RESUMO
OBJECTIVE: To characterize 2 unrelated patients with either asymmetric or unilateral muscle weakness at the clinical, genetic, histologic, and ultrastructural level. METHODS: The patients underwent thorough clinical examination, whole-body MRI, and exome sequencing. Muscle morphology was assessed by histology and electron microscopy. RESULTS: Both patients presented with early-onset hypotonia, delayed motor milestones, scoliosis, and reduced pulmonary function. Patient P1 manifested unilateral muscle weakness exclusively affecting the left side of the body; the asymmetry was less pronounced in patient P2. Muscle biopsies from both patients showed nemaline rods as the main histopathologic hallmark, and MRI revealed major fatty infiltrations in selective head, proximal, and distal muscles, correlating with the degree of muscle weakness asymmetry. Exome sequencing on blood DNA from both patients identified de novo ACTA1 missense mutations in a small number of reads, suggesting mutation mosaicism. Subsequent Sanger sequencing confirmed the presence of the mutations on muscle DNA, while they were barely detectable on blood DNA. CONCLUSIONS: De novo mutations can occur anytime during embryonic development and may result in a mosaic pattern of affected cells and tissues and lead to the development of an asymmetric clinical picture. The present study points out that mosaic mutations might not be easily detectable on leukocyte DNA and thereby escape routine genetic analysis, and possibly account for a significant number of molecularly undiagnosed patients.
Assuntos
Actinas/genética , Mosaicismo , Debilidade Muscular/diagnóstico , Debilidade Muscular/genética , Debilidade Muscular/fisiopatologia , Biópsia , Criança , Eletromiografia , Humanos , Imageamento por Ressonância Magnética , Mutação de Sentido Incorreto , Linhagem , Sequenciamento do ExomaRESUMO
PURPOSE: Malignant hyperthermia (MH) is a pharmacogenetic disorder arising from uncontrolled muscle calcium release due to an abnormality in the sarcoplasmic reticulum (SR) calcium-release mechanism triggered by halogenated inhalational anesthetics. However, the molecular mechanisms involved are still incomplete. METHODS: We aimed to identify transient receptor potential vanilloid 1 (TRPV1) variants within the entire coding sequence in patients who developed sensitivity to MH of unknown etiology. In vitro and in vivo functional studies were performed in heterologous expression system, trpv1-/- mice, and a murine model of human MH. RESULTS: We identified TRPV1 variants in two patients and their heterologous expression in muscles of trpv1-/- mice strongly enhanced calcium release from SR upon halogenated anesthetic stimulation, suggesting they could be responsible for the MH phenotype. We confirmed the in vivo significance by using mice with a knock-in mutation (Y524S) in the type I ryanodine receptor (Ryr1), a mutation analogous to the Y522S mutation associated with MH in humans. We showed that the TRPV1 antagonist capsazepine slows the heat-induced hypermetabolic response in this model. CONCLUSION: We propose that TRPV1 contributes to MH and could represent an actionable therapeutic target for prevention of the pathology and also be responsible for MH sensitivity when mutated.
Assuntos
Sinalização do Cálcio , Predisposição Genética para Doença , Hipertermia Maligna/genética , Canais de Cátion TRPV/genética , Anestésicos/farmacologia , Animais , Cálcio , Modelos Animais de Doenças , Feminino , Expressão Gênica/efeitos dos fármacos , Técnicas de Introdução de Genes , Células HEK293 , Homeostase , Humanos , Masculino , Hipertermia Maligna/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Canais de Cátion TRPV/metabolismoRESUMO
We report a novel and particularly unusual type of mutation, namely, large deletion in the RYR1 gene, in a Belgian family with myopathy: Patients were found to be compound heterozygous and presented a clinico-pathological phenotype characterized by late-onset and recessive myopathy with cores. We depict the clinical, electrophysiological, pathological and molecular genetic characteristics of family members. To date, large deletions in the RYR1 gene have been reported in only two cases. Both involved different mutations and, in sharp contrast to our cases, presented with a very early-onset, neonatal, and a very severe or lethal phenotype. Overview of reported clinico-pathologic phenotypes, also highlights the rarity of combined late-onset/recessive co-occurrence in this group of myopathies with cores. Finally, this report underlines the broadening spectrum in this group of myopathologic disorders and highlights the concept of 'RYR1-associated/related core myopathies'.
