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The substitution for Arg168His (R168H) in γ-tropomyosin (TPM3 gene, Tpm3.12 isoform) is associated with congenital muscle fiber type disproportion (CFTD) and muscle weakness. It is still unclear what molecular mechanisms underlie the muscle dysfunction seen in CFTD. The aim of this work was to study the effect of the R168H mutation in Tpm3.12 on the critical conformational changes that myosin, actin, troponin, and tropomyosin undergo during the ATPase cycle. We used polarized fluorescence microscopy and ghost muscle fibers containing regulated thin filaments and myosin heads (myosin subfragment-1) modified with the 1,5-IAEDANS fluorescent probe. Analysis of the data obtained revealed that a sequential interdependent conformational-functional rearrangement of tropomyosin, actin and myosin heads takes place when modeling the ATPase cycle in the presence of wild-type tropomyosin. A multistep shift of the tropomyosin strands from the outer to the inner domain of actin occurs during the transition from weak to strong binding of myosin to actin. Each tropomyosin position determines the corresponding balance between switched-on and switched-off actin monomers and between the strongly and weakly bound myosin heads. At low Ca2+, the R168H mutation was shown to switch some extra actin monomers on and increase the persistence length of tropomyosin, demonstrating the freezing of the R168HTpm strands close to the open position and disruption of the regulatory function of troponin. Instead of reducing the formation of strong bonds between myosin heads and F-actin, troponin activated it. However, at high Ca2+, troponin decreased the amount of strongly bound myosin heads instead of promoting their formation. Abnormally high sensitivity of thin filaments to Ca2+, inhibition of muscle fiber relaxation due to the appearance of the myosin heads strongly associated with F-actin, and distinct activation of the contractile system at submaximal concentrations of Ca2+ can lead to muscle inefficiency and weakness. Modulators of troponin (tirasemtiv and epigallocatechin-3-gallate) and myosin (omecamtiv mecarbil and 2,3-butanedione monoxime) have been shown to more or less attenuate the negative effects of the tropomyosin R168H mutant. Tirasemtiv and epigallocatechin-3-gallate may be used to prevent muscle dysfunction. [ABSTRACT FROM AUTHOR]

Congenital myopathies are a vast group of genetic muscle diseases. Among the causes are mutations in the MYH2 gene resulting in truncated type IIa myosin heavy chains (MyHCs). The precise cellular and molecular mechanisms by which these mutations induce skeletal muscle symptoms remain obscure. Hence, in the present study, we aimed to explore whether such genetic defects would alter the presence as well as the post-translational modifications of MyHCs and the functionality of myosin molecules. For this, we dissected muscle fibers from four myopathic patients with MYH2 truncating mutations and from five human healthy controls. We then assessed 1) MyHCs presence/post-translational modifications using LC/MS; 2) relaxed myosin conformation and concomitant ATP consumption with a loaded Mant-ATP chase setup; 3) myosin activation with an unloaded in vitro motility assay; and 4) cellular force production with a myofiber mechanical setup. Interestingly, the type IIa MyHC with one additional acetylated lysine (Lys35-Ac) was present in the patients. This was accompanied by 1) a higher ATP demand of myosin heads in the disordered-relaxed conformation; 2) faster actomyosin kinetics; and 3) reduced muscle fiber force. Overall, our findings indicate that MYH2 truncating mutations impact myosin presence/functionality in human adult mature myofibers by disrupting the ATPase activity and actomyosin complex. These are likely important molecular pathological disturbances leading to the myopathic phenotype in patients. [ABSTRACT FROM AUTHOR]

Introduction: Spinal fusion for children with neuromuscular scoliosis has been known to improve sitting balance and quality of life as well as for high caregiver satisfaction. However, most studies performed were single surveys, and it remains unclear whether high satisfaction levels are maintained. Thus, in this article, we report the short- and medium-term improvements in caregiver standing assessment after neuromuscular scoliosis surgery in children with Gross Motor Function Classification System (GMFCS) level IV or V. Methods: In total, 18 patients with GMFCS levels IV and V were included in this study. The underlying diseases were typical cerebral palsy in 12 cases, chromosomal abnormalities in 5 cases, and congenital myopathy in 1 case. The median age at the time of surgery was 14.5 years. The medians for the first and second follow-up surveys were after 1.4 and 5.9 years, respectively. All the patients had undergone posterior spinal fusion, whereas 12 had undergone pelvic fixation. These patients were assessed using a caregiver questionnaire, in addition to patient demographic data and radiographic assessments. Results: The median BMI was 15.4 kg/m2 preoperatively, 16.6 kg/m2 at the first survey, and 17.1 kg/m2 at the second survey. The main Cobb angles were 97.5°, 36.5°, and 37.0° and the spino-pelvic obliquity angles were 22.5°, 6.0°, and 6.5° preoperatively, at the first survey and at the second survey, respectively. In the questionnaire, most domains were rated similarly in the first and second surveys, but the ratings for the “children's QOL” and “digestion and defecation” domains were noted to increase, while that for the “transfer” and “satisfaction with treatment” domains have decreased. Conclusions: Neuromuscular scoliosis surgery in children has been associated with extremely high treatment satisfaction in the early postoperative period. However, some caregivers showed a decline in the “transfer” and “treatment satisfaction” domains over time.

Abstract Nemaline myopathy (NM) is a muscle disorder with broad clinical and genetic heterogeneity. The clinical presentation of affected individuals ranges from severe perinatal muscle weakness to milder childhood-onset forms, and the disease course and prognosis depends on the gene and mutation type. To date, 14 causative genes have been identified, and ACTA1 accounts for more than half of the severe NM cases. ACTA1 encodes α-actin, one of the principal components of the contractile units in skeletal muscle. We established a homogenous cohort of ten unreported families with severe NM, and we provide clinical, genetic, histological, and ultrastructural data. The patients manifested antenatal or neonatal muscle weakness requiring permanent respiratory assistance, and most deceased within the first months of life. DNA sequencing identified known or novel ACTA1 mutations in all. Morphological analyses of the muscle biopsy specimens showed characteristic features of NM histopathology including cytoplasmic and intranuclear rods, cytoplasmic bodies, and major myofibrillar disorganization. We also detected structural anomalies of the perinuclear space, emphasizing a physiological contribution of skeletal muscle α-actin to nuclear shape. In-depth investigations of the nuclei confirmed an abnormal localization of lamin A/C, Nesprin-1, and Nesprin-2, forming the main constituents of the nuclear lamina and the LINC complex and ensuring nuclear envelope integrity. To validate the relevance of our findings, we examined muscle samples from three previously reported ACTA1 cases, and we identified the same set of structural aberrations. Moreover, we measured an increased expression of cardiac α-actin in the muscle samples from the patients with longer lifespan, indicating a potential compensatory effect. Overall, this study expands the genetic and morphological spectrum of severe ACTA1-related nemaline myopathy, improves molecular diagnosis, highlights the enlargement of the perinuclear space as an ultrastructural hallmark, and indicates a potential genotype/phenotype correlation.

