RESUMEN
This study establishes PYROXD1 variants as a cause of early-onset myopathy and uses biospecimens and cell lines, yeast, and zebrafish models to elucidate the fundamental role of PYROXD1 in skeletal muscle. Exome sequencing identified recessive variants in PYROXD1 in nine probands from five families. Affected individuals presented in infancy or childhood with slowly progressive proximal and distal weakness, facial weakness, nasal speech, swallowing difficulties, and normal to moderately elevated creatine kinase. Distinctive histopathology showed abundant internalized nuclei, myofibrillar disorganization, desmin-positive inclusions, and thickened Z-bands. PYROXD1 is a nuclear-cytoplasmic pyridine nucleotide-disulphide reductase (PNDR). PNDRs are flavoproteins (FAD-binding) and catalyze pyridine-nucleotide-dependent (NAD/NADH) reduction of thiol residues in other proteins. Complementation experiments in yeast lacking glutathione reductase glr1 show that human PYROXD1 has reductase activity that is strongly impaired by the disease-associated missense mutations. Immunolocalization studies in human muscle and zebrafish myofibers demonstrate that PYROXD1 localizes to the nucleus and to striated sarcomeric compartments. Zebrafish with ryroxD1 knock-down recapitulate features of PYROXD1 myopathy with sarcomeric disorganization, myofibrillar aggregates, and marked swimming defect. We characterize variants in the oxidoreductase PYROXD1 as a cause of early-onset myopathy with distinctive histopathology and introduce altered redox regulation as a primary cause of congenital muscle disease.
Asunto(s)
Núcleo Celular/genética , Miopatías Distales/genética , Variación Genética , Miopatías Estructurales Congénitas/genética , Oxidorreductasas/genética , Secuencia de Aminoácidos , Animales , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Estudios de Cohortes , Creatina Quinasa/genética , Creatina Quinasa/metabolismo , Citoplasma/metabolismo , Miopatías Distales/patología , Proteína 4 Similar a ELAV/genética , Proteína 4 Similar a ELAV/metabolismo , Femenino , Flavoproteínas/metabolismo , Eliminación de Gen , Estudio de Asociación del Genoma Completo , Glutatión Reductasa/genética , Glutatión Reductasa/metabolismo , Células HEK293 , Humanos , Masculino , Músculo Esquelético/patología , Mutación Missense , Miopatías Estructurales Congénitas/patología , Oxidorreductasas/metabolismo , Linaje , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Pez Cebra/genéticaRESUMEN
OBJECTIVE: Nemaline myopathy (NM) is one of the most common congenital nondystrophic myopathies and is characterized by muscle weakness, often from birth. Mutations in ACTA1 are a frequent cause of NM (ie, NEM3). ACTA1 encodes alpha-actin 1, the main constituent of the sarcomeric thin filament. The mechanisms by which mutations in ACTA1 contribute to muscle weakness in NEM3 are incompletely understood. We hypothesized that sarcomeric dysfunction contributes to muscle weakness in NEM3 patients. METHODS: To test this hypothesis, we performed contractility measurements in individual muscle fibers and myofibrils obtained from muscle biopsies of 14 NEM3 patients with different ACTA1 mutations. To identify the structural basis for impaired contractility, low angle X-ray diffraction and stimulated emission-depletion microscopy were applied. RESULTS: Our findings reveal that muscle fibers of NEM3 patients display a reduced maximal force-generating capacity, which is caused by dysfunctional sarcomere contractility in the majority of patients, as revealed by contractility measurements in myofibrils. Low angle X-ray diffraction and stimulated emission-depletion microscopy indicate that dysfunctional sarcomere contractility in NEM3 patients involves a lower number of myosin heads binding to actin during muscle activation. This lower number is not the result of reduced thin filament length. Interestingly, the calcium sensitivity of force is unaffected in some patients, but decreased in others. INTERPRETATION: Dysfunctional sarcomere contractility is an important contributor to muscle weakness in the majority of NEM3 patients. This information is crucial for patient stratification in future clinical trials. Ann Neurol 2018;83:269-282.
