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Background and Objectives: Omigapil is a small molecule which inhibits the GAPDH-Siah1-mediated apoptosis pathway. Apoptosis is a pathomechanism underlying the congenital muscular dystrophy subtypes LAMA2-related dystrophy (LAMA2-RD) and COL6-related dystrophy (COL6-RD). Studies of omigapil in the (dyw/dyw) LAMA2-RD mouse model demonstrated improved survival, and studies in the (dy2J/dy2J) LAMA2-RD mouse model and the (Col6a1-/-) COL6-RD mouse model demonstrated decreased apoptosis. Methods: A phase 1 open-label, sequential group, ascending oral dose, cohort study of omigapil in patients with LAMA2-RD or COL6-RD ages 5-16 years was performed (1) to establish the pharmacokinetic (PK) profile of omigapil at a range of doses, (2) to evaluate the safety and tolerability of omigapil at a range of doses, and (3) to establish the feasibility of conducting disease-relevant clinical assessments. Patients were enrolled in cohorts of size 4, with each patient receiving 4 weeks of vehicle run-in and 12 weeks of study drug (at daily doses ranging from 0.02 to 0.08 mg/kg). PK data from each cohort were analyzed before each subsequent dosing cohort was enrolled. A novel, adaptive dose-finding method (stochastic approximation with virtual observation recursion) was used to allow for dose escalation/reduction between cohorts based on PK data. Results: Twenty patients were enrolled at the NIH (LAMA2-RD: N = 10; COL6-RD: N = 10). Slightly greater than dose-proportional increases in systemic exposure to omigapil were seen at doses 0.02-0.08 mg/kg/d. The dose which achieved patient exposure within the pre-established target area under the plasma concentration-vs-time curve (AUC0-24h) range was 0.06 mg/kg/d. In general, omigapil was safe and well tolerated. No consistent changes were seen in the disease-relevant clinical assessments during the duration of the study. Discussion: This study represents the thus far only clinical trial of a therapeutic small molecule for LAMA2-RD and COL6-RD, completed with an adaptive trial design to arrive at dose adjustments. The trial met its primary end point and established that the PK profile of omigapil is suitable for further development in pediatric patients with LAMA2-RD or COL6-RD, the most common forms of congenital muscular dystrophy. While within the short duration of the study disease-relevant clinical assessments did not demonstrate significant changes, this study establishes the feasibility of performing interventional clinical trials in these rare disease patient populations. Classification of Evidence: This study provides Class IV evidence of omigapil in a dose-finding phase 1 study. Trial Registration Information: Clinical Trials NCT01805024.
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Collagen VI-related dystrophies (COL6-RDs) manifest with a spectrum of clinical phenotypes, ranging from Ullrich congenital muscular dystrophy (UCMD), presenting with prominent congenital symptoms and characterised by progressive muscle weakness, joint contractures and respiratory insufficiency, to Bethlem muscular dystrophy, with milder symptoms typically recognised later and at times resembling a limb girdle muscular dystrophy, and intermediate phenotypes falling between UCMD and Bethlem muscular dystrophy. Despite clinical and immunohistochemical features highly suggestive of COL6-RD, some patients had remained without an identified causative variant in COL6A1, COL6A2 or COL6A3. With combined muscle RNA-sequencing and whole-genome sequencing we uncovered a recurrent, de novo deep intronic variant in intron 11 of COL6A1 (c.930+189C>T) that leads to a dominantly acting in-frame pseudoexon insertion. We subsequently identified and have characterised an international cohort of forty-four patients with this COL6A1 intron 11 causative variant, one of the most common recurrent causative variants in the collagen VI genes. Patients manifest a consistently severe phenotype characterised by a paucity of early symptoms followed by an accelerated progression to a severe form of UCMD, except for one patient with somatic mosaicism for this COL6A1 intron 11 variant who manifests a milder phenotype consistent with Bethlem muscular dystrophy. Characterisation of this individual provides a robust validation for the development of our pseudoexon skipping therapy. We have previously shown that splice-modulating antisense oligomers applied in vitro effectively decreased the abundance of the mutant pseudoexon-containing COL6A1 transcripts to levels comparable to the in vivo scenario of the somatic mosaicism shown here, indicating that this therapeutic approach carries significant translational promise for ameliorating the severe form of UCMD caused by this common recurrent COL6A1 causative variant to a Bethlem muscular dystrophy phenotype.