Skeletal muscles are a highly structured tissue responsible for movement and metabolic regulation, which can be broadly subdivided into fast and slow twitch muscles with each type expressing common as well as specific sets of proteins. Congenital myopathies are a group of muscle diseases leading to a weak muscle phenotype caused by mutations in a number of genes including RYR1. Patients carrying recessive RYR1 mutations usually present from birth and are generally more severely affected, showing preferential involvement of fast twitch muscles as well as extraocular and facial muscles. In order to gain more insight into the pathophysiology of recessive RYR1-congential myopathies, we performed relative and absolute quantitative proteomic analysis of skeletal muscles from wild-type and transgenic mice carrying p.Q1970fsX16 and p.A4329D RyR1 mutations which were identified in a child with a severe congenital myopathy. Our in-depth proteomic analysis shows that recessive RYR1 mutations not only decrease the content of RyR1 protein in muscle, but change the expression of 1130, 753, and 967 proteins EDL, soleus and extraocular muscles, respectively. Specifically, recessive RYR1 mutations affect the expression level of proteins involved in calcium signaling, extracellular matrix, metabolism and ER protein quality control. This study also reveals the stoichiometry of major proteins involved in excitation contraction coupling and identifies novel potential pharmacological targets to treat RyR1-related congenital myopathies.

DNAJ/HSP40 co-chaperones are integral to the chaperone network, bind client proteins and recruit them to HSP70 for folding. We performed exome sequencing on patients with a presumed hereditary muscle disease and no genetic diagnosis. This identified four individuals from three unrelated families carrying an unreported homozygous stop gain (c.856A > T; p.Lys286Ter), or homozygous missense variants (c.74G > A; p.Arg25Gln and c.785 T > C; p.Leu262Ser) in DNAJB4. Affected patients presented with axial rigidity and early respiratory failure requiring ventilator support between the 1st and 4th decade of life. Selective involvement of the semitendinosus and biceps femoris muscles was seen on MRI scans of the thigh. On biopsy, muscle was myopathic with angular fibers, protein inclusions and occasional rimmed vacuoles. DNAJB4 normally localizes to the Z-disc and was absent from muscle and fibroblasts of affected patients supporting a loss of function. Functional studies confirmed that the p.Lys286Ter and p.Leu262Ser mutant proteins are rapidly degraded in cells. In contrast, the p.Arg25Gln mutant protein is stable but failed to complement for DNAJB function in yeast, disaggregate client proteins or protect from heat shock-induced cell death consistent with its loss of function. DNAJB4 knockout mice had muscle weakness and fiber atrophy with prominent diaphragm involvement and kyphosis. DNAJB4 knockout muscle and myotubes had myofibrillar disorganization and accumulated Z-disc proteins and protein chaperones. These data demonstrate a novel chaperonopathy associated with DNAJB4 causing a myopathy with early respiratory failure. DNAJB4 loss of function variants may lead to the accumulation of DNAJB4 client proteins resulting in muscle dysfunction and degeneration. [ABSTRACT FROM AUTHOR]

KBTBD13 variants cause nemaline myopathy type 6 (NEM6). The majority of NEM6 patients harbors the Dutch founder variant, c.1222C>T, p.Arg408Cys (KBTBD13 p.R408C). Although KBTBD13 is expressed in cardiac muscle, cardiac involvement in NEM6 is unknown. Here, we constructed pedigrees of three families with the KBTBD13 p.R408C variant. In 65 evaluated patients, 12% presented with left ventricle dilatation, 29% with left ventricular ejection fraction< 50%, 8% with atrial fibrillation, 9% with ventricular tachycardia, and 20% with repolarization abnormalities. Five patients received an implantable cardioverter defibrillator, three cases of sudden cardiac death were reported. Linkage analysis confirmed cosegregation of the KBTBD13 p.R408C variant with the cardiac phenotype. Mouse studies revealed that (1) mice harboring the Kbtbd13 p.R408C variant display mild diastolic dysfunction; (2) Kbtbd13‐deficient mice have systolic dysfunction. Hence, (1) KBTBD13 is associated with cardiac dysfunction and cardiomyopathy; (2) KBTBD13 should be added to the cardiomyopathy gene panel; (3) NEM6 patients should be referred to the cardiologist. [ABSTRACT FROM AUTHOR]

ACTN2 encodes alpha‐actinin‐2, a protein expressed in human cardiac and skeletal muscle. The protein, located in the sarcomere Z‐disk, functions as a link between the anti‐parallel actin filaments. This important structural protein also binds N‐terminal titins, and thus contributes to sarcomere stability. Previously, ACTN2 mutations have been solely associated with cardiomyopathy, without skeletal muscle disease. Recently, however, ACTN2 mutations have been associated with novel congenital and distal myopathy. Previously reported variants are in varying locations across the gene, but the potential clustering effect of pathogenic locations is not clearly understood. Further, the genotype‐phenotype correlations of these variants remain unclear. Here we review the previously reported ACTN2‐related molecular and clinical findings and present an additional variant, c.1840‐2A>T, that further expands the mutation and phenotypic spectrum. Our results show a growing body of clinical, genetic, and functional evidence, which underlines the central role of ACTN2 in the muscle tissue and myopathy. However, limited segregation and functional data are available to support the pathogenicity of most previously reported missense variants and clear‐cut genotype‐phenotype correlations are currently only demonstrated for some ACTN2‐related myopathies. [ABSTRACT FROM AUTHOR]

Objective. To present a clinical case of a patient with spinal deformity associated with myopathy, which was initially undiagnosed and as- sessed as secondary myopathy after performed spinal surgeries. Material and Methods. Study design: clinical observation and review of the literature. A 51-year-old female patient has been repeated- ly operated on for scoliotic deformity of the spine with progressing neurological disorders. Clinical and complex radiological assessment (spinal telemetry in sitting position, CT, MRI) of the pathology was performed. In connection with the atypical course of the disease, a pathohistological study of muscle tissue fragments was performed with their fixation in a solution of 10 % neutral formalin. After histo- logical processing, the fragments were embedded in paraffin and stained with hematoxylin-eosin by the Masson trichromic method and by the PTAH method. The preparations were examined using the Pannoramic MIDI II BF hardware and software complex to obtain digital images using the Whole slide imaging technology in the Single layer and Extended focus modes and an AxioScope.A1 stereomicroscope with a digital camera. Results. Two patterns of distribution of nemaline rods of various density and configuration stained dark blue (by PTAH) were identified in sarcoplasm of parts of muscular tissues: diffusely throughout the myocyte and forming clusters of various sizes in transverse sections, and as filamentous structures – in longitudinal ones. Extensive fields of fatty degeneration, transformation of fibers into lipocytes, con- tracturely changed fibers, pictures of myophaga and areas of fibrosis of the interstitial space associated with residual myocytes were noted. Intramuscular nervous conductors were subjected to complete involution, singular nervous fibers were deformed, internal space was fibrotic. Neuromuscular spindles had unusually increased connective tissue capsule filled with lipocytes. Arterial vessels had fibrosis of t. adventicia and t. media, narrowed or obliterated lumen, and venous vessels were thin-walled and tortuous, which causes their permeability and can cause hemorrhage. The revealed changes in myocytes with the presence of nemaline rods are characteristic of primary nemaline myopathy. Conclusion. The atypicality of clinical manifestations, primarily of the neurological status in scoliosis, requires excluding neuromuscular disease by conducting a histopathological examination of the surgical material.

