title: Impact of BRCA1 and BRCA2 variants on splicing: clues from an allelic imbalance study.
abstract: Nearly one-half of BRCA1 and BRCA2 sequence variations are variants of uncertain significance (VUSs) and are candidates for splice alterations for example, by disrupting/creating splice sites. As out-of-frame splicing defects lead to a marked reduction of the level of the mutant mRNA cleared through nonsense-mediated mRNA decay, a cDNA-based test was developed to show the resulting allelic imbalance (AI). Fifty-four VUSs identified in 53 hereditary breast/ovarian cancer (HBOC) patients without BRCA1/2 mutation were included in the study. Two frequent exonic single-nucleotide polymorphisms on both BRCA1 and BRCA2 were investigated by using a semiquantitative single-nucleotide primer extension approach and the cDNA allelic ratios obtained were corrected using genomic DNA ratios from the same sample. A total of five samples showed AI. Subsequent transcript analyses ruled out the implication of VUS on AI and identified a deletion encompassing BRCA2 exons 12 and 13 in one sample. No sequence abnormality was found in the remaining four samples, suggesting implication of cis- or trans-acting factors in allelic expression regulation that might be disease causative in these HBOC patients. Overall, this study showed that AI screening is a simple way to detect deleterious splicing defects and that a major role for VUSs and deep intronic mutations in splicing anomalies is unlikely in BRCA1/2 genes. Methods to analyze gene expression and identify regulatory elements in BRCA1/2 are now needed to complement standard approaches to mutational analysis.
authors.affiliation: Institut Curie Hôpital, Paris, France.
authors.forename: Virginie
authors.initials: V
authors.lastname: Caux-Moncoutier
authors.forename: Sabine
authors.initials: S
authors.lastname: Pagès-Berhouet
authors.forename: Dorothée
authors.initials: D
authors.lastname: Michaux
authors.forename: Bernard
authors.initials: B
authors.lastname: Asselain
authors.forename: Laurent
authors.initials: L
authors.lastname: Castéra
authors.forename: Antoine
authors.initials: A
authors.lastname: De Pauw
authors.forename: Bruno
authors.initials: B
authors.lastname: Buecher
authors.forename: Marion
authors.initials: M
authors.lastname: Gauthier-Villars
authors.forename: Dominique
authors.initials: D
authors.lastname: Stoppa-Lyonnet
authors.forename: Claude
authors.initials: C
authors.lastname: Houdayer
mesh_terms: D022981:Allelic Imbalance; D001943:Breast Neoplasms / Q000235:genetics*; D005260:Female; D019398:Genes, BRCA1; D024522:Genes, BRCA2; D006801:Humans; D009154:Mutation; D010051:Ovarian Neoplasms / Q000235:genetics*; D012326:RNA Splicing; D050436:Regulatory Elements, Transcriptional