Characterisation of unclassified variants in the BRCA1/2 genes with a putative effect on splicing

Rita Dias Brandao*, Kees van Roozendaal, Demis Tserpelis, Encarna Gomez Garcia, Marinus J. Blok

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review


A subset of the unclassified variants (UVs) identified during genetic screening of BRCA1/2 genes may affect splicing. We assessed at RNA level the effect of four BRCA1 and ten BRCA2 UVs with a putative splice effect, as predicted in silico. The variants selected for this study were beyond the positions -1, -2 or +1, +2 from the exon, and were not previously described (n = 8) or their effect on splicing was not assessed previously (n = 6). Lymphocytes from UV carriers and healthy controls were cultured and treated with puromycin to prevent nonsense-mediated mRNA decay. The relative contribution of each allele to the various transcripts was assessed using combinations of allele-specific and transcript-specific primers. BRCA2 c.425G > T, c.7976+3_7976+4del and c.8754+3G > C give rise to aberrant transcripts BRCA2 Delta 4, BRCA2 Delta 17 and retention of 46nt of intron 21, respectively, and were considered pathogenic. BRCA1 c.4987-3C > G gives rise to BRCA1 Delta 17 that is likely pathogenic; however, residual expression of the full-length transcript from the variant allele could not be excluded. BRCA1 c.692C > T, c.693G > A and BRCA2 c.6935A > T, besides expressing the full-length transcript, increased expression of BRCA1 Delta 11 and BRCA2 Delta 12, respectively. As these are naturally occurring isoforms, also observed in controls, the clinical relevance is unclear. The seven remaining UVs did not affect splicing and three intronic variants were therefore classified as neutral. In conclusion, the RNA analysis results clarified the clinical relevance of 6 of the 14 studied UVs and thereby greatly improve the genetic counselling of high-risk breast/ovarian cancer patients carrying these classified variants.
Original languageEnglish
Pages (from-to)971-982
JournalBreast Cancer Research and Treatment
Issue number3
Publication statusPublished - Oct 2011


  • RNA splicing
  • BRCA1 gene
  • BRCA2 gene
  • RNA
  • Breast cancer
  • Exonic splice enhancer motifs

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