Abstract Background Congenital myopathies are a group of rare neuromuscular diseases characterized by specific histopathological features. The relationship between the pathologies and the genetic causes is complex, and the prevalence of myopathy-causing genes varies among patients from different ethnic groups. The aim of the present study was to characterize congenital myopathies with infancy onset among patients registered at our institution. Method This retrospective study enrolled 56 patients based on the pathological and/or genetic diagnosis. Clinical, histopathological and genetic features of the patients were analysed with long-term follow-up. Results Twenty-six out of 43 patients who received next-generation sequencing had genetic confirmation, and RYR1 variations (12/26) were the most prevalent. Eighteen novel variations were identified in 6 disease-causing genes, including RYR1, NEB, TTN, TNNT1, DNM2 and ACTA1. Nemaline myopathy (17/55) was the most common histopathology. The onset ages ranged from birth to 1 year. Thirty-one patients were followed for 3.83 ± 3.05 years (ranging from 3 months to 11 years). No patient died before 1 year. Two patients died at 5 years and 8 years respectively. The motor abilities were stable or improved in 23 patients and deteriorated in 6 patients. Ten (10/31) patients developed respiratory involvement, and 9 patients (9/31) had mildly abnormal electrocardiograms and/or echocardiograms. Conclusion The severity of congenital myopathies in the neonatal/infantile period may vary in patients from different ethnic groups. More concern should be given to cardiac monitoring in patients with congenital myopathies even in those with static courses.

Background Integrin α7β1 is a major laminin receptor in skeletal and cardiac muscle. In skeletal muscle, integrin α7β1 plays an important role during muscle development and has been described as an important modifier of skeletal muscle diseases. The integrin α7β1 is also highly expressed in the heart, but its precise role in cardiac function is unknown. Mutations in the integrin α7 gene (ITGA7) have been reported in children with congenital myopathy. Methods and Results In this study, we described skeletal and cardiac muscle pathology in Itga7−/− mice and 5 patients from 2 unrelated families with ITGA7 mutations. Proband in family 1 presented a homozygous c.806_818del [p.S269fs] variant, and proband in family 2 was identified with 2 intron variants in the ITGA7 gene. The complete absence of the integrin α7 protein in muscle supports the ITGA7 mutations are pathogenic. We performed electrocardiography, echocardiography, or cardiac magnetic resonance imaging, and histological biopsy analyses in patients with ITGA7 deficiency and Itga7−/− mice. The patients exhibited cardiac dysrhythmia and dysfunction from the third decade of life and late‐onset respiratory insufficiency, but with relatively mild limb muscle involvement. Mice demonstrated corresponding abnormalities in cardiac conduction and contraction as well as diaphragm muscle fibrosis. Conclusions Our data suggest that loss of integrin α7 causes a novel form of adult‐onset cardiac dysfunction indicating a critical role for the integrin α7β1 in normal cardiac function and highlights the need for long‐term cardiac monitoring in patients with ITGA7‐related congenital myopathy.

In recent years, increasing attention has been paid to titin (TTN) and its mutations. Heterozygous TTN truncating variants (TTNtv) increase the risk of a cardiomyopathy. At the same time, TTNtv and few missense variants have been identified in patients with mainly recessive skeletal muscle diseases. The pathogenic mechanisms underlying titin‐related diseases are still partly unknown. Similarly, the titin mechanical and functional role in the muscle contraction are far from being exhaustively clarified. In the last few years, several animal models carrying variants in the titin gene have been developed and characterized to study the structural and mechanical properties of specific titin domains or to mimic patients' mutations. This review describes the main animal models so far characterized, including eight mice models and three fish models (Medaka and Zebrafish) and discusses the useful insights provided by a thorough characterization of the cell‐, tissue‐ and organism‐phenotypes in these models. [ABSTRACT FROM AUTHOR]

• First study to investigate VT effectiveness in congenital myopathy. • VT is safe and feasible for children/adolescents with congenital myopathy. • VT improved motor function in children with congenital myopathy. • Home-based VT was acceptable to children and families. This pilot study explored the feasibility and effectiveness of vibration therapy (VT) on muscle and bone health, motor performance, and respiratory function in patients with congenital myopathy (CM). Eleven participants with CM (11.5 ± 2.8 years) underwent 12 weeks of side-alternating VT at 20 Hz for nine minutes per session, four days a week. VT was preceded by a 12-week control period. Assessments included dual-energy X-ray absorptiometry scans, 6-minute walk and 10-meter run tests, muscle function and motor performance assessment, dynamometry, and pulmonary function. VT was well-tolerated, with occasional mild itchiness reported. The median compliance level with VT treatment was 75%. 12 weeks of VT improved the total score of motor function performance by 2.4 units (p=0.006) and velocity rise maximum of the chair rising test by 0.11 m/s (p=0.029). VT was shown to be feasible, safe, and associated with improving motor function performance. Our findings support further exploration of VT's potential health benefits to patients with CM in larger studies involving a longer intervention period. [ABSTRACT FROM AUTHOR]

Home mechanical ventilation is classically used to treat neuromuscular patients with chronic respiratory insufficiency. Since the heart and lungs are localized in the thorax, intrathoracic pressure may directly affect heart function. Here, we report the direct cyclic effects of mechanical ventilation on the right and left ventricular systolic and diastolic function in serial cases. These cases highlight the importance of performing Doppler echocardiography in patients with neuromuscular disorders, focusing on the right and left ventricular systolic and diastolic functions in the context of ventilation setting modifications or introduction. [ABSTRACT FROM AUTHOR]

Nemaline myopathy (NM), a structural congenital myopathy, presents a significant clinical and genetic heterogeneity. Here, we compiled molecular and clinical data of 30 Brazilian patients from 25 unrelated families. Next-generation sequencing was able to genetically classify all patients: sixteen families (64%) with mutation in NEB, five (20%) in ACTA1, two (8%) in KLHL40, and one in TPM2 (4%) and TPM3 (4%). In the NEB-related families, 25 different variants, 11 of them novel, were identified; splice site (10/25) and frame shift (9/25) mutations were the most common. Mutation c.24579 G>C was recurrent in three unrelated patients from the same region, suggesting a common ancestor. Clinically, the "typical" form was the more frequent and caused by mutations in the different NM genes. Phenotypic heterogeneity was observed among patients with mutations in the same gene. Respiratory involvement was very common and often out of proportion with limb weakness. Muscle MRI patterns showed variability within the forms and genes, which was related to the severity of the weakness. Considering the high frequency of NEB mutations and the complexity of this gene, NGS tools should be combined with CNV identification, especially in patients with a likely non-identified second mutation. [ABSTRACT FROM AUTHOR]

