title: Analysis of 30 putative BRCA1 splicing mutations in hereditary breast and ovarian cancer families identifies exonic splice site mutations that escape in silico prediction.
abstract: Screening for pathogenic mutations in breast and ovarian cancer genes such as BRCA1/2, CHEK2 and RAD51C is common practice for individuals from high-risk families. However, test results may be ambiguous due to the presence of unclassified variants (UCV) in the concurrent absence of clearly cancer-predisposing mutations. Especially the presence of intronic or exonic variants within these genes that possibly affect proper pre-mRNA processing poses a challenge as their functional implications are not immediately apparent. Therefore, it appears necessary to characterize potential splicing UCV and to develop appropriate classification tools. We investigated 30 distinct BRCA1 variants, both intronic and exonic, regarding their spliceogenic potential by commonly used in silico prediction algorithms (HSF, MaxEntScan) along with in vitro transcript analyses. A total of 25 variants were identified spliceogenic, either causing/enhancing exon skipping or activation of cryptic splice sites, or both. Except from a single intronic variant causing minor effects on BRCA1 pre-mRNA processing in our analyses, 23 out of 24 intronic variants were correctly predicted by MaxEntScan, while HSF was less accurate in this cohort. Among the 6 exonic variants analyzed, 4 severely impair correct pre-mRNA processing, while the remaining two have partial effects. In contrast to the intronic alterations investigated, only half of the spliceogenic exonic variants were correctly predicted by HSF and/or MaxEntScan. These data support the idea that exonic splicing mutations are commonly disease-causing and concurrently prone to escape in silico prediction, hence necessitating experimental in vitro splicing analysis.
authors.affiliation: Division of Molecular Gynaeco-Oncology, Department of Gynaecology and Obstetrics, University Hospital of Cologne, Cologne, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Wappenschmidt
authors.forename: Alexandra A
authors.initials: AA
authors.lastname: Becker
authors.forename: Jan
authors.initials: J
authors.lastname: Hauke
authors.forename: Ute
authors.initials: U
authors.lastname: Weber
authors.forename: Stefanie
authors.initials: S
authors.lastname: Engert
authors.forename: Juliane
authors.initials: J
authors.lastname: Köhler
authors.forename: Karin
authors.initials: K
authors.lastname: Kast
authors.forename: Norbert
authors.initials: N
authors.lastname: Arnold
authors.forename: Kerstin
authors.initials: K
authors.lastname: Rhiem
authors.forename: Eric
authors.initials: E
authors.lastname: Hahnen
authors.forename: Alfons
authors.initials: A
authors.lastname: Meindl
authors.forename: Rita K
authors.initials: RK
authors.lastname: Schmutzler
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D019313:BRCA1 Protein / Q000097:blood / Q000235:genetics*; D024682:BRCA2 Protein / Q000097:blood / Q000235:genetics; D001943:Breast Neoplasms / Q000097:blood / Q000235:genetics*; D003198:Computer Simulation; D005091:Exons / Q000235:genetics; D005260:Female; D006801:Humans; D007438:Introns / Q000235:genetics; D009154:Mutation; D010051:Ovarian Neoplasms / Q000097:blood / Q000235:genetics*; D012322:RNA Precursors / Q000096:biosynthesis / Q000235:genetics; D022821:RNA Splice Sites / Q000235:genetics
chemical_list: D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D024682:BRCA2 Protein; D012322:RNA Precursors; D022821:RNA Splice Sites