title: A high proportion of DNA variants of BRCA1 and BRCA2 is associated with aberrant splicing in breast/ovarian cancer patients.
abstract: OBJECTIVE
  Most BRCA1/2 mutations are of unknown clinical relevance. An increasing amount of evidence indicates that there can be deleterious effects through the disruption of the splicing process. We have investigated the effect of aberrant splicing of BRCA1/2 on hereditary breast/ovarian cancer (HBOC).
  
  
  METHODS
  DNA variants were analyzed with splicing prediction programs to select putative splicing mutations. Splicing assays of 57 genetic variants were done by lymphocyte reverse transcription-PCR and/or hybrid minigenes in HeLa and nontumor breast epithelial cells.
  
  
  RESULTS
  Twenty-four BRCA1/2 variants of Spanish HBOC patients were bioinformatically preselected. Functional assays showed that 12 variants induced anomalous splicing patterns, 6 of which accounted for 58.5% of BRCA1 families. To further evaluate the defective splicing of BRCA1/2, we analyzed 31 Breast Cancer Information Core Database (BIC) and two artificial variants that were generated by mutagenesis. Sixteen variants induced different degrees of aberrant splicing. Altogether, anomalous splicing was caused by 28 BRCA1/2 variants of all types, indicating that any DNA change can disrupt pre-mRNA processing. We show that a wide range of regulatory elements can be involved, including the canonical and cryptic splice sites, the polypyrimidine tract, and splicing enhancers/silencers. Twenty mutations were predicted to truncate the BRCA proteins and/or to delete essential domains, thus supporting a role in HBOC.
  
  
  CONCLUSIONS
  An important fraction of DNA variants of BRCA1/2 presents splicing aberrations that may represent a relevant disease-causing mechanism in HBOC. The identification of splicing disruptions by functional assays is a valuable tool to discriminate between benign polymorphisms and pathogenic mutations.
authors.affiliation: Grupo de Genetica del Cancer, Instituto de Biologia y Genetica Molecular, Consejo Superior de Investigaciones Cientificas-Universidad de Valladolid, Valladolid, Spain.
authors.forename: David J
authors.initials: DJ
authors.lastname: Sanz
authors.forename: Alberto
authors.initials: A
authors.lastname: Acedo
authors.forename: Mar
authors.initials: M
authors.lastname: Infante
authors.forename: Mercedes
authors.initials: M
authors.lastname: Durán
authors.forename: Lucía
authors.initials: L
authors.lastname: Pérez-Cabornero
authors.forename: Eva
authors.initials: E
authors.lastname: Esteban-Cardeñosa
authors.forename: Enrique
authors.initials: E
authors.lastname: Lastra
authors.forename: Franco
authors.initials: F
authors.lastname: Pagani
authors.forename: Cristina
authors.initials: C
authors.lastname: Miner
authors.forename: Eladio A
authors.initials: EA
authors.lastname: Velasco
mesh_terms: D017668:Age of Onset; D017398:Alternative Splicing; D019313:BRCA1 Protein / Q000235:genetics*; D024682:BRCA2 Protein / Q000235:genetics*; D001943:Breast Neoplasms / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000235:genetics; D005260:Female; D020022:Genetic Predisposition to Disease; D014644:Genetic Variation / Q000235:genetics*; D006801:Humans; D008214:Lymphocytes; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010051:Ovarian Neoplasms / Q000235:genetics*; D010641:Phenotype; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics
chemical_list: D019313:BRCA1 Protein; D024682:BRCA2 Protein; D017931:DNA Primers; D004273:DNA, Neoplasm; D012333:RNA, Messenger; D012334:RNA, Neoplasm