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Troponin I (TnI) regulates thin filament activation and muscle contraction. Two isoforms, TnI-fast (TNNI2) and TnI-slow (TNNI1), are predominantly expressed in fast- and slow-twitch myofibers, respectively. TNNI2 variants are a rare cause of arthrogryposis, whereas TNNI1 variants have not been conclusively established to cause skeletal myopathy. We identified recessive loss-of-function TNNI1 variants as well as dominant gain-of-function TNNI1 variants as a cause of muscle disease, each with distinct physiological consequences and disease mechanisms. We identified three families with biallelic TNNI1 variants (F1: p.R14H/c.190-9G>A, F2 and F3: homozygous p.R14C), resulting in loss of function, manifesting with early-onset progressive muscle weakness and rod formation on histology. We also identified two families with a dominantly acting heterozygous TNNI1 variant (F4: p.R174Q and F5: p.K176del), resulting in gain of function, manifesting with muscle cramping, myalgias, and rod formation in F5. In zebrafish, TnI proteins with either of the missense variants (p.R14H; p.R174Q) incorporated into thin filaments. Molecular dynamics simulations suggested that the loss-of-function p.R14H variant decouples TnI from TnC, which was supported by functional studies showing a reduced force response of sarcomeres to submaximal [Ca2+] in patient myofibers. This contractile deficit could be reversed by a slow skeletal muscle troponin activator. In contrast, patient myofibers with the gain-of-function p.R174Q variant showed an increased force to submaximal [Ca2+], which was reversed by the small-molecule drug mavacamten. Our findings demonstrated that TNNI1 variants can cause muscle disease with variant-specific pathomechanisms, manifesting as either a hypo- or a hypercontractile phenotype, suggesting rational therapeutic strategies for each mechanism.
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Doenças Musculares , Sarcômeros , Animais , Humanos , Cálcio/metabolismo , Contração Muscular , Músculo Esquelético/metabolismo , Doenças Musculares/genética , Sarcômeros/metabolismo , Troponina I/genética , Troponina I/metabolismo , Peixe-Zebra/metabolismoRESUMO
Pathogenic variants in the DES gene clinically manifest as progressive skeletal muscle weakness, cardiomyopathy with associated severe arrhythmias, and respiratory insufficiency, and are collectively known as desminopathies. While most DES pathogenic variants act via a dominant mechanism, recessively acting variants have also been reported. Currently, there are no effective therapeutic interventions for desminopathies of any type. Here, we report an affected individual with rapidly progressive dilated cardiomyopathy, requiring heart transplantation at age 13 years, in the setting of childhood-onset skeletal muscle weakness. We identified biallelic DES variants (c.640-13 T>A and c.1288+1 G>A) and show aberrant DES gene splicing in the affected individual's muscle. Through the generation of an inducible lentiviral system, we transdifferentiated fibroblast cultures derived from the affected individual into myoblasts and validated this system using RNA sequencing. We tested rationally designed, custom antisense oligonucleotides to screen for splice correction in these transdifferentiated cells and a functional minigene splicing assay. However, rather than correctly redirecting splicing, we found them to induce undesired exon skipping. Our results indicate that, while an individual precision-based molecular therapeutic approach to splice-altering pathogenic variants is promising, careful preclinical testing is imperative for each novel variant to test the feasibility of this type of approach for translation.