Assuntos
Doenças Musculares/genética , Doenças Musculares/patologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Bélgica , Família , Feminino , Deleção de Genes , Humanos , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Transtornos de Início Tardio/genética , Transtornos de Início Tardio/patologia , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , População Branca , Adulto JovemRESUMO
BACKGROUND: Hereditary angioedema (HAE) with normal C1 inhibitor (C1-INH) is a rare disorder. Mutations of the gene encoding coagulation factor XII have been identified in a subset of patients with this condition. Our aim was to investigate mutations in the F12 gene in patients with HAE with normal C1-INH from Brazil. METHODS: We studied 5 Brazilian families with index female patients who presented with recurrent angioedema with normal C1-INH and C4 levels. Genomic DNA was isolated from whole blood and PCR was performed. Mutations were detected by the sequencing of exon 9 of the F12 gene and allelic discrimination. RESULTS: The c.983C>A (p.Thr328Lys) mutation was identified in 16 subjects, from 4 of the 5 families studied, including 8 patients with symptoms of HAE with normal C1-INH (87.5% women) and 8 subjects asymptomatic for HAE (25% women). Mean age at onset of symptoms among the FXII-HAE patients was 13.8 years (range 6-25 years). Recurrent abdominal pain (100%) and subcutaneous angioedema (87.5%) were the most frequent clinical presentations. Two patients presented with associated laryngeal edema. In keeping with previous observations in patients with both C1-INH-HAE and HAE with normal C1-INH, all 7 women with FXII-HAE reported triggering or worsening of symptoms upon intake of estrogen-containing oral contraceptives and/or pregnancy. CONCLUSIONS: We report for the first time in Brazil a mutation in the F12 gene as a likely cause of HAE with normal C1-INH in patients with recurrent attacks of angioedema and/or abdominal pain. A higher frequency of abdominal pain attacks and onset of symptoms at a younger age were observed among Brazilian patients when compared to those from other parts of the world.
Assuntos
Angioedemas Hereditários/genética , Proteínas Inativadoras do Complemento 1/imunologia , Fator XII/genética , Mutação Puntual , Adolescente , Adulto , Idade de Início , Idoso , Alelos , Angioedemas Hereditários/sangue , Angioedemas Hereditários/imunologia , Brasil , Proteína Inibidora do Complemento C1 , DNA/química , DNA/genética , Fator XII/imunologia , Feminino , Humanos , Pessoa de Meia-Idade , Linhagem , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Adulto JovemRESUMO
BACKGROUND: Central Core Disease (CCD) is a congenital myopathy often resulting from a mutation in RYR1 gene. Mutations in RyR1 can increase or decrease channel activity, or induce a reduction in the amount of protein. The consequences of a single mutation are sometimes multiple and the analysis of the functional effects is complex. OBJECTIVE: The consequences of the p.Y4864H mutation identified in a CCD patient have been studied regarding both RyR1 function and amount. METHODS: The amount of RyR1 in human and mouse muscles was evaluated using qRT-PCR and quantitative Western blot, and calcium release was studied using calcium imaging on primary cultures. The results were compared between human and mouse. RESULTS: The p.Y4864H mutation induced an alteration of calcium release, and in addition was associated to a reduction in the amount of RyR1 in the patient's muscle. This suggests two possible pathophysiological mechanisms: the alteration of calcium release could result from a modification of the channel properties of RyR1 or from a RyR1 reduction. In order to discriminate between the two hypotheses, we used the heterozygous RyR1 knockout (RyR1+/-) mouse model showing a comparable RyR1 protein reduction. No reduction in calcium release was observed in primary muscle culture from these mice, and no muscle weakness was measured. CONCLUSIONS: Because the reduction in the amount of RyR1 protein has no functional consequences in the murine model, the muscle weakness observed in the patient is most likely the result of a modification of the calcium channel function of RyR1 due to the p.Y4864H mutation.