Background: FLNC is one of the few genes associated with all types of cardiomyopathies, but it also underlies neuromuscular phenotype. The combination of concomitant neuromuscular and cardiac involvement is not often observed in filaminopathies and the impact of this on the disease prognosis has hitherto not been analyzed.Results: Here we provide a detailed clinical, genetic, and structural prediction analysis of distinct FLNC-associated phenotypes based on twelve pediatric cases. They include early-onset restrictive cardiomyopathy (RCM) in association with congenital myopathy. In all patients the initial diagnosis was established during the first year of life and in five out of twelve (41.7%) patients the first symptoms were observed at birth. RCM was present in all patients, often in combination with septal defects. No ventricular arrhythmias were noted in any of the patients presented here. Myopathy was confirmed by neurological examination, electromyography, and morphological studies. Arthrogryposes was diagnosed in six patients and remained clinically meaningful with increasing age in three of them. One patient underwent successful heart transplantation at the age of 18 years and two patients are currently included in the waiting list for heart transplantation. Two died due to congestive heart failure. One patient had ICD instally as primary prevention of SCD. In ten out of twelve patients the disease was associated with missense variants and only in two cases loss of function variants were detected. In half of the described cases, an amino acid substitution A1186V, altering the structure of IgFLNc10, was found.Conclusions: The present description of twelve cases of early-onset restrictive cardiomyopathy with congenital myopathy and FLNC mutation, underlines a distinct unique phenotype that can be suggested as a separate clinical form of filaminopathies. Amino acid substitution A1186V, which was observed in half of the cases, defines a mutational hotspot for the reported combination of myopathy and cardiomyopathy. Several independent molecular mechanisms of FLNC mutations linked to filamin structure and function can explain the broad spectrum of FLNC-associated phenotypes. Early disease presentation and unfavorable prognosis of heart failure demanding heart transplantation make awareness of this clinical form of filaminopathy of great clinical importance. [ABSTRACT FROM AUTHOR]

Introduction  ORAI-1 is a plasma membrane calcium release-activated calcium channel that plays a crucial role in the excitation-contraction of skeletal muscles. Loss-of-function mutations of ORAI-1 cause severe combined immunodeficiency, nonprogressive muscle hypotonia, and anhidrotic ectodermal dysplasia. Autosomal dominant gain-of-function mutation causes Stormorken's syndrome, which includes tubular aggregate myopathy along with bleeding diathesis. Methods  This is a description of a genetically confirmed case of ORAI-1-associated myopathy with clinical, histopathological, and imaging characteristics and a detailed literature review. Results  We report an 18-year-old woman who presented with 2-and-a-half year history of slowly progressive proximal lower limb weakness and ophthalmoparesis. Her serum creatine kinase levels were normal. Magnetic resonance imaging of the muscle showed predominant fatty infiltration of the glutei and quadriceps femoris. Histopathological analysis of muscle biopsy was suggestive of congenital fiber-type disproportion (CFTD). Clinical exome sequencing showed novel homozygous nonsense pathogenic variant NC&#95;000012.12 (NM&#95;032790.3): c.205G > T (p.Glu69Ter) in ORAI-1 gene. Conclusion  This report expands the phenotypic spectrum of ORAI-1-related myopathy to include congenital myopathy-CFTD with ophthalmoparesis, a novel manifestation., Competing Interests: Conflict of Interest None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. ( https://creativecommons.org/licenses/by/4.0/ ).)

Abstract The ryanodine receptor RyR1 is the main sarcoplasmic reticulum Ca2+ channel in skeletal muscle and acts as a connecting link between electrical stimulation and Ca2+-dependent muscle contraction. Abnormal RyR1 activity compromises normal muscle function and results in various human disorders including malignant hyperthermia, central core disease, and centronuclear myopathy. However, RYR1 is one of the largest genes of the human genome and accumulates numerous missense variants of uncertain significance (VUS), precluding an efficient molecular diagnosis for many patients and families. Here we describe a recurrent RYR1 mutation previously classified as VUS, and we provide clinical, histological, and genetic data supporting its pathogenicity. The heterozygous c.12083C>T (p.Ser4028Leu) mutation was found in thirteen patients from nine unrelated congenital myopathy families with consistent clinical presentation, and either segregated with the disease in the dominant families or occurred de novo. The affected individuals essentially manifested neonatal or infancy-onset hypotonia, delayed motor milestones, and a benign disease course differing from classical RYR1-related muscle disorders. Muscle biopsies showed unspecific histological and ultrastructural findings, while RYR1-typical cores and internal nuclei were seen only in single patients. In conclusion, our data evidence the causality of the RYR1 c.12083C>T (p.Ser4028Leu) mutation in the development of an atypical congenital myopathy with gradually improving motor function over the first decades of life, and may direct molecular diagnosis for patients with comparable clinical presentation and unspecific histopathological features on the muscle biopsy.

Background: Congenital myopathy-13 (CMYP13), also known as Bailey-Bloch congenital myopathy and Native American myopathy (NAM), is a condition caused by biallelic missense pathogenic variants in STAC3, which encodes an important protein necessary for the excitation-relaxation coupling machinery in the muscle. Patients with biallelic pathogenic variants in STAC3 often present with congenital weakness and arthrogryposis, cleft palate, ptosis, myopathic facies, short stature, kyphoscoliosis, and susceptibility to malignant hyperthermia provoked by anesthesia. We present two unrelated cases of Bailey-Bloch congenital myopathy descendants of non-consanguineous parents, which were investigated for delayed psychomotor development and generalized weakness. To the best of our knowledge, these are the first descriptions of CMYP13 in Brazil. In both patients, we found the previously described pathogenic missense variant p.Trp284Ser in homozygosity. Conclusion: We seek to highlight the need for screening for CMYP13 in patients expressing the typical phenotype of the disease even in the absence of Lumbee Native American ancestry, and to raise awareness to possible complications like malignant hyperthermia in Bailey-Bloch congenital myopathy.

Congenital myopathies (CMs) are a group of diseases that primarily affect the muscle fiber, especially the contractile apparatus and the different components that condition its normal functioning. They present as muscle weakness and hypotonia at birth or during the first year of life. Centronuclear CM is characterized by a high incidence of nuclei located centrally and internally in muscle fibers. Clinical case: a 22-year-old male patient with symptoms of muscle weakness since early childhood, with difficulty in performing physical activity according to his age, with the presence of a long face, a waddling gait, and a global decrease in muscle mass. Electromyography was performed, showing a neurogenic pattern and not the expected myopathic one, neuroconduction with reduced amplitude of the motor potential of the peroneal nerve and axonal and myelin damage of the posterior tibial nerves. The microscopic study of the studied striated muscle fragments stained with hematoxylin–eosin and Masson’s trichrome showed the presence of fibers with central nuclei, diagnosing CM. The patient meets most of the description for CM, with involvement of all striated muscles, although it is important to note the neurogenic pattern present in this case, due to the denervation of damaged muscle fibers, which contain terminal axonal segments. Neuroconduction shows the involvement of motor nerves, but with normal sensory studies, axonal polyneuropathy is unlikely, due to normal sensory potentials. Different pathological findings have been described depending on the mutated gene in this disease, but all coincide with the presence of fibers with central nuclei for diagnosis by this means, which is so important in institutions where it is not possible to carry out genetic studies, and allowing early specific treatment, according to the stage through which the patient passes.

