title: Multiple sequence variants of BRCA2 exon 7 alter splicing regulation.
abstract: BACKGROUND
  Exonic variants of unknown biological significance (VUS) identified in patients can affect mRNA splicing, either by changing 5' or 3' splice sites or by modifying splicing regulatory elements. Bioinformatic predictions of these elements are still inaccurate and only few such elements have been functionally mapped in BRCA2. We studied the effect on splicing of eight exon 7 VUS, selected from the French UMD-BRCA2 mutation database.
  
  
  METHODS
  We performed splicing minigene assays and analyses of patient RNA. We also developed a pyrosequencing-based quantitative assay, to measure, in patient RNA, the relative contribution of each allele to the production of exon 7-containing transcripts. Moreover, an exonic splicing enhancer (ESE)-dependent minigene assay was used to evaluate the splicing regulatory properties of wild-type and mutant segments.
  
  
  RESULTS
  Six out of the eight variants induced splicing defects. In the minigene assay, c.517G>T and c.631G>A altered the natural splice sites, c.572A>G created a new 5' splice site, and c.520C>T, c.587G>A and c.617C>G induced exon 7 skipping (66%, 25% and 46%, respectively). Pyrosequencing of patient RNA confirmed these levels of exon skipping for c.520C>T and c.617C>G. Results from the ESE-dependent minigene assay indicated that c.520C>T and c.587G>A disturb splicing regulatory elements.
  
  
  CONCLUSIONS
  BRCA2 exon 7 splicing is regulated by multiple exonic elements and is sensitive to disease-associated sequence variations. Measurements of allelic imbalance in patient-derived RNA and/or quantitative analyses using minigene assays provide valuable estimates of the extent of partial splicing defects. Assessment of pathogenicity of variants with partial splicing effect awaits additional evidence and especially the completion of segregation analyses.
authors.affiliation: Inserm UMR 1079, Rouen, France. pascaline.gaildrat@univ-rouen.fr
authors.forename: Pascaline
authors.initials: P
authors.lastname: Gaildrat
authors.forename: Sophie
authors.initials: S
authors.lastname: Krieger
authors.forename: Daniela
authors.initials: D
authors.lastname: Di Giacomo
authors.forename: Julie
authors.initials: J
authors.lastname: Abdat
authors.forename: Françoise
authors.initials: F
authors.lastname: Révillion
authors.forename: Sandrine
authors.initials: S
authors.lastname: Caputo
authors.forename: Dominique
authors.initials: D
authors.lastname: Vaur
authors.forename: Estelle
authors.initials: E
authors.lastname: Jamard
authors.forename: Elodie
authors.initials: E
authors.lastname: Bohers
authors.forename: Danielle
authors.initials: D
authors.lastname: Ledemeney
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Peyrat
authors.forename: Claude
authors.initials: C
authors.lastname: Houdayer
authors.forename: Etienne
authors.initials: E
authors.lastname: Rouleau
authors.forename: Rosette
authors.initials: R
authors.lastname: Lidereau
authors.forename: Thierry
authors.initials: T
authors.lastname: Frébourg
authors.forename: Agnès
authors.initials: A
authors.lastname: Hardouin
authors.forename: Mario
authors.initials: M
authors.lastname: Tosi
authors.forename: Alexandra
authors.initials: A
authors.lastname: Martins
mesh_terms: D000483:Alleles; D017398:Alternative Splicing; D001483:Base Sequence; D045744:Cell Line, Tumor; D019295:Computational Biology / Q000379:methods; D030561:Databases, Nucleic Acid; D004742:Enhancer Elements, Genetic; D005091:Exons; D005602:France; D015972:Gene Expression Regulation, Neoplastic; D023061:Gene Order; D020868:Gene Silencing; D024522:Genes, BRCA2; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D012313:RNA / Q000235:genetics / Q000378:metabolism; D022821:RNA Splice Sites
chemical_list: D022821:RNA Splice Sites; D012313:RNA