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Cardiomiopatias , Cardiomiopatia Dilatada , Adolescente , Humanos , Cardiomiopatias/genética , Cardiomiopatia Dilatada/genética , Mutação , Splicing de RNA/genéticaRESUMO
Background: The concept of a myopathy with associated tremor ("myogenic tremor") in humans has been previously described for specific MYBPC1 (Myosin-Binding Protein C) variants. Here we report for the first time an individual with tremor who was found to have a de-novo likely pathogenic variant in Myosin Heavy Chain 7 (MYH7).We provide a detailed electrophysiological characterization of the tremor syndrome in a human individual with a myopathy and this pathogenic MYH7 variant to provide further insight in the phenotypic spectrum and pathomechanism of myogenic tremors in skeletal sarcomeric myopathies. Methods: Electromyographic recordings were obtained from facial muscles, as well as bilateral upper and lower extremities. Results: 10 to 11 Hz activity was observed in the face and extremities during recordings with muscle activation. There were intermittent episodes of significant left-right coherence that would modulate across muscle groups throughout the recording, but no coherence between muscles at different levels of the neuraxis. Conclusions: A possible explanation for this phenomenon is that the tremor originates at the sarcomere level within muscles, which is then picked up by muscle spindles and leads to activating input to the neuraxis segment. At the same time, the stability of the tremor frequency does suggest the presence of central oscillators at the segmental level. Thus, further studies will be needed to determine the origin of myogenic tremor and to better understand the pathomechanism.
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INTRODUCTION: Arthrogryposis is characterized by the presence of multiple contractures at birth and can be caused by pathogenic variants in TTN (Titin). Exons and variants that are not expressed in one of the three major isoforms of titin are referred to as "metatranscript-only" and have been considered to be only expressed during fetal development. Recently, the metatranscript-only variant (c.39974-11T > G) in TTN with a second truncating TTN variant has been linked to arthrogryposis multiplex congenita and myopathy. METHODS: Via exome sequencing we identified the TTN c.39974-11T > G splice variant in trans with one of three truncating variants (p.Arg8922*, p.Lys32998Asnfs*63, p.Tyr10345*) in five individuals from three families. Clinical presentation and muscle ultrasound as well as MRI images were analyzed. RESULTS: All five patients presented with generalized muscular hypotonia, reduced muscle bulk, and congenital contractures most prominently affecting the upper limbs and distal joints. Muscular hypotonia persisted and contractures improved over time. One individual, the recipient twin in the setting of twin-to-twin transfusion syndrome, died from severe cardiac hypertrophy 1 day after birth. Ultrasound and MRI imaging studies revealed a recognizable pattern of muscle involvement with striking fibrofatty involvement of the hamstrings and calves, and relative sparing of the femoral adductors and anterior segment of the thighs. CONCLUSION: The recurrent TTN c.39974-11T > G variant consistently causes congenital arthrogryposis and persisting myopathy providing evidence that the metatranscript-only 213 to 217 exons impact muscle elasticity during early development and beyond. There is a recognizable pattern of muscle involvement, which is distinct from other myopathies and provides valuable clues for diagnostic work-up.
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Artrogripose , Contratura , Doenças Musculares , Artrogripose/diagnóstico por imagem , Artrogripose/genética , Conectina/genética , Contratura/diagnóstico por imagem , Contratura/genética , Humanos , Recém-Nascido , Hipotonia Muscular , Mutação , Isoformas de ProteínasRESUMO
Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disease of the lower and upper motor neurons with sporadic or hereditary occurrence. Age of onset, pattern of motor neuron degeneration and disease progression vary widely among individuals with ALS. Various cellular processes may drive ALS pathomechanisms, but a monogenic direct metabolic disturbance has not been causally linked to ALS. Here we show SPTLC1 variants that result in unrestrained sphingoid base synthesis cause a monogenic form of ALS. We identified four specific, dominantly acting SPTLC1 variants in seven families manifesting as childhood-onset ALS. These variants disrupt the normal homeostatic regulation of serine palmitoyltransferase (SPT) by ORMDL proteins, resulting in unregulated SPT activity and elevated levels of canonical SPT products. Notably, this is in contrast with SPTLC1 variants that shift SPT amino acid usage from serine to alanine, result in elevated levels of deoxysphingolipids and manifest with the alternate phenotype of hereditary sensory and autonomic neuropathy. We custom designed small interfering RNAs that selectively target the SPTLC1 ALS allele for degradation, leave the normal allele intact and normalize sphingolipid levels in vitro. The role of primary metabolic disturbances in ALS has been elusive; this study defines excess sphingolipid biosynthesis as a fundamental metabolic mechanism for motor neuron disease.