• The novel variant of uncertain significance p.Glu237Lys in TPM3 gene is reported • Muscle biopsy featured a combination of congenital fiber type disproportion with cap disease, and no nemaline rods • Clinically the patient showed moderate myopathic features with disproportionate respiratory involvement • Muscle MRI demonstrated a generalized involvement including the tongue • The clinical, MRI and histological findings demonstrate the mutation's pathogenicity Tropomyosin 3 (TPM3) gene mutations associate with autosomal dominant and recessive nemaline myopathy 1 (NEM1), congenital fiber type disproportion myopathy (CFTD) and cap myopathy (CAPM1), and a combination of caps and nemaline bodies. We report on a 47-year-old man with polyglobulia, restricted vital capacity and mild apnea hypopnea syndrome, requiring noninvasive ventilation. Physical assessment revealed bilateral ptosis and facial paresis, with high arched palate and retrognathia; global hypotonia and diffuse axial weakness, including neck and upper and lower limb girdle and foot dorsiflexion weakness. Whole body MRI showed a diffuse fatty replacement with an unspecific pattern. A 122 gene NGS neuromuscular disorders panel revealed the heterozygous VUS c.709G>A (p.Glu237Lys) on exon 8 of TMP3. A deltoid muscle biopsy showed a novel histological pattern combining fiber type disproportion and caps. Our findings support the pathogenicity of the novel TPM3 variant and widen the phenotypic gamut of TMP3 -related congenital myopathy. [ABSTRACT FROM AUTHOR]

Background: Congenital myopathies (CM) were traditionally classified according to the muscle histopathological features, but in recent years, molecular diagnosis has become increasingly important. CM may present a wide phenotype variability, and while adult‐onset CM have been increasingly recognized, substantial diagnostic delays are still reported. Objectives: To describe a cohort of adult CM patients, including clinical, genetic, and histopathological features, and further characterize the subgroup of adult‐diagnosed patients. Materials and Methods: We performed a retrospective observational cohort study to characterize the CM patients evaluated in our adult Neuromuscular outpatient clinic, including the subgroup of adult‐diagnosed patients. Results: We identified 19 CM patients with compatible molecular and/or histological diagnoses, of which 14 were diagnosed in adulthood. Eleven adult‐diagnosed patients had symptoms since childhood and 9 had a family history of myopathy. The median age of symptoms' onset was 4 years old and the median age at diagnosis was 37 years old. The most common causative gene was RYR1, followed by TTN and MYH7. Three patients had non‐specific features on muscle biopsy, all diagnosed during adulthood. Conclusions: In our cohort, the majority of CM were diagnosed in adulthood, despite most having pediatric‐onset symptoms and positive family history. The diagnostic delay may be associated with mild presentation, slow course, atypical muscle histology, and lack of awareness of adult‐onset CM. Studies with larger populations are needed. [ABSTRACT FROM AUTHOR]

Nemaline myopathy (NM) is a muscle disorder with broad clinical and genetic heterogeneity. The clinical presentation of affected individuals ranges from severe perinatal muscle weakness to milder childhood-onset forms, and the disease course and prognosis depends on the gene and mutation type. To date, 14 causative genes have been identified, and ACTA1 accounts for more than half of the severe NM cases. ACTA1 encodes α-actin, one of the principal components of the contractile units in skeletal muscle. We established a homogenous cohort of ten unreported families with severe NM, and we provide clinical, genetic, histological, and ultrastructural data. The patients manifested antenatal or neonatal muscle weakness requiring permanent respiratory assistance, and most deceased within the first months of life. DNA sequencing identified known or novel ACTA1 mutations in all. Morphological analyses of the muscle biopsy specimens showed characteristic features of NM histopathology including cytoplasmic and intranuclear rods, cytoplasmic bodies, and major myofibrillar disorganization. We also detected structural anomalies of the perinuclear space, emphasizing a physiological contribution of skeletal muscle α-actin to nuclear shape. In-depth investigations of the nuclei confirmed an abnormal localization of lamin A/C, Nesprin-1, and Nesprin-2, forming the main constituents of the nuclear lamina and the LINC complex and ensuring nuclear envelope integrity. To validate the relevance of our findings, we examined muscle samples from three previously reported ACTA1 cases, and we identified the same set of structural aberrations. Moreover, we measured an increased expression of cardiac α-actin in the muscle samples from the patients with longer lifespan, indicating a potential compensatory effect. Overall, this study expands the genetic and morphological spectrum of severe ACTA1-related nemaline myopathy, improves molecular diagnosis, highlights the enlargement of the perinuclear space as an ultrastructural hallmark, and indicates a potential genotype/phenotype correlation. [ABSTRACT FROM AUTHOR]

Generalized weakness in the pediatric and adolescent population is caused by many disorders that affect the neuromuscular axis. As next-generation sequencing (NGS) is becoming of high yield in replacing more invasive procedures, that is, muscle and nerve biopsy, more previously undiagnosed diseases of the muscles are now labeled with specific pathogenicity. A 16-year-old-girl diagnosed with nemaline myopathy but previously was misdiagnosed with congenital myasthenia and put-on unnecessary medications. Clinicians should be aware of congenital diseases that affect the muscles and know the importance of the NGS in reaching the correct diagnosis more so when there is a history of consanguinity.

Abstract Background Dermatomyositis is an inflammatory muscle disease caused by immune-mediated muscle injury, and central core disease (CCD) is a congenital myopathy associated with disturbed intracellular calcium homeostasis and excitation-contraction coupling. To date, CCD has not been reported to have autoantibodies or coexist with inflammatory myopathy. Case presentation Here, we described the case of a 25-year-old woman who had progressive proximal muscle weakness, myalgia, pruritic macular rash, skin ulcers, and calcinosis. Dermatomyositis was initially suspected based on the clinical symptoms accompanied by elevated muscle enzyme levels, electromyography abnormalities, and a positive antinuclear antibody test. However, the patient’s muscle biopsy revealed the characteristic findings of both dermatomyositis and CCD, suggesting that dermatomyositis occurred in this patient with previously asymptomatic CCD. The patient did not have any pathogenic gene mutations associated with congenital myopathy, including RYR1 and SEPN1 in targeted next-generation sequencing. She received high-dose glucocorticoid therapy and azathioprine with a significant improvement in muscle strength. Conclusions We present a case of rare coexistence of dermatomyositis and CCD. Clinicians should be aware that patients with CCD may have inflammatory myopathy that responds well to immunosuppressive therapy.

We present the anaesthetic management of a parturient with myogenic differentiation-1 gene-related congenital myopathy who presented for urgent caesarean section due to pre-eclampsia and respiratory failure. The challenges we faced include chronic respiratory failure with diaphragmatic dysfunction, difficulties with neuraxial anaesthesia, potential risk of triggering malignant hyperthermia in an unknown myopathy, and the complexities of multidisciplinary team involvement. As there is limited medical literature regarding this condition, we believe this is the first case report describing the anaesthetic care of a pregnant patient with this rare congenital myopathy.

