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


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.


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



Allele-specific PCR


Acceptor splice site


Donor splice site


Exonic splice enhancer


Nonsense-mediated mRNA decay


Premature termination codon


Unclassified variants


Wild type



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


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)


  1. 1.
    The Breast Information Core Database. Accessed 23 Nov 2010
  2. 2.
    Cartegni L, Chew SL, Krainer AR (2002) Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet 3(4):285–298PubMedCrossRefGoogle Scholar
  3. 3.
    Shapiro MB, Senapathy P (1987) RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res 15(17):7155–7174PubMedCrossRefGoogle Scholar
  4. 4.
    Yeo G, Burge CB (2004) Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J Comput Biol 11(2–3):377–394. doi: 10.1089/1066527041410418 PubMedCrossRefGoogle Scholar
  5. 5.
    Reese M, Eeckman F, Kulp D, Haussler D (1997) Improved splice site detection in Genie. J Comput Biol 4(3):311–323PubMedCrossRefGoogle Scholar
  6. 6.
    Pertea M, Lin X, Salzberg SL (2001) GeneSplicer: a new computational method for splice site prediction. Nucleic Acids Res 29(5):1185–1190. doi: 10.1093/nar/29.5.1185 PubMedCrossRefGoogle Scholar
  7. 7.
    Cartegni L, Wang J, Zhu Z, Zhang MQ, Krainer AR (2003) ESEfinder: a Web resource to identify exonic splicing enhancers. Nucleic Acids Res 31(13):3568–3571. doi: 10.1093/nar/gkg616 PubMedCrossRefGoogle Scholar
  8. 8.
    Fairbrother WG, Yeo GW, Yeh R, Goldstein P, Mawson M, Sharp PA, Burge CB (2004) RESCUE-ESE identifies candidate exonic splicing enhancers in vertebrate exons. Nucleic Acids Res 32 (Web Server Issue):W187–190. doi: 10.1093/nar/gkh393
  9. 9.
    Gómez García E, Ambergen T, Blok MJ, Wijngaard A (2005) Patients with an unclassified genetic variant in the BRCA 1 or BRCA2 genes show different clinical features from those with a mutation. J Clin Oncol 23:2185–2190PubMedCrossRefGoogle Scholar
  10. 10.
    LOVD—Leiden Open Variation Database. Accessed 23 Nov 2010
  11. 11.
    JTd Dunnen, Antonarakis SE (2000) Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mutat 15(1):7–12. doi: 10.1002/(sici)1098-1004(200001)15:1<7:aid-humu4>;2-n CrossRefGoogle Scholar
  12. 12.
    Carter MS, Doskow J, Morris P, Li S, Nhim RP, Sandstedt S, Wilkinson MF (1995) A regulatory mechanism that detects premature nonsense codons in T-cell receptor transcripts in vivo is reversed by protein synthesis inhibitors in vitro. J Biol Chem 270(48):28995–29003. doi: 10.1074/jbc.270.48.28995 PubMedCrossRefGoogle Scholar
  13. 13.
    Yu C-E, Devlin B, Galloway N, Loomis E, Schellenberg GD (2004) ADLAPH: A molecular haplotyping method based on allele-discriminating long-range PCR. Genomics 84(3):600–612PubMedCrossRefGoogle Scholar
  14. 14.
    Thakur S, Zhang HB, Peng Y, Le H, Carroll B, Ward T, Yao J, Farid LM, Couch FJ, Wilson RB, Weber BL (1997) Localization of BRCA1 and a splice variant identifies the nuclear localization signal. Mol Cell Biol 17(1):444–452PubMedGoogle Scholar
  15. 15.
    Bieche I, Lidereau R (1999) Increased level of exon 12 alternatively spliced BRCA2 transcripts in tumor breast tissue compared with normal tissue. Cancer Res 59(11):2546–2550PubMedGoogle Scholar
  16. 16.
    Bonatti F, Pepe C, Tancredi M, Lombardi G, Aretini P, Sensi E, Falaschi E, Cipollini G, Bevilacqua G, Caligo MA (2006) RNA-based analysis of BRCA1 and BRCA2 gene alterations. Cancer Genet Cytogenet 170(2):93–101PubMedCrossRefGoogle Scholar
  17. 17.
    Hansen TVO, Bisgaard ML, Jønson L, Albrechtsen A, Filtenborg-Barnkob B, Eiberg H, Ejlertsen B, Nielsen FC (2008) Novel de novo BRCA2 mutation in a patient with a family history of breast cancer BMC Med Genet. doi: 10.1186/1471-2350-9-58
  18. 18.