RESUMO
Distal limb contractures (DLC) represent a heterogeneous clinical and genetic condition. Overall, 20-25% of the DLC are caused by mutations in genes encoding the muscle contractile apparatus. Large interstitial deletions of the 3p have already been diagnosed by standard chromosomal analysis, but not associated with a specific phenotype. We report on four patients with syndromic DLC presenting with a de novo 3p14.1p13 microdeletion. The clinical features associated multiple contractures, feeding problems, developmental delay, and intellectual disability. Facial dysmorphism was constant with low-set posteriorly rotated ears and blepharophimosis. Review of previously reported cases with a precise mapping of the deletions, documented a 250 kb smallest region of overlap (SRO) necessary for DLC. This region contained one gene, EIF4E3, the first three exons of the FOXP1 gene, and an intronic enhancer of FOXP1 named hs1149. Sanger sequencing and locus quantification of hs1149, EIF4E3, and FOXP1 in a cohort of 11 French patients affected by DLC appeared normal. In conclusion, we delineate a new microdeletion syndrome involving the 3p14.1p13 locus and associated with DLC and severe developmental delay.
Assuntos
Artrogripose/epidemiologia , Aberrações Cromossômicas , Cromossomos Humanos Par 3/genética , Contratura/epidemiologia , Contratura/genética , Extremidades/patologia , Animais , Proteínas de Transporte/genética , Hibridização Genômica Comparativa , Contratura/patologia , Feminino , Fatores de Transcrição Forkhead/genética , França/epidemiologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Proteínas Repressoras/genética , SíndromeRESUMO
BACKGROUND: Autosomal dominant (AD) central core disease (CCD) is a congenital myopathy characterised by the presence of cores in the muscle fibres which correspond to broad areas of myofibrils disorganisation, Z-line streaming and lack of mitochondria. Heterozygous mutations in the RYR1 gene were observed in the large majority of AD-CCD families; however, this gene was excluded in some of AD-CCD families. OBJECTIVE: To enlarge the genetic spectrum of AD-CCD demonstrating mutations in an additional gene. PATIENTS AND METHODS: Four affected AD family members over three generations, three of whom were alive and participate in the study: the mother and two of three siblings. The symptoms began during the early childhood with mild delayed motor development. Later they developed mainly tibialis anterior weakness, hypertrophy of calves and significant weakness (amyotrophic) of quadriceps. No cardiac or ocular involvement was noted. RESULTS: The muscle biopsies sections showed a particular pattern: eccentric cores in type 1 fibres, associated with type 1 predominance. Most cores have abrupt borders. Electron microscopy confirmed the presence of both unstructured and structured cores. Exome sequencing analysis identified a novel heterozygous missense mutation p.Leu1723Pro in MYH7 segregating with the disease and affecting a conserved residue in the myosin tail domain. CONCLUSIONS: We describe MYH7 as an additional causative gene for AD-CCD. These findings have important implications for diagnosis and future investigations of AD-congenital myopathies with cores, without cardiomyopathy, but presenting a particular involvement of distal and quadriceps muscles.
Assuntos
Miosinas Cardíacas/genética , Predisposição Genética para Doença/genética , Mutação de Sentido Incorreto/genética , Miopatia da Parte Central/genética , Cadeias Pesadas de Miosina/genética , Adulto , Idoso , Feminino , Heterozigoto , Humanos , Masculino , Fibras Musculares de Contração Lenta/diagnóstico por imagem , Fibras Musculares de Contração Lenta/patologia , Fibras Musculares de Contração Lenta/ultraestrutura , Miopatia da Parte Central/diagnóstico por imagem , Miopatia da Parte Central/patologia , Linhagem , RadiografiaRESUMO
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.
