RESUMEN
X-linked myopathy with excessive autophagy (XMEA) is a genetic disease associated with weakness of the proximal muscles. It is caused by mutations in the VMA21 gene, coding for a chaperone that functions in the vacuolar ATPase (v-ATPase) assembly. Mutations associated with lower content of assembled v-ATPases lead to an increase in lysosomal pH, culminating in partial blockage of macroautophagy, with accumulation of vacuoles of undigested content. Here, we studied a 5-year-old boy affected by XMEA, caused by a small indel in the VMA21 gene. Detection of sarcoplasmic Lc3 (also known as MAP1LC3B)-positive vacuoles in his muscle biopsy confirmed an autophagy defect. To understand how autophagy is regulated in XMEA myogenesis, we used patient-derived muscle cells to evaluate autophagy during in vitro muscle differentiation. An increase in lysosomal pH was observed in the patient's cells, compatible with predicted functional defect of his mutation. Additionally, there was an increase in autophagic flux in XMEA myotubes. Interestingly, we observed that differentiation of XMEA myoblasts was altered, with increased myotube formation observed through a higher fusion index, which was not dependent on lysosomal acidification. Moreover, no variation in the expression of myogenic factors nor the presence of regenerating fibers in the patient's muscle were observed. Myoblast fusion is a tightly regulated process; therefore, the uncontrolled fusion of XMEA myoblasts might generate cells that are not as functional as normal muscle cells. Our data provide new evidence on the reason for predominant muscle involvement in the context of the XMEA phenotype.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Diferenciación Celular , Enfermedades Genéticas Ligadas al Cromosoma X/patología , Músculo Esquelético/patología , Enfermedades Musculares/patología , Autofagia , Secuencia de Bases , Biopsia , Brasil , Proliferación Celular , Preescolar , Femenino , Regulación de la Expresión Génica , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Humanos , Recién Nacido , Lisosomas/metabolismo , Masculino , Fusión de Membrana , Desarrollo de Músculos/genética , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Enfermedades Musculares/genética , Mioblastos/metabolismo , Mioblastos/patología , Linaje , ARN Mensajero/genética , ARN Mensajero/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo , Vacuolas/patología , Vacuolas/ultraestructuraRESUMEN
Repeats in coding and non-coding regions have increasingly been associated with many human genetic disorders, such as Richieri-Costa-Pereira syndrome (RCPS). RCPS, mostly characterized by midline cleft mandible, Robin sequence and limb defects, is an autosomal-recessive acrofacial dysostosis mainly reported in Brazilian patients. This disorder is caused by decreased levels of EIF4A3, mostly due to an increased number of repeats at the EIF4A3 5'UTR. EIF4A3 5'UTR alleles are CG-rich and vary in size and organization of three types of motifs. An exclusive allelic pattern was identified among affected individuals, in which the CGCA-motif is the most prevalent, herein referred as "disease-associated CGCA-20nt motif." The origin of the pathogenic alleles containing the disease-associated motif, as well as the functional effects of the 5'UTR motifs on EIF4A3 expression, to date, are entirely unknown. Here, we characterized 43 different EIF4A3 5'UTR alleles in a cohort of 380 unaffected individuals. We identified eight heterozygous unaffected individuals harboring the disease-associated CGCA-20nt motif and our haplotype analyses indicate that there are more than one haplotype associated with RCPS. The combined analysis of number, motif organization and haplotypic diversity, as well as the observation of two apparently distinct haplotypes associated with the disease-associated CGCA-20nt motif, suggest that the RCPS alleles might have arisen from independent unequal crossing-over events between ancient alleles at least twice. Moreover, we have shown that the number and sequence of motifs in the 5'UTR region is associated with EIF4A3 repression, which is not mediated by CpG methylation. In conclusion, this study has shown that the large number of repeats in EIF4A3 does not represent a dynamic mutation and RCPS can arise in any population harboring alleles with the CGCA-20nt motif. We also provided further evidence that EIF4A3 5'UTR is a regulatory region and the size and sequence type of the repeats at 5'UTR may contribute to clinical variability in RCPS.