Asunto(s)
Actinas/genética , Contracción Muscular/fisiología , Debilidad Muscular/genética , Miopatías Estructurales Congénitas/fisiopatología , Sarcómeros/patología , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Debilidad Muscular/fisiopatología , Músculo Esquelético/patología , Miopatías Estructurales Congénitas/genética , Sarcómeros/fisiología , Adulto JovenRESUMEN
Dominant mutations in TPM3, encoding α-tropomyosinslow, cause a congenital myopathy characterized by generalized muscle weakness. Here, we used a multidisciplinary approach to investigate the mechanism of muscle dysfunction in 12 TPM3-myopathy patients. We confirm that slow myofibre hypotrophy is a diagnostic hallmark of TPM3-myopathy, and is commonly accompanied by skewing of fibre-type ratios (either slow or fast fibre predominance). Patient muscle contained normal ratios of the three tropomyosin isoforms and normal fibre-type expression of myosins and troponins. Using 2D-PAGE, we demonstrate that mutant α-tropomyosinslow was expressed, suggesting muscle dysfunction is due to a dominant-negative effect of mutant protein on muscle contraction. Molecular modelling suggested mutant α-tropomyosinslow likely impacts actin-tropomyosin interactions and, indeed, co-sedimentation assays showed reduced binding of mutant α-tropomyosinslow (R168C) to filamentous actin. Single fibre contractility studies of patient myofibres revealed marked slow myofibre specific abnormalities. At saturating [Ca(2+)] (pCa 4.5), patient slow fibres produced only 63% of the contractile force produced in control slow fibres and had reduced acto-myosin cross-bridge cycling kinetics. Importantly, due to reduced Ca(2+)-sensitivity, at sub-saturating [Ca(2+)] (pCa 6, levels typically released during in vivo contraction) patient slow fibres produced only 26% of the force generated by control slow fibres. Thus, weakness in TPM3-myopathy patients can be directly attributed to reduced slow fibre force at physiological [Ca(2+)], and impaired acto-myosin cross-bridge cycling kinetics. Fast myofibres are spared; however, they appear to be unable to compensate for slow fibre dysfunction. Abnormal Ca(2+)-sensitivity in TPM3-myopathy patients suggests Ca(2+)-sensitizing drugs may represent a useful treatment for this condition.
Asunto(s)
Fibras Musculares de Contracción Lenta/metabolismo , Atrofia Muscular/metabolismo , Enfermedades Musculares/metabolismo , Miosinas/metabolismo , Tropomiosina/genética , Actinas/genética , Actinas/metabolismo , Adolescente , Adulto , Calcio/metabolismo , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Contracción Muscular/fisiología , Debilidad Muscular/genética , Debilidad Muscular/metabolismo , Atrofia Muscular/genética , Enfermedades Musculares/genética , Mutación , Miosinas/genética , Isoformas de Proteínas , Tropomiosina/metabolismoRESUMEN
Glycosylphosphatidylinositol (GPI)-anchored proteins are ubiquitously expressed in the human body and are important for various functions at the cell surface. Mutations in many GPI biosynthesis genes have been described to date in patients with multi-system disease and together these constitute a subtype of congenital disorders of glycosylation. We used whole exome sequencing in two families to investigate the genetic basis of disease and used RNA and cellular studies to investigate the functional consequences of sequence variants in the PIGY gene. Two families with different phenotypes had homozygous recessive sequence variants in the GPI biosynthesis gene PIGY. Two sisters with c.137T>C (p.Leu46Pro) PIGY variants had multi-system disease including dysmorphism, seizures, severe developmental delay, cataracts and early death. There were significantly reduced levels of GPI-anchored proteins (CD55 and CD59) on the surface of patient-derived skin fibroblasts (â¼20-50% compared with controls). In a second, consanguineous family, two siblings had moderate development delay and microcephaly. A homozygous PIGY promoter variant (c.-540G>A) was detected within a 7.7 Mb region of autozygosity. This variant was predicted to disrupt a SP1 consensus binding site and was shown to be associated with reduced gene expression. Mutations in PIGY can occur in coding and non-coding regions of the gene and cause variable phenotypes. This article contributes to understanding of the range of disease phenotypes and disease genes associated with deficiencies of the GPI-anchor biosynthesis pathway and also serves to highlight the potential importance of analysing variants detected in 5'-UTR regions despite their typically low coverage in exome data.
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Glicosilfosfatidilinositoles/deficiencia , Proteínas de la Membrana/genética , Mutación , Antígenos CD55/biosíntesis , Antígenos CD59/biosíntesis , Línea Celular Tumoral , Preescolar , Análisis Mutacional de ADN , Femenino , Expresión Génica , Glicosilfosfatidilinositoles/genética , Humanos , Lactante , Recién Nacido , Masculino , Fenotipo , Convulsiones , TransfecciónRESUMEN
OBJECTIVE: To evaluate the diagnostic outcomes in a large cohort of congenital muscular dystrophy (CMD) patients using traditional and next generation sequencing (NGS) technologies. METHODS: A total of 123 CMD patients were investigated using the traditional approaches of histology, immunohistochemical analysis of muscle biopsy, and candidate gene sequencing. Undiagnosed patients available for further testing were investigated using NGS. RESULTS: Muscle biopsy and immunohistochemical analysis found deficiencies of laminin α2, α-dystroglycan, or collagen VI in 50% of patients. Candidate gene sequencing and chromosomal microarray established a genetic diagnosis in 32% (39 of 123). Of 85 patients presenting in the past 20 years, 28 of 51 who lacked a confirmed genetic diagnosis (55%) consented to NGS studies, leading to confirmed diagnoses in a further 11 patients. Using the combination of approaches, a confirmed genetic diagnosis was achieved in 51% (43 of 85). The diagnoses within the cohort were heterogeneous. Forty-five of 59 probands with confirmed or probable diagnoses had variants in genes known to cause CMD (76%), and 11 of 59 (19%) had variants in genes associated with congenital myopathies, reflecting overlapping features of these conditions. One patient had a congenital myasthenic syndrome, and 2 had microdeletions. Within the cohort, 5 patients had variants in novel (PIGY and GMPPB) or recently published genes (GFPT1 and MICU1), and 7 had variants in TTN or RYR1, large genes that are technically difficult to Sanger sequence. INTERPRETATION: These data support NGS as a first-line tool for genetic evaluation of patients with a clinical phenotype suggestive of CMD, with muscle biopsy reserved as a second-tier investigation. Ann Neurol 2016;80:101-111.