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Esclerose Lateral Amiotrófica/metabolismo , Esfingolipídeos/biossíntese , Adolescente , Adulto , Alelos , Sequência de Aminoácidos , Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/genética , Sistemas CRISPR-Cas , Criança , Feminino , Genes Dominantes , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Adulto JovemRESUMO
The extracellular matrix comprises a network of macromolecules such as collagens, proteoglycans and glycoproteins. VWA1 (von Willebrand factor A domain containing 1) encodes a component of the extracellular matrix that interacts with perlecan/collagen VI, appears to be involved in stabilizing extracellular matrix structures, and demonstrates high expression levels in tibial nerve. Vwa1-deficient mice manifest with abnormal peripheral nerve structure/function; however, VWA1 variants have not previously been associated with human disease. By interrogating the genome sequences of 74 180 individuals from the 100K Genomes Project in combination with international gene-matching efforts and targeted sequencing, we identified 17 individuals from 15 families with an autosomal-recessive, non-length dependent, hereditary motor neuropathy and rare biallelic variants in VWA1. A single disease-associated allele p.(G25Rfs*74), a 10-bp repeat expansion, was observed in 14/15 families and was homozygous in 10/15. Given an allele frequency in European populations approaching 1/1000, the seven unrelated homozygote individuals ascertained from the 100K Genomes Project represents a substantial enrichment above expected. Haplotype analysis identified a shared 220 kb region suggesting that this founder mutation arose >7000 years ago. A wide age-range of patients (6-83 years) helped delineate the clinical phenotype over time. The commonest disease presentation in the cohort was an early-onset (mean 2.0 ± 1.4 years) non-length-dependent axonal hereditary motor neuropathy, confirmed on electrophysiology, which will have to be differentiated from other predominantly or pure motor neuropathies and neuronopathies. Because of slow disease progression, ambulation was largely preserved. Neurophysiology, muscle histopathology, and muscle MRI findings typically revealed clear neurogenic changes with single isolated cases displaying additional myopathic process. We speculate that a few findings of myopathic changes might be secondary to chronic denervation rather than indicating an additional myopathic disease process. Duplex reverse transcription polymerase chain reaction and immunoblotting using patient fibroblasts revealed that the founder allele results in partial nonsense mediated decay and an absence of detectable protein. CRISPR and morpholino vwa1 modelling in zebrafish demonstrated reductions in motor neuron axonal growth, synaptic formation in the skeletal muscles and locomotive behaviour. In summary, we estimate that biallelic variants in VWA1 may be responsible for up to 1% of unexplained hereditary motor neuropathy cases in Europeans. The detailed clinical characterization provided here will facilitate targeted testing on suitable patient cohorts. This novel disease gene may have previously evaded detection because of high GC content, consequential low coverage and computational difficulties associated with robustly detecting repeat-expansions. Reviewing previously unsolved exomes using lower QC filters may generate further diagnoses.
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Proteínas da Matriz Extracelular/genética , Neuropatia Hereditária Motora e Sensorial/genética , Adulto , Idoso , Animais , Comportamento Animal/fisiologia , Criança , Feminino , Neuropatia Hereditária Motora e Sensorial/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Mutação , Linhagem , Adulto Jovem , Peixe-ZebraRESUMO
The myosin-directed chaperone UNC-45B is essential for sarcomeric organization and muscle function from Caenorhabditis elegans to humans. The pathological impact of UNC-45B in muscle disease remained elusive. We report ten individuals with bi-allelic variants in UNC45B who exhibit childhood-onset progressive muscle weakness. We identified a common UNC45B variant that acts as a complex hypomorph splice variant. Purified UNC-45B mutants showed changes in folding and solubility. In situ localization studies further demonstrated reduced expression of mutant UNC-45B in muscle combined with abnormal localization away from the A-band towards the Z-disk of the sarcomere. The physiological relevance of these observations was investigated in C. elegans by transgenic expression of conserved UNC-45 missense variants, which showed impaired myosin binding for one and defective muscle function for three. Together, our results demonstrate that UNC-45B impairment manifests as a chaperonopathy with progressive muscle pathology, which discovers the previously unknown conserved role of UNC-45B in myofibrillar organization.