Assuntos
Estudos de Associação Genética , Doenças Musculares/congênito , Doenças Musculares/genética , Mutação , Tropomiosina/genética , Actinas/metabolismo , Adolescente , Adulto , Sequência de Aminoácidos , Criança , Pré-Escolar , Bases de Dados Genéticas , Feminino , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Doenças Musculares/diagnóstico , Fenótipo , Fosforilação , Ligação Proteica , Alinhamento de Sequência , Tropomiosina/química , Tropomiosina/metabolismo , Adulto JovemRESUMO
Non-syndromic arthrogryposis multiplex congenita (AMC) is characterized by multiple congenital contractures resulting from reduced fetal mobility. Genetic mapping and whole exome sequencing (WES) were performed in 31 multiplex and/or consanguineous undiagnosed AMC families. Although this approach identified known AMC genes, we here report pathogenic mutations in two new genes. Homozygous frameshift mutations in CNTNAP1 were found in four unrelated families. Patients showed a marked reduction in motor nerve conduction velocity (<10 m/s) and transmission electron microscopy (TEM) of sciatic nerve in the index cases revealed severe abnormalities of both nodes of Ranvier width and myelinated axons. CNTNAP1 encodes CASPR, an essential component of node of Ranvier domains which underlies saltatory conduction of action potentials along the myelinated axons, an important process for neuronal function. A homozygous missense mutation in adenylate cyclase 6 gene (ADCY6) was found in another family characterized by a lack of myelin in the peripheral nervous system (PNS) as determined by TEM. Morpholino knockdown of the zebrafish orthologs led to severe and specific defects in peripheral myelin in spite of the presence of Schwann cells. ADCY6 encodes a protein that belongs to the adenylate cyclase family responsible for the synthesis of cAMP. Elevation of cAMP can mimic axonal contact in vitro and upregulates myelinating signals. Our data indicate an essential and so far unknown role of ADCY6 in PNS myelination likely through the cAMP pathway. Mutations of genes encoding proteins of Ranvier domains or involved in myelination of Schwann cells are responsible for novel and severe human axoglial diseases.
Assuntos
Adenilil Ciclases/genética , Artrogripose/genética , Artrogripose/patologia , Moléculas de Adesão Celular Neuronais/genética , Axônios/patologia , Axônios/ultraestrutura , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Mutação/genética , Bainha de Mielina/patologia , Sistema Nervoso Periférico/patologia , Sistema Nervoso Periférico/ultraestrutura , Gravidez , Células de Schwann/metabolismoRESUMO
Congenital myopathies are severe muscle disorders affecting adults as well as children in all populations. The diagnosis of congenital myopathies is constrained by strong clinical and genetic heterogeneity. Moreover, the majority of patients present with unspecific histological features, precluding purposive molecular diagnosis and demonstrating the need for an alternative and more efficient diagnostic approach. We used exome sequencing complemented by histological and ultrastructural analysis of muscle biopsies to identify the causative mutations in eight patients with clinically different skeletal muscle pathologies, ranging from a fatal neonatal myopathy to a mild and slowly progressive myopathy with adult onset. We identified RYR1 (ryanodine receptor) mutations in six patients and NEB (nebulin) mutations in two patients. We found novel missense and nonsense mutations, unraveled small insertions/deletions and confirmed their impact on splicing and mRNA/protein stability. Histological and ultrastructural findings of the muscle biopsies of the patients validated the exome sequencing results. We provide the evidence that an integrated strategy combining exome sequencing with clinical and histopathological investigations overcomes the limitations of the individual approaches to allow a fast and efficient diagnosis, accelerating the patient's access to a better healthcare and disease management. This is of particular interest for the diagnosis of congenital myopathies, which involve very large genes like RYR1 and NEB as well as genetic and phenotypic heterogeneity.