Asunto(s)
Predisposición Genética a la Enfermedad/genética , Distrofias Musculares/diagnóstico , Distrofias Musculares/genética , Adolescente , Adulto , Niño , Preescolar , Colágeno Tipo VI/deficiencia , Distroglicanos/deficiencia , Variación Genética/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Laminina/deficiencia , Músculo Esquelético/metabolismo , Adulto JovenRESUMEN
OBJECTIVE: Thin filament myopathies are among the most common nondystrophic congenital muscular disorders, and are caused by mutations in genes encoding proteins that are associated with the skeletal muscle thin filament. Mechanisms underlying muscle weakness are poorly understood, but might involve the length of the thin filament, an important determinant of force generation. METHODS: We investigated the sarcomere length-dependence of force, a functional assay that provides insights into the contractile strength of muscle fibers as well as the length of the thin filaments, in muscle fibers from 51 patients with thin filament myopathy caused by mutations in NEB, ACTA1, TPM2, TPM3, TNNT1, KBTBD13, KLHL40, and KLHL41. RESULTS: Lower force generation was observed in muscle fibers from patients of all genotypes. In a subset of patients who harbor mutations in NEB and ACTA1, the lower force was associated with downward shifted force-sarcomere length relations, indicative of shorter thin filaments. Confocal microscopy confirmed shorter thin filaments in muscle fibers of these patients. A conditional Neb knockout mouse model, which recapitulates thin filament myopathy, revealed a compensatory mechanism; the lower force generation that was associated with shorter thin filaments was compensated for by increasing the number of sarcomeres in series. This allowed muscle fibers to operate at a shorter sarcomere length and maintain optimal thin-thick filament overlap. INTERPRETATION: These findings might provide a novel direction for the development of therapeutic strategies for thin filament myopathy patients with shortened thin filament lengths. Ann Neurol 2016;79:959-969.
Asunto(s)
Citoesqueleto/genética , Proteínas Musculares/genética , Enfermedades Musculares/genética , Enfermedades Musculares/fisiopatología , Sarcómeros/genética , Actinas/genética , Animales , Estudios de Casos y Controles , Citoesqueleto/fisiología , Humanos , Ratones Noqueados , Contracción Muscular/genética , Contracción Muscular/fisiología , Proteínas Musculares/metabolismo , Proteínas Musculares/fisiología , Músculo Esquelético/metabolismo , Mutación , Sarcómeros/fisiologíaRESUMEN
Dominant congenital spinal muscular atrophy (DCSMA) is a disorder of developing anterior horn cells and shows lower-limb predominance and clinical overlap with hereditary spastic paraplegia (HSP), a lower-limb-predominant disorder of corticospinal motor neurons. We have identified four mutations in bicaudal D homolog 2 (Drosophila) (BICD2) in six kindreds affected by DCSMA, DCSMA with upper motor neuron features, or HSP. BICD2 encodes BICD2, a key adaptor protein that interacts with the dynein-dynactin motor complex, which facilitates trafficking of cellular cargos that are critical to motor neuron development and maintenance. We demonstrate that mutations resulting in amino acid substitutions in two binding regions of BICD2 increase its binding affinity for the cytoplasmic dynein-dynactin complex, which might result in the perturbation of BICD2-dynein-dynactin-mediated trafficking, and impair neurite outgrowth. These findings provide insight into the mechanism underlying both the static and the slowly progressive clinical features and the motor neuron pathology that characterize BICD2-associated diseases, and underscore the importance of the dynein-dynactin transport pathway in the development and survival of both lower and upper motor neurons.