RESUMEN
Exon skipping uses chemically modified antisense oligonucleotides to modulate RNA splicing. Therapeutically, exon skipping can bypass mutations and restore reading frame disruption by generating internally truncated, functional proteins to rescue the loss of native gene expression. Limb-girdle muscular dystrophy type 2C is caused by autosomal recessive mutations in the SGCG gene, which encodes the dystrophin-associated protein γ-sarcoglycan. The most common SGCG mutations disrupt the transcript reading frame abrogating γ-sarcoglycan protein expression. In order to treat most SGCG gene mutations, it is necessary to skip 4 exons in order to restore the SGCG transcript reading frame, creating an internally truncated protein referred to as Mini-Gamma. Using direct reprogramming of human cells with MyoD, myogenic cells were tested with 2 antisense oligonucleotide chemistries, 2'-O-methyl phosphorothioate oligonucleotides and vivo-phosphorodiamidate morpholino oligomers, to induce exon skipping. Treatment with vivo-phosphorodiamidate morpholino oligomers demonstrated efficient skipping of the targeted exons and corrected the mutant reading frame, resulting in the expression of a functional Mini-Gamma protein. Antisense-induced exon skipping of SGCG occurred in normal cells and those with multiple distinct SGCG mutations, including the most common 521ΔT mutation. These findings demonstrate a multiexon-skipping strategy applicable to the majority of limb-girdle muscular dystrophy 2C patients.
Asunto(s)
Morfolinos/genética , Sarcoglicanopatías/genética , Sarcoglicanopatías/terapia , Sarcoglicanos/genética , Células Cultivadas , Reprogramación Celular , Exones , Fibroblastos/metabolismo , Terapia Genética , Humanos , Microscopía Fluorescente , Mutación , Cultivo Primario de Células , Empalme del ARN , Sistemas de Lectura , Sarcoglicanopatías/metabolismo , Sarcoglicanos/metabolismo , Transducción Genética , Orina/citologíaRESUMEN
BACKGROUND: Central core disease is a congenital myopathy, characterized by presence of central core-like areas in muscle fibers. Patients have mild or moderate weakness, hypotonia and motor developmental delay. The disease is caused by mutations in the human ryanodine receptor gene (RYR1), which encodes a calcium-release channel. Since the RYR1 gene is huge, containing 106 exons, mutation screening has been limited to three 'hot spots', with particular attention to the C-terminal region. Recent next-generation sequencing methods are now identifying multiple numbers of variants in patients, in which interpretation and phenotype prevision is difficult. CASE PRESENTATION: In a Brazilian Caucasian family, clinical, histopathological and molecular analysis identified a new case of central core disease in a 48-year female. Sanger sequencing of the C-terminal region of the RYR1 gene identified two different missense mutations: c.14256 A > C polymorphism in exon 98 and c.14693 T > C in exon 102, which have already been described as pathogenic. Trans-position of the 2 mutations was confirmed because patient's daughter, mother and sister carried only the exon 98's mutation, a synonymous variant that was subsequently found in the frequency of 013-0,05 of alleles. Further next generation sequencing study of the whole RYR1 gene in the patient revealed the presence of additional 5 common silent polymorphisms in homozygosis and 8 polymorphisms in heterozygosis. CONCLUSIONS: Considering that patient's relatives showed no pathologic phenotype, and the phenotype presented by the patient is within the range observed in other central core disease patients with the same mutation, it was concluded that the c.14256 A > C polymorphism alone is not responsible for disease, and the associated additional silent polymorphisms are not acting as modifiers of the primary pathogenic mutation in the affected patient. The case described above illustrates the present reality where new methods for wide genome screening are becoming more accessible and able to identify a great variety of mutations and polymorphisms of unknown function in patients and their families.
Asunto(s)
Predisposición Genética a la Enfermedad , Mutación/genética , Miopatía del Núcleo Central/genética , Polimorfismo de Nucleótido Simple/genética , Canal Liberador de Calcio Receptor de Rianodina/genética , Exones/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Músculos/metabolismo , Músculos/patología , Linaje , FenotipoRESUMEN
BACKGROUND: Congenital muscular dystrophy (CMD) type 1A (MDC1A) is caused by recessive mutations in laminin-α2 (LAMA2) gene. Laminin-211, a heterotrimeric glycoprotein that contains the α2 chain, is crucial for muscle stability establishing a bond between the sarcolemma and the extracellular matrix. More than 215 mutations are listed in the locus specific database (LSDB) for LAMA2 gene (May 2014). OBJECTIVE: A limited number of large deletions/duplications have been reported in LAMA2. Our main objective was the identification of additional large rearrangements in LAMA2 found in CMD patients and a systematic review of cases in the literature and LSDB. METHODS: In four of the fifty-two patients studied over the last 10 years, only one heterozygous mutation was identified, after sequencing and screening for a frequent LAMA2 deletion. Initial screening of large mutations was performed by multiplex ligation-dependent probe application (MLPA). Further characterization implied several techniques: long-range PCR, cDNA and Southern-blot analysis. RESULTS: Three novel large deletions in LAMA2 and the first pathogenic large duplication were successfully identified, allowing a definitive molecular diagnosis, carrier screening and prenatal diagnosis. A total of fifteen deletions and two duplications previously reported were also reviewed. Two possible mutational "hotspots" for deletions may exist, the first encompassing exons 3 and 4 and second in the 3' region (exons 56 to 65) of LAMA2. CONCLUSIONS: Our findings show that this type of mutation is fairly frequent (18.4% of mutated alleles) and is underestimated in the literature. It is important to include the screening of large deletions/duplications as part of the genetic diagnosis strategy.
