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1.
Hum Mol Genet ; 32(21): 3029-3039, 2023 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-37070754

RESUMO

Recessive mutations in the DNAJB2 gene, encoding the J-domain co-chaperones DNAJB2a and DNAJB2b, have previously been reported as the genetic cause of progressive peripheral neuropathies, rarely involving pyramidal signs, parkinsonism and myopathy. We describe here a family with the first dominantly acting DNAJB2 mutation resulting in a late-onset neuromyopathy phenotype. The c.832 T > G p.(*278Glyext*83) mutation abolishes the stop codon of the DNAJB2a isoform resulting in a C-terminal extension of the protein, with no direct effect predicted on the DNAJB2b isoform of the protein. Analysis of the muscle biopsy showed reduction of both protein isoforms. In functional studies, the mutant protein mislocalized to the endoplasmic reticulum due to a transmembrane helix in the C-terminal extension. The mutant protein underwent rapid proteasomal degradation and also increased the turnover of co-expressed wild-type DNAJB2a, potentially explaining the reduced protein amount in the patient muscle tissue. In line with this dominant negative effect, both wild-type and mutant DNAJB2a were shown to form polydisperse oligomers.


Assuntos
Doenças Neuromusculares , Doenças do Sistema Nervoso Periférico , Humanos , Chaperonas Moleculares/genética , Mutação , Isoformas de Proteínas/genética , Proteínas Mutantes/genética , Proteínas de Choque Térmico HSP40/genética
2.
Acta Neuropathol ; 142(2): 375-393, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33974137

RESUMO

Using deep phenotyping and high-throughput sequencing, we have identified a novel type of distal myopathy caused by mutations in the Small muscle protein X-linked (SMPX) gene. Four different missense mutations were identified in ten patients from nine families in five different countries, suggesting that this disease could be prevalent in other populations as well. Haplotype analysis of patients with similar ancestry revealed two different founder mutations in Southern Europe and France, indicating that the prevalence in these populations may be higher. In our study all patients presented with highly similar clinical features: adult-onset, usually distal more than proximal limb muscle weakness, slowly progressing over decades with preserved walking. Lower limb muscle imaging showed a characteristic pattern of muscle involvement and fatty degeneration. Histopathological and electron microscopic analysis of patient muscle biopsies revealed myopathic findings with rimmed vacuoles and the presence of sarcoplasmic inclusions, some with amyloid-like characteristics. In silico predictions and subsequent cell culture studies showed that the missense mutations increase aggregation propensity of the SMPX protein. In cell culture studies, overexpressed SMPX localized to stress granules and slowed down their clearance.


Assuntos
Miopatias Distais/patologia , Proteínas Musculares/genética , Músculo Esquelético/patologia , Mutação de Sentido Incorreto/genética , Adulto , Miopatias Distais/genética , Humanos , Corpos de Inclusão/patologia , Pessoa de Meia-Idade , Debilidade Muscular/patologia , Linhagem , Grânulos de Estresse
3.
Genet Med ; 22(12): 2029-2040, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32778822

RESUMO

PURPOSE: High throughput sequencing analysis has facilitated the rapid analysis of the entire titin (TTN) coding sequence. This has resulted in the identification of a growing number of recessive titinopathy patients. The aim of this study was to (1) characterize the causative genetic variants and clinical features of the largest cohort of recessive titinopathy patients reported to date and (2) to evaluate genotype-phenotype correlations in this cohort. METHODS: We analyzed clinical and genetic data in a cohort of patients with biallelic pathogenic or likely pathogenic TTN variants. The cohort included both previously reported cases (100 patients from 81 unrelated families) and unreported cases (23 patients from 20 unrelated families). RESULTS: Overall, 132 causative variants were identified in cohort members. More than half of the cases had hypotonia at birth or muscle weakness and a delayed motor development within the first 12 months of life (congenital myopathy) with causative variants located along the entire gene. The remaining patients had a distal or proximal phenotype and a childhood or later (noncongenital) onset. All noncongenital cases had at least one pathogenic variant in one of the final three TTN exons (362-364). CONCLUSION: Our findings suggest a novel association between the location of nonsense variants and the clinical severity of the disease.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Hipotonia Muscular , Criança , Conectina/genética , Estudos de Associação Genética , Humanos , Mutação , Fenótipo
4.
Hum Mol Genet ; 24(13): 3718-31, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25877298

