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1.
Hum Mol Genet ; 26(20): 3989-3994, 2017 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-29016857

RESUMEN

Arthrogryposis multiplex congenita (AMC) is a developmental condition characterized by multiple joint contractures resulting from reduced or absent fetal movements. Homozygosity mapping of disease loci combined with whole exome sequencing in a consanguineous family presenting with lethal AMC allowed the identification of a homozygous frameshift deletion in UNC50 gene (c.750_751del:p.Cys251Phefs*4) in the index case. To assess the effect of the mutation, an equivalent mutation in the Caenorhabditis elegans orthologous gene was created using CRISPR/Cas9. We demonstrated that unc-50(kr331) modification caused the loss of acetylcholine receptor (AChR) expression in C. elegans muscle. unc-50(kr331) animals were as resistant to the cholinergic agonist levamisole as unc-50 null mutants suggesting that AChRs were no longer expressed in this animal model. This was confirmed by using a knock-in strain in which a red fluorescent protein was inserted into the AChR locus: no signal was detected in unc-50(kr331) background, suggesting that UNC-50, a protein known to be involved in AChR trafficking, was no longer functional. These data indicate that biallelic mutation in the UNC50 gene underlies AMC through a probable loss of AChR expression at the neuromuscular junction which is essential for the cholinergic transmission during human muscle development.


Asunto(s)
Artrogriposis/genética , Artrogriposis/metabolismo , Mutación del Sistema de Lectura , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Receptores Colinérgicos/metabolismo , Alelos , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Unión Neuromuscular/genética , Unión Neuromuscular/metabolismo , Linaje , Transporte de Proteínas , Receptores Colinérgicos/genética , Mortinato/genética
2.
Nat Commun ; 14(1): 6669, 2023 10 20.
Artículo en Inglés | MEDLINE | ID: mdl-37863903

RESUMEN

Atypical teratoid rhabdoid tumors (ATRT) are divided into MYC, TYR and SHH subgroups, suggesting diverse lineages of origin. Here, we investigate the imaging of human ATRT at diagnosis and the precise anatomic origin of brain tumors in the Rosa26-CreERT2::Smarcb1flox/flox model. This cross-species analysis points to an extra-cerebral origin for MYC tumors. Additionally, we clearly distinguish SHH ATRT emerging from the cerebellar anterior lobe (CAL) from those emerging from the basal ganglia (BG) and intra-ventricular (IV) regions. Molecular characteristics point to the midbrain-hindbrain boundary as the origin of CAL SHH ATRT, and to the ganglionic eminence as the origin of BG/IV SHH ATRT. Single-cell RNA sequencing on SHH ATRT supports these hypotheses. Trajectory analyses suggest that SMARCB1 loss induces a de-differentiation process mediated by repressors of the neuronal program such as REST, ID and the NOTCH pathway.


Asunto(s)
Neoplasias Encefálicas , Tumor Rabdoide , Teratoma , Humanos , Tumor Rabdoide/genética , Multiómica , Proteína SMARCB1/genética , Factores de Transcripción/genética , Neoplasias Encefálicas/genética , Diagnóstico por Imagen , Teratoma/patología , Proteínas Hedgehog/genética
3.
Methods Mol Biol ; 1828: E1, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30488375

RESUMEN

The original version of this book was published with the following errors: "2'MOE" have been corrected into "2'MOEPS" in figure.6 - Chapter 35, multiple typo errors in page numbers: 532, 533, 534, 537, 542, 548 and 549. These errors has been updated.

4.
Methods Mol Biol ; 1828: 531-552, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30171566

RESUMEN

Numerous genetic disorders are caused by loss-of-function mutations that disrupt the open reading frame of the gene either by nonsense or by frameshift (insertion, deletion, indel, or splicing) mutations. Most of the time, the result is the absence of functional protein synthesis due to mRNA degradation by nonsense-mediated mRNA decay, or rapid degradation of a truncated protein. Antisense-based splicing modulation is a powerful tool that has the potential to treat genetic disorders by restoring the open reading frame through selective removal of the mutated exon, or by restoring correct splicing.We have developed this approach for a severe skin genetic disorder, recessive dystrophic epidermolysis bullosa, caused by mutations in the COL7A1 gene encoding type VII collagen. This gene is particularly suited for exon skipping approaches due to its unique genomic structure. It is composed of 118 exons, 83 of which are in frame. Moreover, these exons encode a single repetitive collagenous domain.Using this gene as an example, we describe general methods that demonstrate the feasibility and efficacy of the antisense-mediated exon skipping strategy to reframe transcripts.