Abstract The MYH2 gene encodes the skeletal muscle myosin heavy chain IIA (MyHC-IIA) isoform, which is expressed in the fast twitch type 2A fibers. Autosomal dominant or recessive pathogenic variants in MYH2 lead to congenital myopathy clinically featured by ophthalmoparesis and predominantly proximal weakness. MYH2-myopathy is pathologically characterized by loss and atrophy of type 2A fibers. Additional myopathological abnormalities have included rimmed vacuoles containing small p62 positive inclusions, 15–20 nm tubulofilaments, minicores and dystrophic changes. We report an adult patient with late-pediatric onset MYH2-myopathy caused by two heterozygous pathogenic variants: c.3331C>T, p.Gln1111* predicted to result in truncation of the proximal tail region of MyHC-IIA, and c.1546T>G, p.Phe516Val, affecting a highly conserved amino acid within the highly conserved catalytic motor head relay loop. This missense variant is predicted to result in a less compact loop domain and in turn could affect the protein affinity state. The patient’s genotype is accompanied by a novel myopathological phenotype characterized by centralized large myofilamentous tangles associated with clusters of nemaline rods, and ring fibers, in addition to the previously reported rimmed vacuoles, paucity and atrophy of type 2A fibers. Electron microscopy demonstrated wide areas of disorganized myofibrils which were oriented in various planes of direction and entrapped multiple nemaline rods, as corresponding to the large tangles with rods seen on light microscopy. Nemaline rods were rarely observed also in nuclei. We speculate that the mutated MyHC-IIA may influence myofibril disorganization. While nemaline rods have been described in myopathies caused by pathogenic variants in genes encoding several sarcomeric proteins, to our knowledge, nemaline rods have not been previously described in MYH2-myopathy.

Abstract We report a case of neonatal nemaline myopathy with a de novo TPM3 mutation, which has been classified as a likely pathogenic mutation. With the expanding use of genetic testing in congenital myopathies, genotype‐phenotype descriptions of novel variants are important to inform clinical care, diagnosis, genetic counseling, and management of disease.

The mechanoenzyme dynamin 2 (DNM2) is crucial for intracellular organization and trafficking. DNM2 is mutated in dominant centronuclear myopathy (DNM2-CNM), a muscle disease characterized by defects in organelle positioning in myofibers. It remains unclear how the in vivo functions of DNM2 are regulated in muscle. Moreover, there is no therapy for DNM2-CNM to date. Here, we overexpressed human amphiphysin 2 (BIN1), a membrane remodeling protein mutated in other CNM forms, in Dnm2RW/+ and Dnm2RW/RW mice modeling mild and severe DNM2-CNM, through transgenesis or with adeno-associated virus (AAV). Increasing BIN1 improved muscle atrophy and main histopathological features of Dnm2RW/+ mice and rescued the perinatal lethality and survival of Dnm2RW/RW mice. In vitro experiments showed that BIN1 binds and recruits DNM2 to membrane tubules, and that the BIN1-DNM2 complex regulates tubules fission. Overall, BIN1 is a potential therapeutic target for dominant centronuclear myopathy linked to DNM2 mutations. [ABSTRACT FROM AUTHOR]

Monoallelic pathogenic variants in BICD2 are associated with autosomal dominant Spinal Muscular Atrophy Lower Extremity Predominant 2A and 2B (SMALED2A, SMALED2B). As part of the cellular vesicular transport, complex BICD2 facilitates the flow of constitutive secretory cargoes from the trans‐Golgi network, and its dysfunction results in motor neuron loss. The reported phenotypes among patients with SMALED2A and SMALED2B range from a congenital onset disorder of respiratory insufficiency, arthrogryposis, and proximal or distal limb weakness to an adult‐onset disorder of limb weakness and contractures. We report an infant with congenital respiratory insufficiency requiring mechanical ventilation, congenital diaphragmatic paralysis, decreased lung volume, and single finger camptodactyly. The infant displayed appropriate antigravity limb movements but had radiological, electrophysiological, and histopathological evidence of myopathy. Exome sequencing and long‐read whole‐genome sequencing detected a novel de novo BICD2 variant (NM_001003800.1:c.[1543G>A];[=]). This is predicted to encode p.(Glu515Lys); p.Glu515 is located in the coiled‐coil 2 mutation hotspot. We hypothesize that this novel phenotype of diaphragmatic paralysis without clear appendicular muscle weakness and contractures of large joints is a presentation of BICD2‐related disease. [ABSTRACT FROM AUTHOR]

Abstract Background Congenital myopathy (CM) is a group of clinically and genetically heterogeneous muscle disorders, characterized by muscle weakness and hypotonia from birth. Currently, no definite treatment exists for CM. A de novo mutation in Tropomyosin 3-TPM3(E151G) was identified from a boy diagnosed with CM, previously TPM3(E151A) was reported to cause CM. However, the role of TPM3(E151G) in CM is unknown. Methods Histopathological, swimming behavior, and muscle endurance were monitored in TPM3 wild-type and mutant transgenic fish, modelling CM. Gene expression profiling of muscle of the transgenic fish were studied through RNAseq, and mitochondria respiration was investigated. Results While TPM3(WT) and TPM3(E151A) fish show normal appearance, amazingly a few TPM3(E151G) fish display either no tail, a crooked body in both F0 and F1 adults. Using histochemical staining for the muscle biopsy, we found TPM3(E151G) displays congenital fiber type disproportion and TPM3(E151A) resembles nemaline myopathy. TPM3(E151G) transgenic fish dramatically swimming slower than those in TPM3(WT) and TPM3(E151A) fish measured by DanioVision and T-maze, and exhibit weaker muscle endurance by swimming tunnel instrument. Interestingly, l-carnitine treatment on TPM3(E151G) transgenic larvae significantly improves the muscle endurance by restoring the basal respiration and ATP levels in mitochondria. With RNAseq transcriptomic analysis of the expression profiling from the muscle specimens, it surprisingly discloses large downregulation of genes involved in pathways of sodium, potassium, and calcium channels, which can be rescued by l-carnitine treatment, fatty acid metabolism was differentially dysregulated in TPM3(E151G) fish and rescued by l-carnitine treatment. Conclusions These results demonstrate that TPM3(E151G) and TPM3(E151A) exhibit different pathogenicity, also have distinct gene regulatory profiles but the ion channels were downregulated in both mutants, and provides a potential mechanism of action of TPM3 pathophysiology. Our results shed a new light in the future development of potential treatment for TPM3-related CM.

Mutations in DNM2 cause autosomal dominant centronuclear myopathy (ADCNM), a rare disease characterized by skeletal muscle weakness and structural anomalies of the myofibres, including nuclear centralization and mitochondrial mispositioning. Following the clinical report of a Border Collie male with exercise intolerance and histopathological hallmarks of CNM on the muscle biopsy, we identified the c.1393C>T (R465W) mutation in DNM2, corresponding to the most common ADCNM mutation in humans. In order to establish a large animal model for longitudinal and preclinical studies on the muscle disorder, we collected sperm samples from the Border Collie male and generated a dog cohort for subsequent clinical, genetic and histological investigations. Four of the five offspring carried the DNM2 mutation and showed muscle atrophy and a mildly impaired gait. Morphological examinations of transverse muscle sections revealed CNM-typical fibres with centralized nuclei and remodelling of the mitochondrial network. Overall, the DNM2-CNM dog represents a faithful animal model for the human disorder, allows the investigation of ADCNM disease progression, and constitutes a valuable complementary tool to validate innovative therapies established in mice.