Asunto(s)
Proteínas Portadoras/genética , Atrofia Muscular Espinal/genética , Mutación Missense , Paraplejía/genética , Adulto , Anciano , Proteínas Portadoras/metabolismo , Niño , Preescolar , Dineínas Citoplasmáticas/metabolismo , Femenino , Genes Dominantes , Ligamiento Genético , Estudio de Asociación del Genoma Completo , Células HEK293 , Haplotipos , Humanos , Masculino , Proteínas Asociadas a Microtúbulos , Persona de Mediana Edad , Atrofia Muscular Espinal/congénito , Atrofia Muscular Espinal/metabolismo , Paraplejía/metabolismo , Linaje , Polimorfismo de Nucleótido Simple , Unión Proteica , Adulto JovenRESUMEN
Nemaline myopathy (NM) is a rare congenital muscle disorder primarily affecting skeletal muscles that results in neonatal death in severe cases as a result of associated respiratory insufficiency. NM is thought to be a disease of sarcomeric thin filaments as six of eight known genes whose mutation can cause NM encode components of that structure, however, recent discoveries of mutations in non-thin filament genes has called this model in question. We performed whole-exome sequencing and have identified recessive small deletions and missense changes in the Kelch-like family member 41 gene (KLHL41) in four individuals from unrelated NM families. Sanger sequencing of 116 unrelated individuals with NM identified compound heterozygous changes in KLHL41 in a fifth family. Mutations in KLHL41 showed a clear phenotype-genotype correlation: Frameshift mutations resulted in severe phenotypes with neonatal death, whereas missense changes resulted in impaired motor function with survival into late childhood and/or early adulthood. Functional studies in zebrafish showed that loss of Klhl41 results in highly diminished motor function and myofibrillar disorganization, with nemaline body formation, the pathological hallmark of NM. These studies expand the genetic heterogeneity of NM and implicate a critical role of BTB-Kelch family members in maintenance of sarcomeric integrity in NM.
Asunto(s)
Mutación , Miofibrillas/metabolismo , Miopatías Nemalínicas/genética , Miopatías Nemalínicas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas/genética , Transducción de Señal , Ubiquitinación , Adolescente , Animales , Niño , Preescolar , Proteínas del Citoesqueleto , Resultado Fatal , Femenino , Expresión Génica , Orden Génico , Estudios de Asociación Genética , Humanos , Lactante , Recién Nacido , Masculino , Modelos Moleculares , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/ultraestructura , Miopatías Nemalínicas/diagnóstico , Conformación Proteica , Proteínas/química , Pez CebraRESUMEN
Nemaline myopathy (NEM) is a common congenital myopathy. At the very severe end of the NEM clinical spectrum are genetically unresolved cases of autosomal-recessive fetal akinesia sequence. We studied a multinational cohort of 143 severe-NEM-affected families lacking genetic diagnosis. We performed whole-exome sequencing of six families and targeted gene sequencing of additional families. We identified 19 mutations in KLHL40 (kelch-like family member 40) in 28 apparently unrelated NEM kindreds of various ethnicities. Accounting for up to 28% of the tested individuals in the Japanese cohort, KLHL40 mutations were found to be the most common cause of this severe form of NEM. Clinical features of affected individuals were severe and distinctive and included fetal akinesia or hypokinesia and contractures, fractures, respiratory failure, and swallowing difficulties at birth. Molecular modeling suggested that the missense substitutions would destabilize the protein. Protein studies showed that KLHL40 is a striated-muscle-specific protein that is absent in KLHL40-associated NEM skeletal muscle. In zebrafish, klhl40a and klhl40b expression is largely confined to the myotome and skeletal muscle, and knockdown of these isoforms results in disruption of muscle structure and loss of movement. We identified KLHL40 mutations as a frequent cause of severe autosomal-recessive NEM and showed that it plays a key role in muscle development and function. Screening of KLHL40 should be a priority in individuals who are affected by autosomal-recessive NEM and who present with prenatal symptoms and/or contractures and in all Japanese individuals with severe NEM.
Asunto(s)
Proteínas Musculares/metabolismo , Músculo Esquelético/patología , Mutación Missense , Miopatías Nemalínicas/genética , Sustitución de Aminoácidos , Animales , Pueblo Asiatico/genética , Estudios de Cohortes , Mutación del Sistema de Lectura , Genes Recesivos , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Proteínas Musculares/genética , Miopatías Nemalínicas/etnología , Miopatías Nemalínicas/patología , Linaje , Polimorfismo de Nucleótido Simple , Índice de Severidad de la Enfermedad , Pez Cebra/genéticaRESUMEN
Dystroglycanopathies are a heterogeneous group of diseases with a broad phenotypic spectrum ranging from severe disorders with congenital muscle weakness, eye and brain structural abnormalities and intellectual delay to adult-onset limb-girdle muscular dystrophies without mental retardation. Most frequently the disease onset is congenital or during childhood. The exception is FKRP mutations, in which adult onset is a common presentation. Here we report eight patients from five non-consanguineous families where next generation sequencing identified mutations in the GMPPB gene. Six patients presented as an adult or adolescent-onset limb-girdle muscular dystrophy, one presented with isolated episodes of rhabdomyolysis, and one as a congenital muscular dystrophy. This report expands the phenotypic spectrum of GMPPB mutations to include limb-girdle muscular dystrophies with adult onset with or without intellectual disability, or isolated rhabdomyolysis.