RESUMEN
We describe a large Brazilian consanguineous kindred with 3 clinically affected patients with a Thomsen myotonia phenotype. They carry a novel homozygous nonsense mutation in the CLCN1 gene (K248X). None of the 6 heterozygote carriers show any sign of myotonia on clinical evaluation or electromyography. These findings confirm the autosomal recessive inheritance of the novel mutation in this family, as well as the occurrence of phenotypic variability in the autosomal recessive forms of myotonia.
Asunto(s)
Canales de Cloruro/genética , Codón sin Sentido/genética , Miotonía Congénita/genética , Miotonía/genética , Adenosina Trifosfatasas/metabolismo , Adolescente , Brasil , Niño , Consanguinidad , Ecocardiografía , Exones/genética , Salud de la Familia , Femenino , Humanos , Masculino , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Miotonía/patología , Miotonía/fisiopatología , Miotonía Congénita/patología , Miotonía Congénita/fisiopatología , Conducción Nerviosa/genética , FenotipoAsunto(s)
Variación Genética/genética , Hipertrofia/genética , Músculo Esquelético/patología , Miostatina/genética , Miotonía Congénita/genética , Canales de Cloruro/genética , Predisposición Genética a la Enfermedad , Humanos , Hipertrofia/patología , Debilidad Muscular/genética , Mutación , Miotonía Congénita/patología , Linaje , PenetranciaRESUMEN
Facioscapulohumeral muscular dystrophy (FSHD) is a progressive muscle disorder that has been associated with a contraction of 3.3-kb repeats on chromosome 4q35. FSHD is characterized by a wide clinical inter- and intrafamilial variability, ranging from wheelchair-bound patients to asymptomatic carriers. Our study is unique in comparing the gene expression profiles from related affected, asymptomatic carrier, and control individuals. Our results suggest that the expression of genes on chromosome 4q is altered in affected and asymptomatic individuals. Remarkably, the changes seen in asymptomatic samples are largely in products of genes encoding several chemokines, whereas the changes seen in affected samples are largely in genes governing the synthesis of GPI-linked proteins and histone acetylation. Besides this, the affected patient and related asymptomatic carrier share the 4qA161 haplotype. Thus, these polymorphisms by themselves do not explain the pathogenicity of the contracted allele. Interestingly, our results also suggest that the miRNAs might mediate the regulatory network in FSHD. Together, our results support the previous evidence that FSHD may be caused by transcriptional dysregulation of multiple genes, in cis and in trans, and suggest some factors potentially important for FSHD pathogenesis. The study of the gene expression profiles from asymptomatic carriers and related affected patients is a unique approach to try to enhance our understanding of the missing link between the contraction in D4Z4 repeats and muscle disease, while minimizing the effects of differences resulting from genetic background.
Asunto(s)
Regulación de la Expresión Génica/genética , Heterocigoto , Distrofia Muscular Facioescapulohumeral/genética , Transcripción Genética/genética , Estudios de Casos y Controles , Cromosomas Humanos Par 4/genética , Perfilación de la Expresión Génica , Humanos , Polimorfismo Genético/genéticaRESUMEN
Central core disease (CCD) is an autosomal-dominant congenital myopathy, with muscle weakness and malignant hyperthermia (MH) susceptibility. We identified two of nine Brazilian CCD families carrying two mutations in the RYR1 gene. The heterozygous parents were clinically asymptomatic, and patients were mildly affected, differing from the few autosomal-recessive cases described previously. Recessive inheritance in CCD may therefore be more common than previously appreciated, which has important implications for genetic counseling and MH prevention in affected families.
