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Breast Cancer Research and Treatment

, Volume 129, Issue 3, pp 971–982 | Cite as

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

  • Rita Dias Brandão
  • Kees van Roozendaal
  • Demis Tserpelis
  • Encarna Gómez García
  • Marinus J. Blok
Brief Report

Abstract

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 BRCA2Δ4, BRCA2Δ17 and retention of 46nt of intron 21, respectively, and were considered pathogenic. BRCA1 c.4987-3C>G gives rise to BRCA1Δ17 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 BRCA1Δ11 and BRCA2Δ12, 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.

Keywords

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

Abbreviations

ASPCR

Allele-specific PCR

ASS

Acceptor splice site

DSS

Donor splice site

ESE

Exonic splice enhancer

NMD

Nonsense-mediated mRNA decay

PTC

Premature termination codon

UVs

Unclassified variants

WT

Wild type

Notes

Acknowledgments

Rita D. Brandão was supported by Portuguese grants Fundação Calouste Gulbenkian (79117) and Fundação para a Ciência e Tecnologia (SFRH/BD/32386/2006).

Conflict of interests

None.

Supplementary material

10549_2011_1599_MOESM1_ESM.doc (44 kb)
Supplementary material 1 (DOC 44 kb)
10549_2011_1599_MOESM2_ESM.doc (58 kb)
Supplementary material 2 (DOC 58 kb)
10549_2011_1599_MOESM3_ESM.doc (58 kb)
Supplementary material 3 (DOC 58 kb)

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Copyright information

© Springer Science+Business Media, LLC. 2011

Authors and Affiliations

  • Rita Dias Brandão
    • 1
    • 2
  • Kees van Roozendaal
    • 1
  • Demis Tserpelis
    • 1
  • Encarna Gómez García
    • 1
    • 2
  • Marinus J. Blok
    • 1
  1. 1.Department of Clinical GeneticsUniversity Hospital of MaastrichtMaastrichtThe Netherlands
  2. 2.GROW—School for Oncology and Developmental BiologyUniversity Hospital of MaastrichtMaastrichtThe Netherlands

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