title: BRCA2 deep intronic mutation causing activation of a cryptic exon: opening toward a new preventive therapeutic strategy.
abstract: OBJECTIVE
  Diagnostic screening of the BRCA1/2 genes in breast cancer families is mostly done on genomic DNA. For families with a very strong family history and no mutation identified in the coding sequences or the exon-intron boundaries, BRCA1/2 transcripts' analysis is an efficient approach to uncover gene inversion and pre-mRNA splicing defaults missed by conventional DNA-based protocols.
  
  
  METHODS
  We analyzed RNA from patients of negative BRCA families by reverse transcriptase PCR and identified an insertion in one family that we characterized by sequencing and by using a minigene splicing assay. More than 2,000 additional BRCA1/2 negative families were subsequently screened for this mutation using a dedicated PCR approach.
  
  
  RESULTS
  Nine families were found to harbor a BRCA2 mutant transcript containing a 95-nucleotide cryptic exon between exons 12 and 13. This cryptic exon results from a new mutation located deep into intron 12, c.6937+594T > G, which reinforces the strength of a preexisting 5' splice site, turning it into a perfect consensus sequence. It is systematically included in transcripts produced by the mutant allele in cells from mutation carriers or produced by a mutant splicing reporter minigene. The inclusion of the cryptic exon was prevented when we cotransfected the minigene with antisense oligonucleotides complementary to the 3' or mutated 5' splice sites.
  
  
  CONCLUSIONS
  This first deep intronic BRCA mutation emphasizes the importance of analyzing RNA to provide comprehensive BRCA1/2 diagnostic tests and opens the possibility of using antisense therapy in the future as an alternative strategy for cancer prevention.
authors: Genetics of Breast Cancer Team, Cancer Research Centre of Lyon, CNRS UMR5286/Inserm U1052/Université Lyon 1, Centre Léon Bérard, Lyon, France.
authors: Olga
authors: O
authors: Anczuków
authors: Monique
authors: M
authors: Buisson
authors: Mélanie
authors: M
authors: Léoné
authors: Christine
authors: C
authors: Coutanson
authors: Christine
authors: C
authors: Lasset
authors: Alain
authors: A
authors: Calender
authors: Olga M
authors: OM
authors: Sinilnikova
authors: Sylvie
authors: S
authors: Mazoyer
mesh_terms: D017398:Alternative Splicing; D001943:Breast Neoplasms / Q000235:genetics / Q000628:therapy; D045744:Cell Line, Tumor; D005091:Exons; D005260:Female; D019398:Genes, BRCA1; D024522:Genes, BRCA2; D006801:Humans; D007438:Introns; D009154:Mutation; D010051:Ovarian Neoplasms / Q000235:genetics / Q000628:therapy; D010375:Pedigree