Asunto(s)
Genes Recesivos , Miopatía del Núcleo Central/genética , Linaje , Canal Liberador de Calcio Receptor de Rianodina/genética , Heterocigoto , Humanos , Músculos/patología , Mutación , Miopatía del Núcleo Central/patologíaRESUMEN
Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is associated with contractions of D4Z4 repeat on 4q35. It displays a remarkable inter- and intra-familial clinical variability ranging from severe phenotype to asymptomatic carriers. Mosaicism for the contracted FSHD-sized allele is a recurrent finding, but only DNA from lymphocytes had been studied. It is currently not known if mosaicism is unequally distributed between different tissues and if muscle is relatively spared for the presence of the disease allele in mosaic asymptomatic carriers of a disease allele. Here we compare DNA extracted from peripheral blood lymphocytes (PBL), fibroblasts and muscle from a mosaic asymptomatic female carrier and mother of a FSHD patient. PFGE analysis showed a complex allelic segregation: two independent mitotic rearrangement episodes occurred, resulting in mosaicism for a contracted D4Z4 repeat on 4q35 in the mother and mosaicism for an expanded D4Z4 repeat on 10q26 in the affected daughter. The results show that the proportion of mosaicism in PBL and muscle were comparable, while in fibroblasts there was some variation in the mosaicism, which might be caused by culturing artefacts. This finding supports the hypothesis that a mitotic contraction of D4Z4 is an early embryonic event and indicates that the degree of mosaicism in PBL is representative for that of muscle.
Asunto(s)
Mosaicismo , Distrofia Muscular Facioescapulohumeral/patología , Secuencias Repetitivas de Ácidos Nucleicos/genética , Alelos , Cromosomas Humanos Par 4 , ADN/genética , ADN/metabolismo , Desoxirribonucleasa EcoRI/metabolismo , Desoxirribonucleasa HindIII/metabolismo , Electroforesis en Gel de Campo Pulsado , Salud de la Familia , Femenino , Fibroblastos/metabolismo , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Músculos/metabolismo , Distrofia Muscular Facioescapulohumeral/sangre , Distrofia Muscular Facioescapulohumeral/genética , LinajeRESUMEN
Congenital muscular dystrophies (CMD) are characterized by neonatal hypotonia and/or artrogriposis associated with a dystrophic muscle biopsy. The CMD1A form is caused by a deficiency of the alpha2 chain of laminin 2 (LAMA2 gene at 6q2), a protein present in the basal lamina of muscle fibers, in Schwann cells, epidermis, and in fetal trophoblastic tissue. This allows its study for prenatal diagnosis in the chorionic villous (CV), which was performed in a family with one deceased affected CMD1A child. Immunohistochemical analysis of the CV using antibodies against the C- and N-terminal domains of the alpha2-laminin protein showed a normal positive labeling for both antibodies in the "at-risk" CV, which did not differ from the normal control CV. The integrity of the CV membrane was confirmed through the analysis with antibodies against alpha1, beta1, and gamma1 laminins. DNA study using markers flanking the 6q2 region showed that the affected patient and the "at-risk" fetus did not share the same haplotype. Therefore, the fetus was considered normal through both methodologies, which was confirmed after the birth of a clinically normal male baby. As the LAMA2 gene is very large and the spectrum of mutations causing disease is wide, the analysis of the protein in muscle biopsy has been largely used for the diagnosis. Besides, the possibility to detect it in the chorionic villous, mainly using positive markers, also offers a powerful tool for prenatal diagnosis.