RESUMO

Mutations in the extreme C-terminus of titin (TTN), situated in the sarcomeric M-band, cause tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy 2J (LGMD2J). The mutations ultimately cause a loss of C-terminal titin, including a binding site for the protease calpain 3 (CAPN3), and lead to a secondary CAPN3 deficiency in LGMD2J muscle. CAPN3 has been previously shown to bind C-terminal titin and to use it as a substrate in vitro. Interestingly, mutations in CAPN3 underlie limb-girdle muscular dystrophy 2A (LGMD2A). Here, we aimed to clarify the relationship of CAPN3 and M-band titin in normal and pathological muscle. In vitro analyses identified several CAPN3 cleavage sites in C-terminal titin that were defined by protein sequencing. Furthermore, cleavage products were detected in normal muscle extracts by western blotting and in situ by immunofluorescence microscopy. The TMD/LGMD2J mutation FINmaj proved to alter this processing in vitro, while binding of CAPN3 to mutant titin was preserved. Unexpectedly, the pathological loss of M-band titin due to TMD/LGMD2J mutations was found to be independent of CAPN3, whereas the involvement of ubiquitous calpains is likely. We conclude that proteolytic processing of C-terminal titin by CAPN3 may have an important role in normal muscle, and that this process is disrupted in LGMD2A and in TMD/LGMD2J due to CAPN3 deficiency and to the loss of C-terminal titin, respectively.


Assuntos
Calpaína/metabolismo , Conectina/química , Conectina/metabolismo , Miopatias Distais/metabolismo , Proteínas Musculares/metabolismo , Distrofia Muscular do Cíngulo dos Membros/enzimologia , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Motivos de Aminoácidos , Animais , Calpaína/genética , Conectina/genética , Miopatias Distais/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Musculares/genética , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Distrofia Muscular do Cíngulo dos Membros/genética , Ligação Proteica , Proteínas Quinases/genética , Processamento de Proteína Pós-Traducional , Proteólise
5.
Ann Neurol ; 73(4): 500-9, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23401021

RESUMO

OBJECTIVE: A study was undertaken to identify the molecular cause of Welander distal myopathy (WDM), a classic autosomal dominant distal myopathy. METHODS: The genetic linkage was confirmed and defined by microsatellite and single nucleotide polymorphism haplotyping. The whole linked genomic region was sequenced with targeted high-throughput and Sanger sequencing, and coding transcripts were sequenced on the cDNA level. WDM muscle biopsies were studied by Western blotting and immunofluorescence microscopy. Splicing of TIA1 and its target genes in muscle and myoblast cultures was analyzed by reverse transcriptase polymerase chain reaction. Mutant TIA1 was characterized by cell biological studies on HeLa cells, including quantification of stress granules by high content analysis and fluorescence recovery after photobleaching (FRAP) experiments. RESULTS: The linked haplotype at 2p13 was narrowed down to <806 kb. Sequencing by multiple methods revealed only 1 segregating coding mutation, c.1362 G>A (p.E384K) in the RNA-binding protein TIA1, a key component of stress granules. Immunofluorescence microscopy of WDM biopsies showed a focal increase of TIA1 in atrophic and vacuolated fibers. In HeLa cells, mutant TIA1 constructs caused a mild increase in stress granule abundance compared to wild type, and showed slower average fluorescence recovery in FRAP. INTERPRETATION: WDM is caused by mutated TIA1 through a dominant pathomechanism probably involving altered stress granule dynamics.


Assuntos
Miopatias Distais/genética , Predisposição Genética para Doença/genética , Mutação/genética , Proteínas de Ligação a Poli(A)/genética , Células Cultivadas , Feminino , Recuperação de Fluorescência Após Fotodegradação , Ligação Genética , Genótipo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Masculino , Repetições de Microssatélites/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Fotodegradação , Polimorfismo de Nucleotídeo Único/genética , Proteínas/genética , Proteínas/metabolismo , Antígeno-1 Intracelular de Células T , Ubiquitina/metabolismo
6.
Neurol Genet ; 7(6): e632, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34722876