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Distrofia Muscular de Cinturas/diagnóstico , Distrofia Muscular de Cinturas/genética , Mutación/genética , Nucleotidiltransferasas/genética , Fenotipo , Adolescente , Adulto , Anciano , Niño , Preescolar , Distroglicanos/genética , Resultado Fatal , Femenino , Humanos , Masculino , Persona de Mediana Edad , Linaje , Adulto JovenRESUMEN
Spinal muscular atrophy is a disorder of lower motor neurons, most commonly caused by recessive mutations in SMN1 on chromosome 5q. Cases without SMN1 mutations are subclassified according to phenotype. Spinal muscular atrophy, lower extremity-predominant, is characterized by lower limb muscle weakness and wasting, associated with reduced numbers of lumbar motor neurons and is caused by mutations in DYNC1H1, which encodes a microtubule motor protein in the dynein-dynactin complex and one of its cargo adaptors, BICD2. We have now identified 32 patients with BICD2 mutations from nine different families, providing detailed insights into the clinical phenotype and natural history of BICD2 disease. BICD2 spinal muscular atrophy, lower extremity predominant most commonly presents with delayed motor milestones and ankle contractures. Additional features at presentation include arthrogryposis and congenital dislocation of the hips. In all affected individuals, weakness and wasting is lower-limb predominant, and typically involves both proximal and distal muscle groups. There is no evidence of sensory nerve involvement. Upper motor neuron signs are a prominent feature in a subset of individuals, including one family with exclusively adult-onset upper motor neuron features, consistent with a diagnosis of hereditary spastic paraplegia. In all cohort members, lower motor neuron features were static or only slowly progressive, and the majority remained ambulant throughout life. Muscle MRI in six individuals showed a common pattern of muscle involvement with fat deposition in most thigh muscles, but sparing of the adductors and semitendinosus. Muscle pathology findings were highly variable and included pseudomyopathic features, neuropathic features, and minimal change. The six causative mutations, including one not previously reported, result in amino acid changes within all three coiled-coil domains of the BICD2 protein, and include a possible 'hot spot' mutation, p.Ser107Leu present in four families. We used the recently solved crystal structure of a highly conserved region of the Drosophila orthologue of BICD2 to further-explore how the p.Glu774Gly substitution inhibits the binding of BICD2 to Rab6. Overall, the features of BICD2 spinal muscular atrophy, lower extremity predominant are consistent with a pathological process that preferentially affects lumbar lower motor neurons, with or without additional upper motor neuron involvement. Defining the phenotypic features in this, the largest BICD2 disease cohort reported to date, will facilitate focused genetic testing and filtering of next generation sequencing-derived variants in cases with similar features.
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Proteínas Asociadas a Microtúbulos/genética , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patología , Adolescente , Adulto , Anciano , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Músculo Esquelético/patología , Mutación/genética , Linaje , Fenotipo , Unión Proteica , Columna Vertebral/patología , Adulto JovenRESUMEN
Mutations in the LARGE gene have been identified in congenital muscular dystrophy (CMD) patients with brain abnormalities. Both LARGE and its paralog, LARGE2 (also referred to as GYLTL1B) are bifunctional glycosyltransferases with xylosyltransferase (Xyl-T) and glucuronyltransferase (GlcA-T) activities, and are capable of forming polymers consisting of [-3Xyl-α1,3GlcAß1-] repeats. LARGE-dependent modification of α-dystroglycan (α-DG) with these polysaccharides is essential for the ability of α-DG to act as a receptor for ligands in the extracellular matrix. Here we report on the endogenous enzymatic activities of LARGE and LARGE2 in mice and humans, using a newly developed assay for GlcA-T activity. We show that normal mouse and human cultured cells have endogenous LARGE GlcA-T, and that this activity is absent in cells from the Large(myd) (Large-deficient) mouse model of muscular dystrophy, as well as in cells from CMD patients with mutations in the LARGE gene. We also demonstrate that GlcA-T activity is significant in the brain, heart, and skeletal muscle of wild-type and Large2(-/-) mice, but negligible in the corresponding tissues of the Large(myd) mice. Notably, GlcA-T activity is substantial, though reduced, in the kidneys of both the Large(myd) and Large2(-/-) mice, consistent with the observation of α-DG/laminin binding in these contexts. This study is the first to test LARGE activity in samples as small as cryosections and, moreover, provides the first direct evidence that not only LARGE, but also LARGE2, is vital to effective functional modification of α-DG in vivo.