Asunto(s)
Exones , Oligonucleótidos Antisentido/genética , Empalme del ARN , Sistemas de Lectura , Animales , Línea Celular , Clonación Molecular , Colágeno Tipo VII/química , Colágeno Tipo VII/genética , Epidermólisis Ampollosa Distrófica/genética , Fibroblastos/metabolismo , Vectores Genéticos/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Intrones , Ratones , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/química
5.
J Invest Dermatol ; 136(12): 2387-2395, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27498345

RESUMEN

Dystrophic epidermolysis bullosa is a group of orphan genetic skin diseases dominantly or recessively inherited, caused by mutations in COL7A1 encoding type VII collagen, which forms anchoring fibrils. Individuals with recessive dystrophic epidermolysis bullosa develop severe skin and mucosal blistering after mild trauma. The exon skipping strategy consists of modulating splicing of a pre-mRNA to induce skipping of a mutated exon. We have targeted COL7A1 exons 73 and 80, which carry recurrent mutations and whose excision preserves the open reading frame. We first showed the dispensability of these exons for type VII collagen function in vivo. We then showed that transfection of primary recessive dystrophic epidermolysis bullosa keratinocytes and fibroblasts carrying null mutations in exon 73 and/or 80, with 2'-O-methyl antisense oligoribonucleotides, led to efficient ex vivo skipping of these exons (50-95%) and resulted in a significant level (up to 36%) of type VII collagen re-expression. Finally, one or two subcutaneous injections of antisense oligoribonucleotides at doses ranging from 400 µg up to 1 mg restored type VII collagen expression and anchoring fibril formation in vivo in a xenograft model of recessive dystrophic epidermolysis bullosa skin equivalent. This work provides a proof of principle for the treatment of patients with recessive dystrophic epidermolysis bullosa by exon skipping using subcutaneous administration of antisense oligoribonucleotides.


Asunto(s)
Colágeno Tipo VII/genética , Colágeno/genética , Epidermólisis Ampollosa Distrófica/genética , Predisposición Genética a la Enfermedad , Animales , Western Blotting , Células Cultivadas , Complemento C7 , Modelos Animales de Enfermedad , Epidermólisis Ampollosa Distrófica/fisiopatología , Exones/genética , Regulación de la Expresión Génica , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones , Ratones Desnudos , Empalme del ARN/genética , Distribución Aleatoria , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Transfección
6.
Methods Mol Biol ; 867: 221-38, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22454065

RESUMEN

Numerous genetic disorders are caused by loss-of-function mutations that disrupt the open reading frame of the gene either by nonsense or by frameshift (insertion, deletion, indel, or splicing) mutations. Most of the time, the result is the absence of functional protein synthesis due to mRNA degradation by nonsense-mediated mRNA decay, or rapid degradation of a truncated protein. Antisense-based splicing modulation is a powerful tool that has the potential to treat genetic disorders by restoring the open reading frame through selective removal of the mutated exon, or by restoring correct splicing.We have developed this approach for a severe genetic skin disorder, recessive dystrophic epidermolysis bullosa, caused by mutations in the COL7A1 gene encoding type VII collagen. This gene is particularly suited for exon-skipping approaches due to its unique genomic structure. It is composed of 118 exons, 83 of which are in frame. Moreover, these exons encode a single repetitive collagenous domain.Using this gene as an example, we describe general methods that demonstrate the feasibility and efficacy of the antisense-mediated exon-skipping strategy to reframe transcripts.


Asunto(s)
Colágeno Tipo VII/genética , Epidermólisis Ampollosa Distrófica/genética , Exones , Mutación , Oligonucleótidos Antisentido/uso terapéutico , Animales , Línea Celular , Clonación Molecular/métodos , ADN Complementario/genética , Epidermólisis Ampollosa Distrófica/terapia , Fibroblastos/metabolismo , Humanos , Queratinocitos/metabolismo , Oligonucleótidos Antisentido/genética , Reacción en Cadena de la Polimerasa/métodos , ARN/genética , ARN/aislamiento & purificación , Análisis de Secuencia/métodos , Transfección
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