RESUMO

BACKGROUND AND OBJECTIVES: To determine the genetic cause of the disease in the previously reported family with adult-onset autosomal dominant distal myopathy (myopathy, distal, 3; MPD3). METHODS: Continued clinical evaluation including muscle MRI and muscle pathology. A linkage analysis with single nucleotide polymorphism arrays and genome sequencing were used to identify the genetic defect, which was verified by Sanger sequencing. RNA sequencing was used to investigate the transcriptional effects of the identified genetic defect. RESULTS: Small hand muscles (intrinsic, thenar, and hypothenar) were first involved with spread to the lower legs and later proximal muscles. Dystrophic changes with rimmed vacuoles and cytoplasmic inclusions were observed in muscle biopsies at advanced stage. A single nucleotide polymorphism array confirmed the previous microsatellite-based linkage to 8p22-q11 and 12q13-q22. Genome sequencing of three affected family members combined with structural variant calling revealed a small heterozygous deletion of 160 base pairs spanning the second last exon 10 of the heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) gene, which is in the linked region on chromosome 12. Segregation of the mutation with the disease was confirmed by Sanger sequencing. RNA sequencing showed that the mutant allele produces a shorter mutant mRNA transcript compared with the wild-type allele. Immunofluorescence studies on muscle biopsies revealed small p62 and larger TDP-43 inclusions. DISCUSSION: A small exon 10 deletion in the gene HNRNPA1 was identified as the cause of MPD3 in this family. The new HNRNPA1-related phenotype, upper limb presenting distal myopathy, was thus confirmed, and the family displays the complexities of gene identification.

7.
J Neurol Neurosurg Psychiatry ; 81(8): 834-9, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20682716

RESUMO

INTRODUCTION: Two families with autosomal dominant limb girdle muscular dystrophy (LGMD) have previously been linked to a locus on chromosome 7q36 10 years ago. The locus has been termed both LGMD1D and 1E, but because of lack of additional families to narrow down the linked region of interest, this disease has remained elusive. METHODS: A large Finnish family was clinically and genetically investigated. Laboratory parameters were determined, including creatine kinase (CK) value, neurographic and electromyography studies, cardiac and respiratory function examinations, muscle biopsies and muscle imaging by CT or MRI. RESULTS: Patients had onset of muscle weakness in the pelvic girdle between the fourth and sixth decades with an autosomal dominant pattern of inheritance. CK values were slightly elevated and electromyography was myopathic only. Muscle biopsies showed myopathic and/or dystrophic features with very minor rimmed vacuolation and protein aggregation findings. Molecular genetic analysis indicates linkage of the disease to the locus on chromosome 7q36 completely overlapping with the previously reported locus LGMD1D/E. DISCUSSION: Advancement towards the causative gene defect in the 7q36 linked disease needs new additional families to narrow the region of interest. The phenotype in the previously linked families has not been reported in full detail, which may be one reason for the shortage of additional families. A comprehensive clinical and morphological phenotype of chromosome 7q36 linked autosomal dominant LGMD with a restricted and updated 6.4 Mb sized haplotype is reported here.


Assuntos
Cromossomos Humanos Par 7/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Adulto , Idade de Início , Idoso , Creatina Quinase/sangue , Eletromiografia , Família , Feminino , Finlândia , Ligação Genética , Estudo de Associação Genômica Ampla , Humanos , Imuno-Histoquímica , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Debilidade Muscular/etiologia , Debilidade Muscular/fisiopatologia , Músculo Esquelético/patologia , Distrofia Muscular do Cíngulo dos Membros/patologia , Linhagem , Tomografia Computadorizada por Raios X , Adulto Jovem
8.
JAMA Neurol ; 75(5): 557-565, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29435569