Asunto(s)
Distroglicanos/metabolismo , Glicosiltransferasas/metabolismo , Laminina/metabolismo , Distrofias Musculares/enzimología , N-Acetilglucosaminiltransferasas/metabolismo , Animales , Sitios de Unión , Encéfalo/enzimología , Encéfalo/patología , Células Cultivadas , Niño , Modelos Animales de Enfermedad , Distroglicanos/genética , Pruebas de Enzimas , Femenino , Fibroblastos/enzimología , Fibroblastos/patología , Regulación de la Expresión Génica , Glicosiltransferasas/genética , Humanos , Riñón/enzimología , Riñón/patología , Laminina/genética , Ratones , Ratones Noqueados , Músculo Esquelético/enzimología , Músculo Esquelético/patología , Distrofias Musculares/genética , Distrofias Musculares/patología , Miocardio/enzimología , Miocardio/patología , N-Acetilglucosaminiltransferasas/genética , Especificidad de Órganos , Unión ProteicaRESUMEN
A mutation update on the nebulin gene (NEB) is necessary because of recent developments in analysis methodology, the identification of increasing numbers and novel types of variants, and a widening in the spectrum of clinical and histological phenotypes associated with this gigantic, 183 exons containing gene. Recessive pathogenic variants in NEB are the major cause of nemaline myopathy (NM), one of the most common congenital myopathies. Moreover, pathogenic NEB variants have been identified in core-rod myopathy and in distal myopathies. In this update, we present the disease-causing variants in NEB in 159 families, 143 families with NM, and 16 families with NM-related myopathies. Eighty-eight families are presented here for the first time. We summarize 86 previously published and 126 unpublished variants identified in NEB. Furthermore, we have analyzed the NEB variants deposited in the Exome Variant Server (http://evs.gs.washington.edu/EVS/), identifying that pathogenic variants are a minor fraction of all coding variants (â¼7%). This indicates that nebulin tolerates substantial changes in its amino acid sequence, providing an explanation as to why variants in such a large gene result in relatively rare disorders. Lastly, we discuss the difficulties of drawing reliable genotype-phenotype correlations in NEB-associated disease.
Asunto(s)
Proteínas Musculares/genética , Enfermedades Musculares/genética , Mutación , Empalme Alternativo , Animales , Cromosomas Humanos Par 2 , Bases de Datos Genéticas , Exones , Genotipo , Humanos , Modelos Animales , Enfermedades Musculares/clasificación , FenotipoRESUMEN
Mutations affecting skeletal muscle isoforms of the tropomyosin genes may cause nemaline myopathy, cap myopathy, core-rod myopathy, congenital fiber-type disproportion, distal arthrogryposes, and Escobar syndrome. We correlate the clinical picture of these diseases with novel (19) and previously reported (31) mutations of the TPM2 and TPM3 genes. Included are altogether 93 families: 53 with TPM2 mutations and 40 with TPM3 mutations. Thirty distinct pathogenic variants of TPM2 and 20 of TPM3 have been published or listed in the Leiden Open Variant Database (http://www.dmd.nl/). Most are heterozygous changes associated with autosomal-dominant disease. Patients with TPM2 mutations tended to present with milder symptoms than those with TPM3 mutations, DA being present only in the TPM2 group. Previous studies have shown that five of the mutations in TPM2 and one in TPM3 cause increased Ca(2+) sensitivity resulting in a hypercontractile molecular phenotype. Patients with hypercontractile phenotype more often had contractures of the limb joints (18/19) and jaw (6/19) than those with nonhypercontractile ones (2/22 and 1/22), whereas patients with the non-hypercontractile molecular phenotype more often (19/22) had axial contractures than the hypercontractile group (7/19). Our in silico predictions show that most mutations affect tropomyosin-actin association or tropomyosin head-to-tail binding.
Asunto(s)
Estudios de Asociación Genética , Enfermedades Musculares/congénito , Enfermedades Musculares/genética , Mutación , Tropomiosina/genética , Actinas/metabolismo , Adolescente , Adulto , Secuencia de Aminoácidos , Niño , Preescolar , Bases de Datos Genéticas , Femenino , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Enfermedades Musculares/diagnóstico , Fenotipo , Fosforilación , Unión Proteica , Alineación de Secuencia , Tropomiosina/química , Tropomiosina/metabolismo , Adulto JovenRESUMEN
Mutations in the TPM2 gene, which encodes ß-tropomyosin, are an established cause of several congenital skeletal myopathies and distal arthrogryposis. We have identified a TPM2 mutation, p.K7del, in five unrelated families with nemaline myopathy and a consistent distinctive clinical phenotype. Patients develop large joint contractures during childhood, followed by slowly progressive skeletal muscle weakness during adulthood. The TPM2 p.K7del mutation results in the loss of a highly conserved lysine residue near the N-terminus of ß-tropomyosin, which is predicted to disrupt head-to-tail polymerization of tropomyosin. Recombinant K7del-ß-tropomyosin incorporates poorly into sarcomeres in C2C12 myotubes and has a reduced affinity for actin. Two-dimensional gel electrophoresis of patient muscle and primary patient cultured myotubes showed that mutant protein is expressed but incorporates poorly into sarcomeres and likely accumulates in nemaline rods. In vitro studies using recombinant K7del-ß-tropomyosin and force measurements from single dissected patient myofibres showed increased myofilament calcium sensitivity. Together these data indicate that p.K7del is a common recurrent TPM2 mutation associated with mild nemaline myopathy. The p.K7del mutation likely disrupts head-to-tail polymerization of tropomyosin, which impairs incorporation into sarcomeres and also affects the equilibrium of the troponin/tropomyosin-dependent calcium switch of muscle. Joint contractures may stem from chronic muscle hypercontraction due to increased myofibrillar calcium sensitivity while declining strength in adulthood likely arises from other mechanisms, such as myofibre decompensation and fatty infiltration. These results suggest that patients may benefit from therapies that reduce skeletal muscle calcium sensitivity, and we highlight late muscle decompensation as an important cause of morbidity.