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Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiologia , Chaperonas Moleculares/genética , Chaperonas Moleculares/fisiologia , Doenças Musculares/genética , Mutação de Sentido Incorreto , Adolescente , Adulto , Alelos , Animais , Caenorhabditis elegans , Feminino , Variação Genética , Humanos , Mutação com Perda de Função , Masculino , Músculo Esquelético/patologia , Miofibrilas , Miosinas , Sarcômeros/metabolismo , Análise de Sequência de RNA , Transgenes , Sequenciamento do Exoma , Adulto JovemRESUMO
Congenital disorders of muscle most importantly encompass the congenital muscular dystrophies as well as the congenital myopathies. With the broader availability of next generation genetic testing there has been an expansion of phenotypes and genotypes, while the very large genes such as titin, nebulin, and RYR1 have also become accessible to complete sequencing. This development has had considerable diagnostic power while at the same time also creating challenges in the interpretation of the many variants of uncertain significance that will need a solid clinical plausibility test, based on "deep" phenotyping, taking into account clinical, extended clinical, histological, and physiological data. One tool in this context is imaging of skeletal muscle, including by ultrasound. Muscle ultrasound is a useful, noninvasive, child-friendly technique for visualizing normal and pathological skeletal muscle. By virtue of its different mode of image acquisition compared to muscle MRI, it allows for the assessment of different and often earlier changes, also circumventing the need for sedation. Herein we highlight the important role of muscle ultrasound as a diagnostic tool and an extension of the physical exam in the work-up of congenital onset muscle disease, presenting various relevant clinical scenarios. We show how muscle ultrasound can confirm or refute skeletal muscle involvement and yield information about the nature of the involvement (myopathic vs neurogenic). Muscle ultrasound can also guide the appropriate next diagnostic steps and recognize diagnostically important qualitative patterns to help confirm or refute genetic considerations raised by next generation sequencing. We illustrate specific muscle ultrasound involvement patterns, which constitute accessible diagnostic hints and show that muscle ultrasound, in conjunction with the clinical phenotype, the histological appearance of the muscle biopsy (when available), and the ascertained genotype, can be a very powerful tool in integrating all available information into a final accurate and precise diagnosis in the age of next generation sequencing.
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Músculo Esquelético/diagnóstico por imagem , Mutação , Miopatias Congênitas Estruturais/diagnóstico , Ultrassonografia , Análise Mutacional de DNA , Diagnóstico Diferencial , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Miopatias Congênitas Estruturais/diagnóstico por imagem , Miopatias Congênitas Estruturais/genéticaRESUMO
The identification of genes implicated in myopathies is essential for diagnosis and for revealing novel therapeutic targets. Here we characterize a novel subclass of congenital myopathy at the morphological, molecular, and functional level. Through exome sequencing, we identified de novo ACTN2 mutations, a missense and a deletion, in two unrelated patients presenting with progressive early-onset muscle weakness and respiratory involvement. Morphological and ultrastructural analyses of muscle biopsies revealed a distinctive pattern with the presence of muscle fibers containing small structured cores and jagged Z-lines. Deeper analysis of the missense mutation revealed mutant alpha-actinin-2 properly localized to the Z-line in differentiating myotubes and its level was not altered in muscle biopsy. Modelling of the disease in zebrafish and mice by exogenous expression of mutated alpha-actinin-2 recapitulated the abnormal muscle function and structure seen in the patients. Motor deficits were noted in zebrafish, and muscle force was impaired in isolated muscles from AAV-transduced mice. In both models, sarcomeric disorganization was evident, while expression of wild-type alpha-actinin-2 did not result in muscle anomalies. The murine muscles injected with mutant ACTN2 displayed cores and Z-line defects. Dominant ACTN2 mutations were previously associated with cardiomyopathies, and our data demonstrate that specific mutations in the well-known Z-line regulator alpha-actinin-2 can cause a skeletal muscle disorder.