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In Vitro Correction of a Pseudoexon‐Generating Deep Intronic Mutation in LGMD2A by Antisense Oligonucleotides and Modified Small Nuclear RNA s
Author(s) -
Blázquez Lorea,
Aiastui Ana,
Goicoechea Maria,
Martins de Araujo Mafalda,
Avril Aurélie,
Beley Cyriaque,
García Luis,
Valcárcel Juan,
Fortes Puri,
López de Munain Adolfo
Publication year - 2013
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.22379
Subject(s) - biology , minigene , rna splicing , microbiology and biotechnology , intron , exon , genetics , mutation , gene , rna
Limb‐girdle muscular dystrophy type 2A ( LGMD 2 A ) is the most frequent autosomal recessive muscular dystrophy. It is caused by mutations in the calpain‐3 ( CAPN 3 ) 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‐m RNA splicing of the CAPN 3 transcript. We have previously described the first deep intronic mutation in the CAPN 3 gene: c.1782+1072G>C mutation. This mutation causes the pseudoexonization of an intronic sequence of the CAPN 3 gene in the mature m RNA . In the present work, we show that the point mutation generates the inclusion of the pseudoexon in the m RNA 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 RNA s. The best effect was observed with antisense sequences, which induced pseudoexon skipping in both H e L a cells cotransfected with mutant minigene and in fibroblasts from patients. Finally, transfection of antisense sequences and si RNA downregulation of serine/arginine‐rich splicing factor 1 ( SRSF 1) indicate that binding of this factor to splicing enhancer sequences is involved in pseudoexon activation.