Asunto(s)
Calcio/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Mutación/fisiología , Miopatías Nemalínicas/genética , Miopatías Nemalínicas/metabolismo , Tropomiosina/genética , Adolescente , Adulto , Anciano , Secuencia de Aminoácidos , Animales , Línea Celular , Células Cultivadas , Pollos , Femenino , Estudios de Asociación Genética/métodos , Tamización de Portadores Genéticos , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Linaje , Ratas , Prevención Secundaria , PorcinosRESUMEN
BACKGROUND: Nemaline myopathy-the most common non-dystrophic congenital myopathy-is caused by mutations in thin filament genes, of which the nebulin gene is the most frequently affected one. The nebulin gene codes for the giant sarcomeric protein nebulin, which plays a crucial role in skeletal muscle contractile performance. Muscle weakness is a hallmark feature of nemaline myopathy patients with nebulin mutations, and is caused by changes in contractile protein function, including a lower calcium-sensitivity of force generation. To date no therapy exists to treat muscle weakness in nemaline myopathy. Here, we studied the ability of the novel fast skeletal muscle troponin activator, CK-2066260, to augment force generation at submaximal calcium levels in muscle cells from nemaline myopathy patients with nebulin mutations. METHODS: Contractile protein function was determined in permeabilised muscle cells isolated from frozen patient biopsies. The effect of 5 µM CK-2066260 on force production was assessed. RESULTS: Nebulin protein concentrations were severely reduced in muscle cells from these patients compared to controls, while myofibrillar ultrastructure was largely preserved. Both maximal active tension and the calcium-sensitivity of force generation were lower in patients compared to controls. Importantly, CK-2066260 greatly increased the calcium-sensitivity of force generation-without affecting the cooperativity of activation-in patients to levels that exceed those observed in untreated control muscle. CONCLUSIONS: Fast skeletal troponin activation is a therapeutic mechanism to augment contractile protein function in nemaline myopathy patients with nebulin mutations and with other neuromuscular diseases.
Asunto(s)
Imidazoles/farmacología , Proteínas Musculares/genética , Fuerza Muscular/efectos de los fármacos , Mutación/genética , Miopatías Nemalínicas/fisiopatología , Pirazinas/farmacología , Troponina/metabolismo , Adulto , Biopsia , Calcio/metabolismo , Preescolar , Humanos , Imidazoles/administración & dosificación , Lactante , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/patología , Miopatías Nemalínicas/tratamiento farmacológico , Miopatías Nemalínicas/genética , Pirazinas/administración & dosificación , Resultado del Tratamiento , Troponina/efectos de los fármacos , Adulto JovenRESUMEN
PURPOSE OF REVIEW: This article reviews recent advances in the understanding of nemaline myopathy, with a focus on the genetic basis of the disorder, histology, and pathogenesis. RECENT FINDINGS: Pathogenic mutations have been identified in eight genes and there is evidence of further genetic heterogeneity in nemaline myopathy. Clinical presentation, histological features on skeletal muscle biopsy, and pattern of changes on muscle MRI may guide prioritization of molecular genetic testing. It is anticipated that use of new technologies such as whole exome sequencing and comparative genomic hybridization will increase the number of genes associated with nemaline myopathy and the proportion of patients in whom the genetic basis of the disorder is identified. Single fiber studies and animal models continue to add to understanding of the pathogenesis of this disorder. Current management focuses on supportive treatment; however, encouraging advances are emerging for the future. SUMMARY: Recent advances in understanding of nemaline myopathy have important implications for clinical practice and for genetic diagnosis of patients with nemaline myopathy.
