期刊
HUMAN MUTATION
卷 34, 期 10, 页码 1387-1395出版社
WILEY
DOI: 10.1002/humu.22379
关键词
limb-girdle muscular dystrophy; LGMD2A; pseudoexon; exon skipping; antisense oligonucleotide; AON; usnRNA
资金
- Spanish Ministry of Health [FIS PI06/1018, FIS PI09-116, PS09-00660]
- Spanish Ministry of Science and Innovation [BIO2006-13225, BIO 2009/09295, RNAREG CSD2009-00080, PTQ-09-02-02213]
- Ilundain Foundation
- Isabel Gemio Foundation
- Diputacion Foral de Guipuzcoa [DFG09/001]
- Association Francaise contre les Myopathies [12642]
- UTE project CIMA
- Department of Education, University and Research of the Basque Government
Limb-girdle muscular dystrophy type 2A (LGMD2A) is the most frequent autosomal recessive muscular dystrophy. It is caused by mutations in the calpain-3 (CAPN3) gene. The majority of the mutations described to date are located in the coding sequence of the gene. However, it is estimated that 25% of the mutations are present at exon-intron boundaries and modify the pre-mRNA splicing of the CAPN3 transcript. We have previously described the first deep intronic mutation in the CAPN3 gene: c.1782+1072G>C mutation. This mutation causes the pseudoexonization of an intronic sequence of the CAPN3 gene in the mature mRNA. In the present work, we show that the point mutation generates the inclusion of the pseudoexon in the mRNA using a minigene assay. In search of a treatment that restores normal splicing, splicing modulation was induced by RNA-based strategies, which included antisense oligonucleotides and modified small-nuclear RNAs. The best effect was observed with antisense sequences, which induced pseudoexon skipping in both HeLa cells cotransfected with mutant minigene and in fibroblasts from patients. Finally, transfection of antisense sequences and siRNA downregulation of serine/arginine-rich splicing factor 1 (SRSF1) indicate that binding of this factor to splicing enhancer sequences is involved in pseudoexon activation. (C) 2013 Wiley Periodicals, Inc.
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