    Vreeswijk M, Kraan JN, van der Klift HM, Vink GR, Cornelisse CJ, Wijnen JT, Bakker E, van Asperen CJ, Devilee P (2008) Intronic variants in BRCA1 and BRCA2 that affect RNA splicing can be reliably selected by splice-site prediction programs. Hum Mutat 30(1):107–114CrossRefGoogle Scholar
  19. 19.
    Hofmann W, Horn D, Huttner C, Classen E, Scherneck S (2003) The BRCA2 variant 8204G>A is a splicing mutation and results in an in frame deletion of the gene. J Med Genet 40(3):e23. doi: 10.1136/jmg.40.3.e23 PubMedCrossRefGoogle Scholar
  20. 20.
    Farrugia DJ, Agarwal MK, Pankratz VS, Deffenbaugh AM, Pruss D, Frye C, Wadum L, Johnson K, Mentlick J, Tavtigian SV, Goldgar DE, Couch FJ (2008) Functional assays for classification of BRCA2 variants of uncertain significance. Cancer Res 68(9):3523–3531. doi: 10.1158/0008-5472.can-07-1587 PubMedCrossRefGoogle Scholar
  21. 21.
    Wu K, Hinson SR, Ohashi A, Farrugia D, Wendt P, Tavtigian SV, Deffenbaugh A, Goldgar D, Couch FJ (2005) Functional evaluation and cancer risk assessment of BRCA2 unclassified variants. Cancer Res 65(2):417–426PubMedCrossRefGoogle Scholar
  22. 22.
    Bonnet C, Krieger S, Vezain M, Rousselin A, Tournier I, Martins A, Berthet P, Chevrier A, Dugast C, Layet V, Rossi A, Lidereau R, Frebourg T, Hardouin A, Tosi M (2008) Screening BRCA1 and BRCA2 unclassified variants for splicing mutations using reverse transcription PCR on patient RNA and an ex vivo assay based on a splicing reporter minigene. J Med Genet 45(7):438–446. doi: 10.1136/jmg.2007.056895 PubMedCrossRefGoogle Scholar
  23. 23.
    Hurst LD, Pál C (2001) Evidence for purifying selection acting on silent sites in BRCA1. Trends Genet 17(2):62–65PubMedCrossRefGoogle Scholar
  24. 24.
    Orban TI, Olah E (2001) Purifying selection on silent sites: a constraint from splicing regulation? Trends Genet 17(5):252–253PubMedCrossRefGoogle Scholar
  25. 25.
    Easton DF, Deffenbaugh AM, Pruss D, Frye C, Wenstrup RJ, Allen-Brady K, Tavtigian SV, Monteiro ANA, Iversen ES, Couch FJ, Goldgar DE (2007) A systematic genetic assessment of 1,433 sequence variants of unknown clinical significance in the BRCA1 and BRCA2 breast bancer predisposition genes. Am J Hum Genet 81(5):873–883PubMedCrossRefGoogle Scholar
  26. 26.
    Li L, Biswas K, Habib LA, Kuznetsov SG, Hamel N, Kirchhoff T, Wong N, Armel S, Chong G, Narod SA, Claes K, Offit K, Robson ME, Stauffer S, Sharan SK, Foulkes WD (2009) Functional redundancy of exon 12 of BRCA2 revealed by a comprehensive analysis of the c.6853A>G (p.I2285 V) variant. Hum Mutat 30(11):1–8Google Scholar
  27. 27.
    Walker LC, Whiley PJ, Couch FJ, Farrugia DJ, Healey S, Eccles DM, Lin F, Butler SA, Goff SA, Thompson BA, Lakhani SR, Da Silva LM, Tavtigian SV, Goldgar DE, Brown MA, Spurdle AB (2010) Detection of splicing aberrations caused by BRCA1 and BRCA2 sequence variants encoding missense substitutions: implications for prediction of pathogenicity. Hum Mutat 31(6):E1484–E1505. doi: 10.1002/humu.21267 PubMedCrossRefGoogle Scholar
  28. 28.
    Grantham R (1974) Amino acid difference dormula to help explain protein evolution. Science 185(4154):862–864. doi: 10.1126/science.185.4154.862 PubMedCrossRefGoogle Scholar
  29. 29.
    Lovelock P, Spurdle A, Mok M, Farrugia D, Lakhani S, Healey S, Arnold S, Buchanan D, kConFab Investigators, Couch F, Henderson B, Goldgar D, Tavtigian S, Chenevix-Trench G, Brown M (2007) Identification of BRCA1 missense substitutions that confer partial functional activity: potential moderate risk variants? Breast Cancer Res 9(6):R82PubMedCrossRefGoogle Scholar
  30. 30.
    Kote-Jarai Z, Matthews L, Osorio A, Shanley S, Giddings I, Moreews F, Locke I, Evans DG, Eccles D, Carrier Clinic Collaborators, Williams RD, Girolami M, Campbell C, Eeles R (2006) Accurate prediction of BRCA1 and BRCA2 heterozygous genotype using expression profiling after induced DNA damage. Clin Cancer Res 12(13):3896–3901. doi: 10.1158/1078-0432.ccr-05-2805 PubMedCrossRefGoogle Scholar
  31. 31.