RESUMO

Importance: Mutations in the titin gene (TTN) cause a wide spectrum of genetic diseases. The interpretation of the numerous rare variants identified in TTN is a difficult challenge given its large size. Objective: To identify genetic variants in titin in a cohort of patients with muscle disorders. Design, Setting, and Participants: In this case series, 9 patients with titinopathy and 4 other patients with possibly disease-causing variants in TTN were identified. Titin mutations were detected through targeted resequencing performed on DNA from 504 patients with muscular dystrophy, congenital myopathy, or other skeletal muscle disorders. Patients were enrolled from 10 clinical centers in April 2012 to December 2013. All of them had not received a diagnosis after undergoing an extensive investigation, including Sanger sequencing of candidate genes. The data analysis was performed between September 2013 and January 2017. Sequencing data were analyzed using an internal custom bioinformatics pipeline. Main Outcomes and Measures: The identification of novel mutations in the TTN gene and novel patients with titinopathy. We performed an evaluation of putative causative variants in the TTN gene, combining genetic, clinical, and imaging data with messenger RNA and/or protein studies. Results: Of the 9 novel patients with titinopathy, 5 (55.5%) were men and the mean (SD) age at onset was 25 (15.8) years (range, 0-46 years). Of the 4 other patients (3 men and 1 woman) with possibly disease-causing TTN variants, 2 (50%) had a congenital myopathy and 2 (50%) had a slowly progressive distal myopathy with onset in the second decade. Most of the identified mutations were previously unreported. However, all the variants, even the already described mutations, require careful clinical and molecular evaluation of probands and relatives. Heterozygous truncating variants or unique missense changes are not sufficient to make a diagnosis of titinopathy. Conclusions and Relevance: The interpretation of TTN variants often requires further analyses, including a comprehensive evaluation of the clinical phenotype (deep phenotyping) as well as messenger RNA and protein studies. We propose a specific workflow for the clinical interpretation of genetic findings in titin.


Assuntos
Conectina/genética , Conectina/metabolismo , Variação Genética/genética , Doenças Musculares/genética , Doenças Musculares/metabolismo , Adulto , Estudos de Coortes , Análise Mutacional de DNA , Europa (Continente) , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/diagnóstico por imagem , Doenças Musculares/diagnóstico por imagem , Adulto Jovem
9.
Nat Genet ; 44(4): 450-5, S1-2, 2012 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-22366786

RESUMO

Limb-girdle muscular dystrophy type 1D (LGMD1D) was linked to chromosome 7q36 over a decade ago, but its genetic cause has remained elusive. Here we studied nine LGMD-affected families from Finland, the United States and Italy and identified four dominant missense mutations leading to p.Phe93Leu or p.Phe89Ile changes in the ubiquitously expressed co-chaperone DNAJB6. Functional testing in vivo showed that the mutations have a dominant toxic effect mediated specifically by the cytoplasmic isoform of DNAJB6. In vitro studies demonstrated that the mutations increase the half-life of DNAJB6, extending this effect to the wild-type protein, and reduce its protective anti-aggregation effect. Further, we show that DNAJB6 interacts with members of the CASA complex, including the myofibrillar myopathy-causing protein BAG3. Our data identify the genetic cause of LGMD1D, suggest that its pathogenesis is mediated by defective chaperone function and highlight how mutations in a ubiquitously expressed gene can exert effects in a tissue-, isoform- and cellular compartment-specific manner.


Assuntos
Proteínas de Choque Térmico HSP40/genética , Chaperonas Moleculares/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Finlândia , Genótipo , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Itália , Chaperonas Moleculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular do Cíngulo dos Membros/patologia , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/metabolismo , Estados Unidos , Peixe-Zebra/embriologia , Peixe-Zebra/genética
10.
Neuromuscul Disord ; 21(5): 338-44, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21376592

RESUMO

The objective is to refine the clinical and morphological phenotype and the chromosomal region of interest, in the recently reported 7q36 linked autosomal dominant limb-girdle muscular dystrophy (LGMD1 D/E), by describing four new informative Finnish families. Examinations of the patients included serum CK, neurophysiological studies, cardiac and respiratory function examinations, muscle biopsies and muscle imaging. DNA samples were analyzed by genotyping. Patients in all families had very similar phenotypes with onset of muscle weakness in the pelvic girdle muscles between the fourth and sixth decade, later involvement of the shoulder girdle, and marked walking difficulties in the eighth decade. Muscle biopsies showed myopathic and/or dystrophic features. Genotyping confirmed linkage to the same locus at chromosome 7q36 in all families by one identically segregating haplotype. The linked region was narrowed down from <6.3 to <3.4Mb. Sequencing of the genes in the area is ongoing, aiming to identify the genetic defect.


Assuntos
Cromossomos Humanos Par 7/genética , Saúde da Família , Ligação Genética , Fenótipo , Adolescente , Adulto , Criança , Pré-Escolar , Mapeamento Cromossômico , Creatina Quinase/sangue , Eletrocardiografia , Eletromiografia , Feminino , Finlândia/etnologia , Genótipo , Humanos , Masculino , Biologia Molecular/métodos , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/patologia , Adulto Jovem
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