Asunto(s)
Músculo Esquelético/patología , Miopatías Nemalínicas/genética , Miopatías Nemalínicas/patología , Animales , Biopsia/métodos , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Humanos , Músculo Esquelético/fisiopatología , Mutación/genética , Miopatías Nemalínicas/inmunologíaRESUMEN
Neurofibromatosis type 1 (NF1) is a multisystem disease associated with a lifelong risk of debilitating and potentially life-limiting complications, however many adults with NF1 have no regular health surveillance. We interviewed and examined 17 young adults with NF1 between the ages of 25 and 33. Most had not been assessed for NF1-related complications within the previous 8 years, including patients with known serious vascular complications, for example, renal artery stenosis. Acute and/or chronic pain, particularly back and plexiform-related pain were common symptoms, and despite a significant impact on quality of life, was untreated in most instances. Symptom and examination-directed imaging revealed serious complications in 41% of the cohort. These included severe spinal cord compression (two cases), a highly SUV avid lesion suggestive of malignancy (one case), and a Juvenile Pilocytic Astrocytoma in a patient without any previous NF1-related complications. Few study participants had a good understanding of NF1, its associated risks and complications, and many had not sought appropriate medical advice as questions or problems arose. NF1-related cognitive deficits in some participants, and the lack of a clear source of expert medical advice for adults with NF1 likely contributed to poor health surveillance and management in this population. Overall, these findings suggest that many Australian adults with NF1 are at risk of serious and life-threatening medical complications, but are not accessing and receiving adequate health care. Access to multidisciplinary adult clinics that specialize in NF1 may address many of the unmet health needs of young adults with NF1.
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Conocimientos, Actitudes y Práctica en Salud , Accesibilidad a los Servicios de Salud , Neurofibromatosis 1/epidemiología , Adulto , Factores de Edad , Australia/epidemiología , Encéfalo/patología , Comorbilidad , Manejo de la Enfermedad , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Neurofibromatosis 1/complicaciones , Neurofibromatosis 1/diagnóstico , Tomografía de Emisión de Positrones , Médula Espinal/patologíaRESUMEN
Autosomal dominant congenital spinal muscular atrophy is characterized by predominantly lower limb weakness and wasting, and congenital or early-onset contractures of the hip, knee and ankle. Mutations in TRPV4, encoding a cation channel, have recently been identified in one large dominant congenital spinal muscular atrophy kindred, but the genetic basis of dominant congenital spinal muscular atrophy in many families remains unknown. It has been hypothesized that differences in the timing and site of anterior horn cell loss in the central nervous system account for the variations in clinical phenotype between different forms of spinal muscular atrophy, but there has been a lack of neuropathological data to support this concept in dominant congenital spinal muscular atrophy. We report clinical, electrophysiology, muscle magnetic resonance imaging and histopathology findings in a four generation family with typical dominant congenital spinal muscular atrophy features, without mutations in TRPV4, and in whom linkage to other known dominant neuropathy and spinal muscular atrophy genes has been excluded. The autopsy findings in the proband, who died at 14 months of age from an unrelated illness, provided a rare opportunity to study the neuropathological basis of dominant congenital spinal muscular atrophy. There was a reduction in anterior horn cell number in the lumbar and, to a lesser degree, the cervical spinal cord, and atrophy of the ventral nerve roots at these levels, in the absence of additional peripheral nerve pathology or abnormalities elsewhere along the neuraxis. Despite the young age of the child at the time of autopsy, there was no pathological evidence of ongoing loss or degeneration of anterior horn cells suggesting that anterior horn cell loss in dominant congenital spinal muscular atrophy occurs in early life, and is largely complete by the end of infancy. These findings confirm that dominant congenital spinal muscular atrophy is a true form of spinal muscular atrophy caused by a loss of anterior horn cells localized to lumbar and cervical regions early in development.
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Células del Asta Anterior/patología , Salud de la Familia , Ligamiento Genético , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patología , Canales Catiónicos TRPV/genética , Anciano , Autopsia , Niño , Preescolar , Femenino , Humanos , Lactante , Imagen por Resonancia Magnética , Masculino , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Atrofia Muscular Espinal/complicaciones , Miosinas/metabolismo , Fenotipo , Ultrasonografía DopplerRESUMEN
INTRODUCTION: Mutations in the gene that encodes filamin C, FLNC, represent a rare cause of a distinctive type of myofibrillar myopathy (MFM). METHODS: We investigated an Italian patient by means of muscle biopsy, muscle and brain imaging and molecular analysis of MFM genes. RESULTS: The patient harbored a novel 7256C>T, p.Thr2419Met mutation in exon 44 of FLNC. Clinical, pathological and muscle MRI findings were similar to the previously described filaminopathy cases. This patient had, in addition, cerebellar ataxia with atrophy of cerebellum and vermis evident on brain MRI scan. Extensive screening failed to establish a cause of cerebellar atrophy. CONCLUSIONS: We report an Italian filaminopathy patient, with a novel mutation in a highly conserved region. This case raises the possibility that the disease spectrum caused by FLNC may include cerebellar dysfunction.