Assuntos
Doenças Musculares/congênito , Doenças Musculares/diagnóstico , Adulto , Sequência de Bases , Biópsia , Análise Mutacional de DNA , Exoma/genética , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Músculos/patologia , Músculos/ultraestrutura , Doenças Musculares/genética , Mutação/genética , Linhagem , Fenótipo , Análise de Sequência de DNARESUMO
Central core disease is a myopathy often arising from mutations in the type 1 ryanodine receptor (RYR1) gene, encoding the sarcoplasmic reticulum calcium release channel RyR1. No treatment is currently available for this disease. We studied the pathological situation of a severely affected child with two recessive mutations, which resulted in a massive reduction in the amount of RyR1. The paternal mutation induced the inclusion of a new in-frame pseudo-exon in RyR1 mRNA that resulted in the insertion of additional amino acids leading to the instability of the protein. We hypothesized that skipping this additional exon would be sufficient to restore RyR1 expression and to normalize calcium releases. We therefore developed U7-AON lentiviral vectors to force exon skipping on affected primary muscle cells. The efficiency of the exon skipping was evaluated at the mRNA level, at the protein level, and at the functional level using calcium imaging. In these affected cells, we observed a decreased inclusion of the pseudo-exon, an increased RyR1 protein expression, and a restoration of calcium releases of normal amplitude either upon direct RyR1 stimulation or in response to membrane depolarization. This study is the first demonstration of the potential of exon-skipping strategy for the therapy of central core disease, from the molecular to the functional level.
Assuntos
Éxons/genética , Regulação da Expressão Gênica/genética , Terapia Genética/métodos , Miopatia da Parte Central/terapia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Western Blotting , Cálcio/metabolismo , Primers do DNA/genética , Vetores Genéticos/genética , Células HEK293 , Humanos , Lentivirus , Microscopia de Fluorescência , Mutação/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
OBJECTIVE: To report a series of 11 patients on the severe end of the spectrum of ryanodine receptor 1 (RYR1) gene-related myopathy, in order to expand the clinical, histologic, and genetic heterogeneity associated with this group of patients. METHODS: Eleven patients evaluated in the neonatal period with severe neonatal-onset RYR1-associated myopathy confirmed by genetic testing were ascertained. Clinical features, molecular testing results, muscle imaging, and muscle histology are reviewed. RESULTS: Clinical features associated with the severe neonatal presentation of RYR1-associated myopathy included decreased fetal movement, hypotonia, poor feeding, respiratory involvement, arthrogryposis, and ophthalmoplegia in 3 patients, and femur fractures or hip dislocation at birth. Four patients had dominant RYR1 mutations, and 7 had recessive RYR1 mutations. One patient had a cleft palate, and another a congenital rigid spine phenotype-findings not previously described in the literature in patients with early-onset RYR1 mutations. Six patients who underwent muscle ultrasound showed relative sparing of the rectus femoris muscle. Histologically, all patients with dominant mutations had classic central cores on muscle biopsy. Patients with recessive mutations showed great histologic heterogeneity, including fibrosis, variation in fiber size, skewed fiber typing, very small fibers, and nuclear internalization with or without ill-defined cores. CONCLUSIONS: This series confirms and expands the clinical and histologic variability associated with severe congenital RYR1-associated myopathy. Both dominant and recessive mutations of the RYR1 gene can result in a severe neonatal-onset phenotype, but more clinical and histologic heterogeneity has been seen in those with recessive RYR1 gene mutations. Central cores are not obligatory histologic features in recessive RYR1 mutations. Sparing of the rectus femoris muscle on imaging should prompt evaluation for RYR1-associated myopathy in the appropriate clinical context.
Assuntos
Miopatia da Parte Central/genética , Miopatia da Parte Central/patologia , Miopatia da Parte Central/fisiopatologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Anormalidades Múltiplas/fisiopatologia , Pré-Escolar , Feminino , História Antiga , Humanos , Recém-Nascido , Masculino , MutaçãoRESUMO
MYH7 mutations are an established cause of Laing distal myopathy, myosin storage myopathy, and cardiomyopathy, as well as additional myopathy subtypes. We report a novel MYH7 mutation (p.Leu1597Arg) that arose de novo in two unrelated probands. Proband 1 has a myopathy characterized by distal weakness and prominent contractures and histopathology typical of multi-minicore disease. Proband 2 has an axial myopathy and histopathology consistent with congenital fiber type disproportion. These cases highlight the broad spectrum of clinical and histological patterns associated with MYH7 mutations, and provide further evidence that MYH7 is likely responsible for a greater proportion of congenital myopathies than currently appreciated.