To date there are no therapies for patients with congenital myopathies, muscle disorders causing poor quality of life of affected individuals. In approximately 30% of the cases, patients with congenital myopathies carry either dominant or recessive mutations in the ryanodine receptor 1 (RYR1) gene; recessive RYR1 mutations are accompanied by reduction of RyR1 expression and content in skeletal muscles and are associated with fiber hypotrophy and muscle weakness. Importantly, muscles of patients with recessive RYR1 mutations exhibit increased content of class II histone deacetylases and of DNA genomic methylation. We recently created a mouse model knocked-in for the p.Q1970fsX16+ p.A4329D RyR1 mutations, which are isogenic to those carried by a severely affected child suffering from a recessive form of RyR1-related multi-mini core disease. The phenotype of the RyR1 mutant mice recapitulates many aspects of the clinical picture of patients carrying recessive RYR1 mutations. We treated the compound heterozygous mice with a combination of two drugs targeting DNA methylases and class II histone deacetylases. Here, we show that treatment of the mutant mice with drugs targeting epigenetic enzymes improves muscle strength, RyR1 protein content, and muscle ultrastructure. This study provides proof of concept for the pharmacological treatment of patients with congenital myopathies linked to recessive RYR1 mutations.

Background: Congenital myopathies are a group of rare neuromuscular diseases characterized by specific histopathological features. The relationship between the pathologies and the genetic causes is complex, and the prevalence of myopathy-causing genes varies among patients from different ethnic groups. The aim of the present study was to characterize congenital myopathies with infancy onset among patients registered at our institution.Method: This retrospective study enrolled 56 patients based on the pathological and/or genetic diagnosis. Clinical, histopathological and genetic features of the patients were analysed with long-term follow-up.Results: Twenty-six out of 43 patients who received next-generation sequencing had genetic confirmation, and RYR1 variations (12/26) were the most prevalent. Eighteen novel variations were identified in 6 disease-causing genes, including RYR1, NEB, TTN, TNNT1, DNM2 and ACTA1. Nemaline myopathy (17/55) was the most common histopathology. The onset ages ranged from birth to 1 year. Thirty-one patients were followed for 3.83 ± 3.05 years (ranging from 3 months to 11 years). No patient died before 1 year. Two patients died at 5 years and 8 years respectively. The motor abilities were stable or improved in 23 patients and deteriorated in 6 patients. Ten (10/31) patients developed respiratory involvement, and 9 patients (9/31) had mildly abnormal electrocardiograms and/or echocardiograms.Conclusion: The severity of congenital myopathies in the neonatal/infantile period may vary in patients from different ethnic groups. More concern should be given to cardiac monitoring in patients with congenital myopathies even in those with static courses. [ABSTRACT FROM AUTHOR]

Aim: Our goal was to determine the genetic basis of early-onset myopathy in patients from two unrelated families. Materials and Methods: Whole-exome sequencing, autozygosity mapping, and confirmatory targeted Sanger sequencing were performed using genomic DNA extracted from blood samples from three myopathic patients of two unrelated families. Variant filtering and pathogenicity analyses were evaluated according to standard protocols and up-to-date pipelines applied at the King Faisal Specialist Hospital and Research Center. Results: A novel homozygous variant was detected in TTN gene within the first three M-line-encoding exons in a 9-year-old female in the first family who had delayed motor development and proximal weakness. Her 4-year-old affected brother, with the same homozygous variant, could not yet walk without help. This pathogenic nonsense variant is predicted to cause a premature stop during translation. In the second family we identified two novel variants as compound heterozygosites (a deletion and a variant affecting a canonical splice site) in an affected 9-year-old female with weakness that developed at age 3, in the second family. SpliceAI predicted the variants being splice-altering with high probability. These variants were fully segregated in the family. The deletion was found to be on the paternal allele, whereas the splicing variant was on the maternal allele. The patient's echocardiography revealed mitral valve prolapse with mild mitral regurgitation. Muscle histology showed minicores that were also confirmed by electron microscopy. Conclusion: Our study identified novel pathogenic variants in the TTN gene that are likely responsible for the phenotype of early-onset myopathy; hence, expanding genotype–phenotype relationship of titinopathies. [ABSTRACT FROM AUTHOR]

Abstract X-linked myotubular myopathy (XLMTM) is a life-threatening skeletal muscle disease caused by mutations in the MTM1 gene. XLMTM fibres display a population of nuclei mispositioned in the centre. In the present study, we aimed to explore whether positioning and overall distribution of nuclei affects cellular organization and contractile function, thereby contributing to muscle weakness in this disease. We also assessed whether gene therapy alters nuclear arrangement and function. We used tissue from human patients and animal models, including XLMTM dogs that had received increasing doses of recombinant AAV8 vector restoring MTM1 expression (rAAV8-cMTM1). We then used single isolated muscle fibres to analyze nuclear organization and contractile function. In addition to the expected mislocalization of nuclei in the centre of muscle fibres, a novel form of nuclear mispositioning was observed: irregular spacing between those located at the fibre periphery, and an overall increased number of nuclei, leading to dramatically smaller and inconsistent myonuclear domains. Nuclear mislocalization was associated with decreases in global nuclear synthetic activity, contractile protein content and intrinsic myofilament force production. A contractile deficit originating at the myofilaments, rather than mechanical interference by centrally positioned nuclei, was supported by experiments in regenerated mouse muscle. Systemic administration of rAAV8-cMTM1 at doses higher than 2.5 × 1013 vg kg−1 allowed a full rescue of all these cellular defects in XLMTM dogs. Altogether, these findings identify previously unrecognized pathological mechanisms in human and animal XLMTM, associated with myonuclear defects and contractile filament function. These defects can be reversed by gene therapy restoring MTM1 expression in dogs with XLMTM.

Abstract Ovine congenital progressive muscular dystrophy (OCPMD) was first described in Merino sheep flocks in Queensland and Western Australia in the 1960s and 1970s. The most prominent feature of the disease is a distinctive gait with stiffness of the hind limbs that can be seen as early as 3 weeks after birth. The disease is progressive. Histopathological examination had revealed dystrophic changes specifically in type I (slow) myofibres, while electron microscopy had demonstrated abundant nemaline bodies. Therefore, it was never certain whether the disease was a dystrophy or a congenital myopathy with dystrophic features. In this study, we performed whole genome sequencing of OCPMD sheep and identified a single base deletion at the splice donor site (+ 1) of intron 13 in the type I myofibre-specific TNNT1 gene (KT218690 c.614 + 1delG). All affected sheep were homozygous for this variant. Examination of TNNT1 splicing by RT-PCR showed intron retention and premature termination, which disrupts the highly conserved 14 amino acid C-terminus. The variant did not reduce TNNT1 protein levels or affect its localization but impaired its ability to modulate muscle contraction in response to Ca2+ levels. Identification of the causative variant in TNNT1 finally clarifies that the OCPMD sheep is in fact a large animal model of TNNT1 congenital myopathy. This model could now be used for testing molecular or gene therapies.

