Small-scale high-throughput sequencing-based identification of new therapeutic tools in cystic fibrosis.
Genet Med
; 17(10): 796-806, 2015 Oct.
Article
em En
| MEDLINE
| ID: mdl-25569440
ABSTRACT
PURPOSE:
Although 97-99% of CFTR mutations have been identified, great efforts must be made to detect yet-unidentified mutations.METHODS:
We developed a small-scale next-generation sequencing approach for reliably and quickly scanning the entire gene, including noncoding regions, to identify new mutations. We applied this approach to 18 samples from patients suffering from cystic fibrosis (CF) in whom only one mutation had hitherto been identified.RESULTS:
Using an in-house bioinformatics pipeline, we could rapidly identify a second disease-causing CFTR mutation for 16 of 18 samples. Of them, c.1680-883A>G was found in three unrelated CF patients. Analysis of minigenes and patients' transcripts showed that this mutation results in aberrantly spliced transcripts because of the inclusion of a pseudoexon. It is located only three base pairs from the c.1680-886A>G mutation (1811+1.6kbA>G), the fourth most frequent mutation in southwestern Europe. We next tested the effect of antisense oligonucleotides targeting splice sites on these two mutations on pseudoexon skipping. Oligonucleotide transfection resulted in the restoration of the full-length, in-frame CFTR transcript, demonstrating the effect of antisense oligonucleotide-induced pseudoexon skipping in CF.CONCLUSION:
Our data confirm the importance of analyzing noncoding regions to find unidentified mutations, which is essential to designing targeted therapeutic approaches.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Regulador de Condutância Transmembrana em Fibrose Cística
/
Fibrose Cística
/
Sequenciamento de Nucleotídeos em Larga Escala
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
Limite:
Humans
/
Male
Idioma:
En
Revista:
Genet Med
Assunto da revista:
GENETICA MEDICA
Ano de publicação:
2015
Tipo de documento:
Article
País de afiliação:
França