Assuntos
Miosinas Cardíacas/genética , Doenças Musculares/patologia , Mutação/genética , Cadeias Pesadas de Miosina/genética , Adolescente , Adulto , Feminino , Predisposição Genética para Doença , Humanos , Doenças Musculares/diagnóstico , Doenças Musculares/genéticaRESUMO
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.
Assuntos
Cálcio/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Mutação/fisiologia , Miopatias da Nemalina/genética , Miopatias da Nemalina/metabolismo , Tropomiosina/genética , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas , Galinhas , Feminino , Estudos de Associação Genética/métodos , Triagem de Portadores Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Linhagem , Ratos , Prevenção Secundária , SuínosRESUMO
Distal arthrogryposis (DA) is a heterogeneous subgroup of arthrogryposis multiplex congenita (AMC), a large family of disorders characterized by multiple congenital joint limitations due to reduced fetal movements. DA is mainly characterized by contractures afflicting especially the distal extremities without overt muscular or neurological signs. Although a limited number of genes mostly implicated in the contractile apparatus have been identified in DA, most patients failed to show mutations in currently known genes. Using a pangenomic approach, we demonstrated linkage of DA to chromosome 2q37 in two consanguineous families and the endothelin-converting enzyme like 1 (ECEL1) gene present in this region was associated with DA. Screening of a panel of 20 families with non-specific DA identified seven homozygous or compound heterozygous mutations of ECEL1 in a total of six families. Mutations resulted mostly in the absence of protein. ECEL1 is a neuronal endopeptidase predominantly expressed in the central nervous system and brain structures during fetal life in mice and human. ECEL1 plays a major role in intramuscular axonal branching of motor neurons in skeletal muscle during embryogenesis. A detailed review of clinical findings of DA patients with ECEL1 mutations revealed a homogeneous and recognizable phenotype characterized by limited knee flexion, flexed third to fifth fingers and severe muscle atrophy predominant on lower limbs and tongue that suggested a common pathogenic mechanism. We described a new and homogenous phenotype of DA associated with ECEL1 that resulted in symptoms involving rather the peripheral than the central nervous system and suggesting a developmental dysfunction.
Assuntos
Artrogripose/genética , Desenvolvimento Embrionário/genética , Metaloendopeptidases/genética , Animais , Artrogripose/embriologia , Artrogripose/patologia , Sistema Nervoso Central/patologia , Mapeamento Cromossômico , Consanguinidade , Genes Recessivos , Ligação Genética , Homozigoto , Humanos , Camundongos , Neurônios Motores/patologia , Mutação , Linhagem , FenótipoRESUMO
Beta-tropomyosin 2 (TPM2) gene mutations are a rare cause of congenital myopathy with variable clinical and histological features. We describe muscle involvement using Whole-Body muscle Magnetic Resonance Imaging (WBMRI) in 8 individuals with genetically proven TPM2 mutations and different clinical and histological features (nemaline myopathy, 'cap disease', Bethlem-like phenotype, arthrogryposis). Most patients shared a recognizable MRI pattern with the involvement of masticatory and distal lower leg muscles. The lower leg showed constant soleus muscle involvement, and often also involvement of peroneus, tibialis anterior, and toe flexor muscles. Pelvic and shoulder girdles, and upper limbs muscles were quite spared. Two adult subjects (a patient and a paucisymptomatic parent) had a more diffuse involvement with striking fat infiltration of the rectus femoris muscle. Two children showed variant findings: one presented with masseter involvement associated with severe axial fat infiltration, the second had masticatory and distal leg muscle involvement (soleus and gastrocnemius muscles). Our study suggests that, independently of the clinical and histological presentation, most patients with TPM2 mutations show a predominant involvement of masticatory and distal leg muscles with the other regions relatively spared. More spread involvement may be observed. This cephalic-distal MRI pattern is not frequent in other known myopathies.
