title: Small-scale high-throughput sequencing-based identification of new therapeutic tools in cystic fibrosis.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  Our data confirm the importance of analyzing noncoding regions to find unidentified mutations, which is essential to designing targeted therapeutic approaches.
authors.affiliation: INSERM U827, Laboratoire de Génétique de Maladies Rares, Montpellier, France.|Université Montpellier I, UFR de Médecine, Montpellier, France.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Bonini
authors.affiliation: INSERM U827, Laboratoire de Génétique de Maladies Rares, Montpellier, France.|Laboratoire de Génétique Moléculaire, CHU Montpellier, Montpellier, France.
authors.forename: Jessica
authors.initials: J
authors.lastname: Varilh
authors.affiliation: INSERM U827, Laboratoire de Génétique de Maladies Rares, Montpellier, France.|Laboratoire de Génétique Moléculaire, CHU Montpellier, Montpellier, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Raynal
authors.affiliation: INSERM U827, Laboratoire de Génétique de Maladies Rares, Montpellier, France.|Laboratoire de Génétique Moléculaire, CHU Montpellier, Montpellier, France.
authors.forename: Corinne
authors.initials: C
authors.lastname: Thèze
authors.affiliation: INSERM U827, Laboratoire de Génétique de Maladies Rares, Montpellier, France.|Laboratoire de Génétique Moléculaire, CHU Montpellier, Montpellier, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Beyne
authors.affiliation: Laboratoire de Génétique Moléculaire et d'Histocompatibilité, CHRU, Brest, France.
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Audrezet
authors.affiliation: Laboratoire de Génétique Moléculaire et d'Histocompatibilité, CHRU, Brest, France.
authors.forename: Claude
authors.initials: C
authors.lastname: Ferec
authors.affiliation: AP-HP, Service de Biochimie et Génétique Moléculaires, Groupe Hospitalier Cochin Broca Hôtel Dieu, Paris, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Bienvenu
authors.affiliation: AP-HP, Service de Biochimie et Génétique Moléculaires, Groupe Hospitalier Cochin Broca Hôtel Dieu, Paris, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Girodon
authors.affiliation: INSERM U827, Laboratoire de Génétique de Maladies Rares, Montpellier, France.|Université Montpellier I, UFR de Médecine, Montpellier, France.
authors.forename: Sylvie
authors.identifier: http://orcid.org/0000-0001-9369-7780
authors.initials: S
authors.lastname: Tuffery-Giraud
authors.affiliation: INSERM U827, Laboratoire de Génétique de Maladies Rares, Montpellier, France.|Laboratoire de Génétique Moléculaire, CHU Montpellier, Montpellier, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Des Georges
authors.affiliation: INSERM U827, Laboratoire de Génétique de Maladies Rares, Montpellier, France.|Université Montpellier I, UFR de Médecine, Montpellier, France.
authors.forename: Mireille
authors.initials: M
authors.lastname: Claustres
authors.affiliation: INSERM U827, Laboratoire de Génétique de Maladies Rares, Montpellier, France.|Université Montpellier I, UFR de Médecine, Montpellier, France.
authors.forename: Magali
authors.initials: M
authors.lastname: Taulan-Cadars
mesh_terms: D017398:Alternative Splicing; D001483:Base Sequence; D002460:Cell Line; D002874:Chromosome Mapping; D002897:Chromosomes, Human, Pair 7; D019295:Computational Biology / Q000379:methods; D003550:Cystic Fibrosis / Q000235:genetics* / Q000628:therapy*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics*; D005091:Exons; D015870:Gene Expression; D023061:Gene Order; D017930:Genes, Reporter; D056426:Genetic Loci; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007438:Introns; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D056510:Position-Specific Scoring Matrices; D016415:Sequence Alignment; D052416:Targeted Gene Repair
chemical_list: D019005:Cystic Fibrosis Transmembrane Conductance Regulator