive contractions. Additionally, no atrophy was seen, muscle strength was generally preserved, and some patients had a remarkable athletic build. Symptomatic treatment was mostly ineffective or produced unacceptable side effects. EMG showed silent contractures in approximately half of the patients and no myotonia. Creatine kinase was normal or mildly elevated, and muscle biopsy showed mild myopathic changes with selective type II atrophy. Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) activity was reduced and western blot analysis showed decreased or absent SERCA1 protein. Based on this cohort, we conclude that Brody disease should be considered in cases of exercise-induced muscle stiffness. When physical examination shows delayed relaxation, and there are no myotonic discharges at electromyography, we recommend direct sequencing of the ATP2A1 gene or next generation sequencing with a myopathy panel. Aside from clinical features, SERCA activity measurement and SERCA1 western blot can assist in proving the pathogenicity of novel ATP2A1 mutations. Finally, patients with Brody disease may be at risk for malignant hyperthermia-like episodes, and therefore appropriate perioperative measures are recommended. This study will help improve understanding and recognition of Brody disease as a distinct myopathy in the broader field of calcium-related myopathies.

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.

Benign acute viral myositis in African migrants: A clinical, serological, and pathological study.

BACKGROUND: Several viruses have been described as causes of acquired inflammatory myopathies; however, the mechanisms by which they cause muscle disease are still unclear. The aim of this study was to describe the laboratory features of benign acute myositis in a small case series. METHODS: A detailed pathological and serological analysis was performed in five African migrants who developed an acute viral myositis complicated by rhabdomyolysis. RESULTS: Muscle biopsies clearly documented an inflammatory myopathy with histological features similar to polymyositis including CD8+ T cells surrounding and invading nonnecrotic muscle fibers, CD68+ macrophages and major histocompatibility complex class I antigen upregulation. In addition, positivity for myositis-specific antibodies (MSA), in particular anti-aminoacyl tRNA synthetases, was found in the serum of two patients. CONCLUSIONS: Our study demonstrated that T-cell mediated injury occurs in muscle of patients with acute viral myositis, and that MSA may be present in the serum of these patients.

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.

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.

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.

Clinical, Histopathologic, and Genetic Features of Patients With Myofibrillary and Distal Myopathies: Experience From the Italian Network.

BACKGROUND AND OBJECTIVES: The diagnostic process for myofibrillar myopathies (MFM) and distal myopathies (DM) is particularly complex because of the large number of causative genes, the existence of still molecularly undefined disease entities, and the overlapping features between the 2 categories. This study aimed to characterize a large cohort of patients affected by MFM and DM and identify the most important diagnostic and prognostic aspects of these diseases. METHODS: Patients with either a myopathological diagnosis of MFM or a clinical diagnosis of DM were included in this retrospective multicentric national study. Demographic, genetic, clinical, and histopathologic data of anonymized patients were collected from the neuromuscular centers of the Italian Association of Myology network. RESULTS: Data regarding 132 patients with MFM (mean age 57.0 ± 15.8 years, 49% female) and 298 patients with DM (mean age 50.7 ± 15.9 years, 40% female) were gathered from 20 neuromuscular centers. 69 patients fulfilled the criteria for both groups (distal myopathies with myofibrillar pathology, DM-MP). Molecular confirmation was achieved in 63% of the patients. Fifty-two percent of the patients with MFM carried pathogenic variants in either DES (n = 30), MYOT (n = 20), or DNAJB6 (n = 18), which were also the most frequent disease-causing genes in DM-MP, while GNE (n = 44) and MYH7 (n = 23) were the genes most commonly carrying pathogenic variants in DM. The mean age at onset varied from <25 years in patients with causative variants in MYH7 and DYSF to 59 years in patients with myotilinopathies. Cardiac involvement was reported in 29% of patients with MFM and 16% of patients with DM, with DES and MYH7 variants significantly associated with the development of cardiomyopathy. Respiratory impairment was more prevalent in patients with TTN and DES variants and rare in other disorders such as GNE myopathy and dysferlinopathies, which were instead associated, together with DNAJB6-related and PLIN4-related myopathies, with the risk of losing ambulation during the disease course. DISCUSSION: The Italian cohort of patients with MFM and DM recapitulates the phenotypic heterogeneity and the partial overlap between the 2 groups. However, in relative contrast to the encountered phenotypic variability, only 5 genes accounted for most of the molecular diagnoses. Specific genetic entities are associated with significantly increased risk of developing cardiorespiratory complications or loss of ambulation, which has relevant prognostic implications.

SERCA1 protein expression in muscle of patients with Brody disease and Brody syndrome and in cultured human muscle fibers.

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.