Assuntos
Músculo Esquelético/patologia , Doenças Musculares/diagnóstico , Doenças Musculares/genética , Mutação/genética , Tropomiosina/genética , Imagem Corporal Total , Adolescente , Adulto , Criança , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto JovemRESUMO
In humans, congenital myopathy-linked tropomyosin mutations lead to skeletal muscle dysfunction, but the cellular and molecular mechanisms underlying such dysfunction remain obscure. Recent studies have suggested a unifying mechanism by which tropomyosin mutations partially inhibit thin filament activation and prevent proper formation and cycling of myosin cross-bridges, inducing force deficits at the fiber and whole-muscle levels. Here, we aimed to verify this mechanism using single membrane-permeabilized fibers from patients with three tropomyosin mutations (TPM2-null, TPM3-R167H and TPM2-E181K) and measuring a broad range of parameters. Interestingly, we identified two divergent, mutation-specific pathophysiological mechanisms. (i) The TPM2-null and TPM3-R167H mutations both decreased cooperative thin filament activation in combination with reductions in the myosin cross-bridge number and force production. The TPM3-R167H mutation also induced a concomitant reduction in thin filament length. (ii) In contrast, the TPM2-E181K mutation increased thin filament activation, cross-bridge binding and force generation. In the former mechanism, modulating thin filament activation by administering troponin activators (CK-1909178 and EMD 57033) to single membrane-permeabilized fibers carrying tropomyosin mutations rescued the thin filament activation defect associated with the pathophysiology. Therefore, administration of troponin activators may constitute a promising therapeutic approach in the future.
Assuntos
Doenças Musculares/congênito , Mutação , Tropomiosina/genética , Citoesqueleto de Actina , Adulto , Idoso , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Quinolinas/farmacologia , Tiadiazinas/farmacologia , Tropomiosina/metabolismoRESUMO
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease so far related to mutations in the cardiac ryanodine receptor (RYR2) or the cardiac calsequestrin (CASQ2) genes. Because mutations in RYR2 or in CASQ2 are not retrieved in all CPVT cases, we searched for mutations in the physiological protein partners of RyR2 and CSQ2 in a large cohort of CPVT patients with no detected mutation in these two genes. Based on a candidate gene approach, we focused our investigations on triadin and junctin, two proteins that link RyR2 and CSQ2. Mutations in the triadin (TRDN) and in the junctin (ASPH) genes were searched in a cohort of 97 CPVT patients. We identified three mutations in triadin which cosegregated with the disease on a recessive mode of transmission in two families, but no mutation was found in junctin. Two TRDN mutations, a 4 bp deletion and a nonsense mutation, resulted in premature stop codons; the third mutation, a p.T59R missense mutation, was further studied. Expression of the p.T59R mutant in COS-7 cells resulted in intracellular retention and degradation of the mutant protein. This was confirmed after in vivo expression of the mutant triadin in triadin knock-out mice by viral transduction. In this work, we identified TRDN as a new gene responsible for an autosomal recessive form of CPVT. The mutations identified in the two families lead to the absence of the protein, thereby demonstrating the importance of triadin for the normal function of the cardiac calcium release complex in humans.
Assuntos
Arritmias Cardíacas/genética , Proteínas de Transporte/genética , Morte Súbita Cardíaca , Proteínas Musculares/genética , Taquicardia Ventricular/genética , Animais , Arritmias Cardíacas/metabolismo , Western Blotting , Células COS , Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , Saúde da Família , Feminino , Genes Recessivos , Predisposição Genética para Doença/genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Proteínas Musculares/metabolismo , Mutação , Miócitos Cardíacos/metabolismo , Linhagem , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/patologiaRESUMO
Large muscle genes are often sequenced using complementary DNA (cDNA) made from muscle messenger RNA (mRNA) to reduce the cost and workload associated with sequencing from genomic DNA. Two potential barriers are the availability of a frozen muscle biopsy, and difficulties in detecting nonsense mutations due to nonsense-mediated mRNA decay (NMD). We present patient examples showing that use of MyoD-transduced fibroblasts as a source of muscle-specific mRNA overcomes these potential difficulties in sequencing large muscle-related genes.