Asunto(s)
Muestra de la Vellosidad Coriónica , Laminina/análisis , Laminina/deficiencia , Músculo Esquelético/fisiología , Distrofias Musculares/diagnóstico , Anticuerpos Monoclonales , Femenino , Genotipo , Haplotipos , Humanos , Inmunohistoquímica , Lactante , Laminina/inmunología , Masculino , Repeticiones de Microsatélite/genética , Distrofias Musculares/congénito , Distrofias Musculares/genéticaRESUMEN
Realizamos análises citogenéticas e moleculares em 55 famílias com a mutaçäo da síndrome do cromossomo X frágil, loco FMR-1 (318 indivíduos e 15 amostras de vilosidade coriônica). Foram estudados 129 indivíduos do sexo masculino, 75 com retardo mental e 54 normais. Entre os 54 normais, 11 eram portadores da pré-mutaçäo e nenhum apresentou o sítio frágil. Foram detectados 73 portadores da mutaçäo completa e 18 por cento eram mosaicos, ou seja, apresentavam também a pré-mutaçäo. Todos expressaram o sítio frágil em pelo menos um dos sistemas de induçäo utilizados. O tamanho da expansäo de trinucleotídeos CGG (delta) e a freqüência de manifestaçäo do sítio frágil apresentaram correlaçäo positiva. Entre as 153 mulheres normais, 85 eram portadoras da pré-mutaçäo e 15 da mutaçäo completa. A freqüência de expressäo do fra(X) foi zero ou extremamente baixa entre as pré-mutadas e essa freqüência näo diferiu da expressäo das näo portadoras da mutaçäo. Portanto a análise citogenética é ineficaz na determinaçäo de indivíduos pré-mutados, homens ou mulheres. Entre as 51 mulheres com a mutaçäo completa, 70 por cento manifestaram algum grau de comprometimento mental. Encontramos também correlaçäo entre o delta e a freqüência de expressäo do fra(X) nessas mulheres. Contudo, a detecçäo citogenética das mulheres com mutaçäo completa foi menos eficiente do que no caso dos homens, pois 14 por cento de falsos negativos foram observados. A análise de segregaçäo confirmou que o risco de prole afetada aumenta com o delta, e o risco médio de prole afetada para todas as heterozigotas foi de 30 por cento. Näo houve indicaçäo de desvio de segregaçäo nas famílias estudadas, pois o número de indivíduos que herdaram a mutaçäo näo diferiu do número daqueles que herdaram os alelos normais. Näo foi detectada nenhuma mutaçäo nova nas 55 genealogias investigadas.
Asunto(s)
Humanos , Masculino , Femenino , Citogenética/métodos , Síndrome del Cromosoma X Frágil/genética , Brasil , Discapacidad Intelectual/complicaciones , Biología Molecular , Mutación/genéticaRESUMEN
A deficiência da proteína Distrofina (locvalizada na membrana das fibras musculares) foi descrita recentemente como provável causa da distrofia muscular tipo Duchenne (DMD). No presente trabalho, foi feita uma reaçäo imuno-histoquímica para a detecçäo da distrofina em cortes histológicos de congelaçäo, obtidos a partir de biópsias musculares. Foram estudadas 56 indivíduos: Em quatro biópsias de musculo normal, ocorreu uma marcaçäo nítida e homogênea em todo o sarcolema das fibras musculares. No musculo de 20 pacientes com DMD, näo se observou marcaçäo na membrana da maioria das fibras, embora em muitos casos tenha ocorrido uma marcaçäo parcial de algumas delas (entre 4% e 30%). Em 10 afetados pela distrofia tipo Becker (DMB), 8 apresentaram marcaçäo normal em todo o sarcolema das fibras e 2 mostraram uma reaçäo aparentemente mais fraca. Nos quatro afetados por distrofia tipo Cinturas (DMC), apesar das alteraçöes histopatológicos típicas de afecçäo muscular primária, a membrana de todas as fibras mostrou-se com forte padräo imuno-reativo. Foram estudadas também 11 heterozigotas certas quanto ao gene da DMD (8 com atividade de enzimas séricas elevadas e 3 com enzimas normais), 4 filhas de heterozigotas certas com enzimas muito elevadas e 3 heterozigotas para o gene da DMB (todas com enzimas normais). Em todas elas observou-se marcaçäo total e homogênea em todas as fibras musculares. estes resultados säo muito importantes para a padronizaçäo de uma metodologia que possibilite um diagnóstico diferencial entre alguns tipos de distrofia, bem como tentar aprimorar a taxa de detecçäo de portadores, para fins de Aconselhamento Genético
Asunto(s)
Diagnóstico Diferencial , Asesoramiento Genético , Histocitoquímica , Inmunoquímica , Distrofias Musculares , Deficiencia de Proteína , SarcolemaRESUMEN
As atividades séricas das enzimas creatino-cinase (CK) e piruvato-cinase (PK) foram determinadas em 146 mulheres beterozigotas certas quanto ao gene da distrofia muscular de Duchenne (DMD) e 187 mäes de casos isolados, pertencentes a dois grupos raciais: caucasóide e negroide. A atividade da CK foi medida em 206 mulheres caucasoides e 127 mulheres negroides e a da PK em 148 caucasoides e 92 negroides. Os resultados desta pesquisa mostraram um aumento da média de atividade enzimática no grupo de mulheres heterozigotes e mäes de casos isolados negroides em comparaçäo ao grupo de mulheres caucasoides. Entretanto, as diferenças foram estatísticamente significantes apenas para a CK sérica. Sugere-se portanto, que os resultados da atividade sérica da CK de mulheres em risco de serem portadoras do gene da DMD sejam comparados com os de mulheres normais de mesmo grupo racial