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Characterization of BRCA1 and BRCA2 splicing variants: a collaborative report by ENIGMA consortium members

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Abstract

Mutations in BRCA1 and BRCA2 predispose carriers to early onset breast and ovarian cancer. A common problem in clinical genetic testing is interpretation of variants with unknown clinical significance. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium was initiated to evaluate and implement strategies to characterize the clinical significance of BRCA1 and BRCA2 variants. As an initial project of the ENIGMA Splicing Working Group, we report splicing and multifactorial likelihood analysis of 25 BRCA1 and BRCA2 variants from seven different laboratories. Splicing analysis was performed by reverse transcriptase PCR or mini gene assay, and sequencing to identify aberrant transcripts. The findings were compared to bioinformatic predictions using four programs. The posterior probability of pathogenicity was estimated using multifactorial likelihood analysis, including co-occurrence with a deleterious mutation, segregation and/or report of family history. Abnormal splicing patterns expected to lead to a non-functional protein were observed for 7 variants (BRCA1 c.441+2T>A, c.4184_4185+2del, c.4357+1G>A, c.4987-2A>G, c.5074G>C, BRCA2 c.316+5G>A, and c.8754+3G>C). Combined interpretation of splicing and multifactorial analysis classified an initiation codon variant (BRCA2 c.3G>A) as likely pathogenic, uncertain clinical significance for 7 variants, and indicated low clinical significance or unlikely pathogenicity for another 10 variants. Bioinformatic tools predicted disruption of consensus donor or acceptor sites with high sensitivity, but cryptic site usage was predicted with low specificity, supporting the value of RNA-based assays. The findings also provide further evidence that clinical RNA-based assays should be extended from analysis of invariant dinucleotides to routinely include all variants located within the donor and acceptor consensus splicing sites. Importantly, this study demonstrates the added value of collaboration between laboratories, and across disciplines, to collate and interpret information from clinical testing laboratories to consolidate patient management.

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Acknowledgments

We thank the families participating in this research, the clinical personnel involved in aspects of recruitment and clinical data collection, and the clinical and research institutions supporting the combined research efforts. Melissa Brown is thanked for collecting the protocols used by the different laboratories. FPGMX: This work was partially supported by grants from the Xunta de Galicia (10PXIB 9101297PR) and FMM Foundation given to A.V. L.F is supported by Isabel Barreto program from Xunta de Galicia and Fondo Social Europeo. ICO: Contract grant sponsor: Spanish Health Research Fund; Carlos III Health Institute; Catalan Health Institute and Autonomous Government of Catalonia. Contract grant numbers: ISCIIIRETIC RD06/0020/1051, PI10/01422 and 2009SGR290. HVH: This work was partially funded by two grants (OD, 2008; SGE, 2008) from Fundación de Investigación Médica Mutua Madrileña. CBCS: We would like to thank the NEYE foundation and Familien Hede Nielsens fond for financial support. ABS is supported by an NHMRC Senior Research Fellowship, and her research on BRCA1/2 variants is funded by an NHMRC project grant. IOV: This study was supported by “Ministero della Salute” (grant numbers RFPS 2006-5-341353, ACC2/R6.9 and “Progetto Tumori Femminili”)

Conflicts of interest

All authors declared no conflicts of interest.

Author information

Authors and Affiliations

  1. Department of Clinical Genetics, Odense University Hospital, Soenderboulevard 29, 5000, Odense C, Denmark

    Mads Thomassen & Torben A. Kruse

  2. Fundación Pública Galega de Medicina Xenómica-SERGAS, Grupo de Medicina Xenómica-USC, CIBERER, IDIS, Santiago de Compostela, Spain

    Ana Blanco, Laura Fachal & Ana Vega

  3. Immunology and Molecular Oncology Unit, Istituto Oncologico Veneto IOV-IRCCS, Via Gattamelata 64, 35128, Padua, Italy

    Marco Montagna, Simona Agata & Silvia Tognazzo

  4. Genomic Medicine, Department of Clinical Biochemistry, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, 2100, Copenhagen, Denmark

    Thomas V. O. Hansen, Ane Y. Steffensen & Lars Jønson

  5. Department of Clinical Biochemistry, Section of Molecular Diagnostics, Aalborg University Hospital, Reberbansgade 15, 9000, Aalborg, Denmark

    Inge S. Pedersen

  6. Oncogenetics Laboratory, Vall d’Hebron Institute of Oncology (VHIO), University Hospital Vall d’Hebron, Barcelona, Spain

    Sara Gutiérrez-Enríquez & Orland Diez

  7. Genetic Diagnosis Unit, Hereditary Cancer Program, Institut Català d’Oncologia, Hospital Duran i Reynals-Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Llobregat, Barcelona, Spain

    Mireia Menéndez, Eva Tornero & Conxi Lázaro

  8. Grupo de Medicina Xenómica-USC, University of Santiago de Compostela, CIBERER, IDIS, Santiago de Compostela, Spain

    Marta Santamariña

  9. Genetics and Population Health Division, Queensland Institute of Medical Research, 300 Herston Rd, Herston, Brisbane, QLD, 4006, Australia

    Phillip Whiley & Amanda B. Spurdle

  10. Department of Clinical Genetics, Aarhus University Hospital, Skejby, Brendstrupgaardsvej 21C, 8200, Aarhus N, Denmark

    Uffe B. Jensen

  11. Medical Oncology Department, University Hospital Vall d’Hebron, Barcelona, Spain

    Judith Balmaña

  12. Department of Dermatology, University of Utah, Salt Lake City, UT, USA

    David E. Goldgar

Authors
  1. Mads Thomassen
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  2. Ana Blanco
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  3. Marco Montagna
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  4. Thomas V. O. Hansen
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  5. Inge S. Pedersen
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  6. Sara Gutiérrez-Enríquez
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  7. Mireia Menéndez
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  8. Laura Fachal
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  9. Marta Santamariña
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  10. Ane Y. Steffensen
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  11. Lars Jønson
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  12. Simona Agata
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  13. Phillip Whiley
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  14. Silvia Tognazzo
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  15. Eva Tornero
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  16. Uffe B. Jensen
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  17. Judith Balmaña
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  18. Torben A. Kruse
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  19. David E. Goldgar
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  20. Conxi Lázaro
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  21. Orland Diez
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  22. Amanda B. Spurdle
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  23. Ana Vega
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Corresponding author

Correspondence to Mads Thomassen.

Additional information

Amanda B. Spurdle and Ana Vega contributing equally to this study.

This study is conducted on behalf of the ENIGMA Consortium Splicing Working Group.

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Thomassen, M., Blanco, A., Montagna, M. et al. Characterization of BRCA1 and BRCA2 splicing variants: a collaborative report by ENIGMA consortium members. Breast Cancer Res Treat 132, 1009–1023 (2012). https://doi.org/10.1007/s10549-011-1674-0

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  • DOI: https://doi.org/10.1007/s10549-011-1674-0

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