Background: Congenital myopathies are a diverse group of diseases that share features from the early onset of symptoms in the first year of life, such as hypotonia, muscle weakness, and developmental delays, and are often associated with respiratory insufficiency and feeding difficulties. Case presentation: Here, we report an 8-year-old boy having hypotonia and signs of respiratory insufficiency that ended with tracheostomy and ventilator-dependent status. Muscle biopsy showed histological findings of congenital fiber-type disproportion myopathy. The whole exome sequencing revealed a novel hemizygous missense variant (c.530A > C p.Gln177Pro) that confirms the diagnosis of FHL1-associated congenital myopathy. Conclusion: The findings in this study help to expand the genetic and mutational spectrum of the FHL1 gene associated with respiratory insufficiency and help in formulating a precise strategy for prognosis and future management of patients. [JBCGenetics 2020; 3(1.000): 45-51]

Abstract Background Pathogenic variations in the gene encoding the skeletal muscle ryanodine receptor (RyR1) are associated with malignant hyperthermia (MH) susceptibility, a life-threatening hypermetabolic condition and RYR1-related myopathies (RYR1-RM), a spectrum of rare neuromuscular disorders. In RYR1-RM, intracellular calcium dysregulation, post-translational modifications, and decreased protein expression lead to a heterogenous clinical presentation including proximal muscle weakness, contractures, scoliosis, respiratory insufficiency, and ophthalmoplegia. Preclinical model systems of RYR1-RM and MH have been developed to better understand underlying pathomechanisms and test potential therapeutics. Methods We conducted a comprehensive scoping review of scientific literature pertaining to RYR1-RM and MH preclinical model systems in accordance with the PRISMA Scoping Reviews Checklist and the framework proposed by Arksey and O’Malley. Two major electronic databases (PubMed and EMBASE) were searched without language restriction for articles and abstracts published between January 1, 1990 and July 3, 2019. Results Our search yielded 5049 publications from which 262 were included in this review. A majority of variants tested in RYR1 preclinical models were localized to established MH/central core disease (MH/CCD) hot spots. A total of 250 unique RYR1 variations were reported in human/rodent/porcine models with 95% being missense substitutions. The most frequently reported RYR1 variant was R614C/R615C (human/porcine total n = 39), followed by Y523S/Y524S (rabbit/mouse total n = 30), I4898T/I4897T/I4895T (human/rabbit/mouse total n = 20), and R163C/R165C (human/mouse total n = 18). The dyspedic mouse was utilized by 47% of publications in the rodent category and its RyR1-null (1B5) myotubes were transfected in 23% of publications in the cellular model category. In studies of transfected HEK-293 cells, 57% of RYR1 variations affected the RyR1 channel and activation core domain. A total of 15 RYR1 mutant mouse strains were identified of which ten were heterozygous, three were compound heterozygous, and a further two were knockout. Porcine, avian, zebrafish, C. elegans, canine, equine, and drosophila model systems were also reported. Conclusions Over the past 30 years, there were 262 publications on MH and RYR1-RM preclinical model systems featuring more than 200 unique RYR1 variations tested in a broad range of species. Findings from these studies have set the foundation for therapeutic development for MH and RYR1-RM.

Abstract Nemaline myopathy (NM) caused by mutations in the gene encoding nebulin (NEB) accounts for at least 50% of all NM cases worldwide, representing a significant disease burden. Most NEB-NM patients have autosomal recessive disease due to a compound heterozygous genotype. Of the few murine models developed for NEB-NM, most are Neb knockout models rather than harbouring Neb mutations. Additionally, some models have a very severe phenotype that limits their application for evaluating disease progression and potential therapies. No existing murine models possess compound heterozygous Neb mutations that reflect the genotype and resulting phenotype present in most patients. We aimed to develop a murine model that more closely matched the underlying genetics of NEB-NM, which could assist elucidation of the pathogenetic mechanisms underlying the disease. Here, we have characterised a mouse strain with compound heterozygous Neb mutations; one missense (p.Tyr2303His), affecting a conserved actin-binding site and one nonsense mutation (p.Tyr935*), introducing a premature stop codon early in the protein. Our studies reveal that this compound heterozygous model, Neb Y2303H, Y935X, has striking skeletal muscle pathology including nemaline bodies. In vitro whole muscle and single myofibre physiology studies also demonstrate functional perturbations. However, no reduction in lifespan was noted. Therefore, Neb Y2303H,Y935X mice recapitulate human NEB-NM and are a much needed addition to the NEB-NM mouse model collection. The moderate phenotype also makes this an appropriate model for studying NEB-NM pathogenesis, and could potentially be suitable for testing therapeutic applications.

A class of genetically based congenital myopathies known as nemaline myopathies is defined by the development of nemaline rods within muscle fibers. We present a case involving an eight-year-old boy who presented with a history of delayed motor development, proximal muscle weakness, and neck flexor weakness. Muscle enzymes were normal, and electrophysiological studies revealed a myopathic pattern. Nemaline myopathy (NM) was diagnosed with the help of clinical exome sequencing, which showed a compound heterozygous mutation with a novel variant in the nebulin (NEB) gene., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Chalipat et al.)

Background: Autosomal-recessive mutations in SPEG (striated muscle preferentially expressed protein kinase) have been linked to centronuclear myopathy with or without dilated cardiomyopathy (CNM5). Loss of SPEG is associated with defective triad formation, abnormal excitation-contraction coupling, calcium mishandling and disruption of the focal adhesion complex in skeletal muscles. To elucidate the underlying molecular pathways, we have utilized multi-omics tools and analysis to obtain a comprehensive view of the complex biological processes and molecular functions., Methods: Skeletal muscles from 2-month-old SPEG-deficient (Speg-CKO) and wild-type (WT) mice were used for RNA sequencing (n = 4 per genotype) to profile transcriptomics and mass spectrometry (n = 4 for WT; n = 3 for Speg-CKO mice) to profile proteomics and phosphoproteomics. In addition, interactomics was performed using the SPEG antibody on pooled muscle lysates (quadriceps, gastrocnemius and triceps) from WT and Speg-CKO mice. Based on the multi-omics results, we performed quantitative real-time PCR, co-immunoprecipitation and immunoblot to verify the findings., Results: We identified that SPEG interacts with myospryn complex proteins CMYA5, FSD2 and RyR1, which are critical for triad formation, and that SPEG deficiency results in myospryn complex abnormalities (protein levels decreased to 22 ± 3% for CMYA5 [P < 0.05] and 18 ± 3% for FSD2 [P < 0.01]). Furthermore, SPEG phosphorylates RyR1 at S2902 (phosphorylation level decreased to 55 ± 15% at S2902 in Speg-CKO mice; P < 0.05), and its loss affects JPH2 phosphorylation at multiple sites (increased phosphorylation at T161 [1.90 ± 0.24-fold], S162 [1.61 ± 0.37-fold] and S165 [1.66 ± 0.13-fold]; decreased phosphorylation at S228 and S231 [39 ± 6%], S234 [50 ± 12%], S593 [48 ± 3%] and S613 [66 ± 10%]; P < 0.05 for S162 and P < 0.01 for other sites). On analysing the transcriptome, the most dysregulated pathways affected by SPEG deficiency included extracellular matrix-receptor interaction (P < 1e -15 ) and peroxisome proliferator-activated receptor signalling (P < 9e -14 )., Conclusions: We have elucidated the critical role of SPEG in the triad as it works closely with myospryn complex proteins (CMYA5, FSD2 and RyR1), it regulates phosphorylation levels of various residues in JPH2 and S2902 in RyR1, and its deficiency is associated with dysregulation of several pathways. The study identifies unique SPEG-interacting proteins and their phosphorylation functions and emphasizes the importance of using a multi-omics approach to comprehensively evaluate the molecular function of proteins involved in various genetic disorders., (© 2024 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by Wiley Periodicals LLC.)