    Brogna S, Wen J (2009) Nonsense-mediated mRNA decay (NMD) mechanisms. Nat Struct Mol Biol 16(2):107–113PubMedCrossRefGoogle Scholar
  32. 32.
    Nicholson P, Yepiskoposyan H, Metze S, Orozco RZ, Kleinschmidt N, Mühlemann O (2010) Nonsense-mediated mRNA decay in human cells: mechanistic insights, functions beyond quality control and the double-life of NMD factors. Cell Mol Life Sci 67(5):677–700PubMedCrossRefGoogle Scholar
  33. 33.
    Perrin-Vidoz L, Sinilnikova OM, Stoppa-Lyonnet D, Lenoir GM, Mazoyer S (2002) The nonsense-mediated mRNA decay pathway triggers degradation of most BRCA1 mRNAs bearing premature termination codons. Hum Mol Genet 11(23):2805–2814. doi: 10.1093/hmg/11.23.2805 PubMedCrossRefGoogle Scholar
  34. 34.
    Anczuków O, Ware MD, Buisson M, Zetoune AB, Stoppa-Lyonnet D, Sinilnikova OM, Mazoyer S (2008) Does the nonsense-mediated mRNA decay mechanism prevent the synthesis of truncated BRCA1, CHK2, and p53 proteins? Hum Mutat 29(1):65–73. doi: 10.1002/humu.20590 PubMedCrossRefGoogle Scholar
  35. 35.
    Ware MD, DeSilva D, Sinilnikova OM, Stoppa-Lyonnet D, Tavtigian SV, Mazoyer S (2006) Does nonsense-mediated mRNA decay explain the ovarian cancer cluster region of the BRCA2 gene? Oncogene 25(2):323–328PubMedGoogle Scholar
  36. 36.
    Orban TI, Olah E (2003) Emerging roles of BRCA1 alternative splicing. Mol Pathol 56(4):191–197. doi: 10.1136/mp.56.4.191 PubMedCrossRefGoogle Scholar
  37. 37.
    Speevak MD, Young SS, Feilotter H, Ainsworth P (2003) Alternatively spliced, truncated human BRCA2 isoforms contain a novel coding exon. Eur J Hum Genet 11(12):951–954PubMedCrossRefGoogle Scholar
  38. 38.
    Tesoriero AA, Wong EM, Jenkins MA, Hopper JL, Brown MA, Chenevix-Trench G, Spurdle AB, Southey MC, kConFab (2005) Molecular characterization and cancer risk associated with BRCA1 and BRCA2 splice site variants identified in multiple-case breast cancer families. Hum Mutat 26(5):495PubMedCrossRefGoogle Scholar
  39. 39.
    Diez O, Gutierrez-Enriquez S, Ramon y Cajal T, Alonso C, Balmana J, Llort G (2007) Caution should be used when interpreting alterations affecting the exon 3 of the BRCA2 gene in breast/ovarian cancer families. J Clin Oncol 25(31):5035–5036. doi: 10.1200/jco.2007.13.4346 PubMedCrossRefGoogle Scholar
  40. 40.
    Ozsolak F, Milos PM (2011) RNA sequencing: advances, challenges and opportunities. Nat Rev Genet 12(2):87–98PubMedCrossRefGoogle Scholar
  41. 41.
    Wu Y, Zhang Y, Zhang J (2005) Distribution of exonic splicing enhancer elements in human genes. Genomics 86(3):329–336PubMedCrossRefGoogle Scholar
  42. 42.
    Anczuków O, Buisson M, Salles M-J, Triboulet S, Longy M, Lidereau R, Sinilnikova OM, Mazoyer S (2008) Unclassified variants identified in BRCA1 exon 11: consequences on splicing. Genes Chromosomes Cancer 47(5):418–426. doi: 10.1002/gcc.20546 PubMedCrossRefGoogle Scholar
  43. 43.
    Whiley P, Pettigrew C, Brewster B, Walker L, kConFab Investigators, Spurdle A, Brown M (2010) Effect of BRCA2 sequence variants predicted to disrupt exonic splice enhancers on BRCA2 transcripts. BMC Med Genet 11(1):80. doi: 10.1186/1471-2350-11-80 PubMedCrossRefGoogle Scholar
  44. 44.
    Gaildrat P, Krieger S, Théry J-C, Killian A, Rousselin A, Berthet P, Frébourg T, Hardouin A, Martins A, Tosi M (2010) The BRCA1 c.5434C>G (p.Pro1812Ala) variant induces a deleterious exon 23 skipping by affecting exonic splicing regulatory elements. J Med Genet 47(6):398–403. doi: 10.1136/jmg.2009.074047 PubMedCrossRefGoogle Scholar
  45. 45.
    Millevoi S, Bernat S, Telly D, Fouque F, Gladieff L, Favre G, Vagner S, Toulas C (2010) The c.5242C>A BRCA1 missense variant induces exon skipping by increasing splicing repressors binding. Breast Cancer Res Treat 120(2):391–399PubMedCrossRefGoogle Scholar
  46. 46.
    Sanz DJ, Acedo A, Infante M, Durán M, Pérez-Cabornero L, Esteban-Cardeñosa E, Lastra E, Pagani F, Miner C, Velasco EA (2010) A high proportion of DNA variants of BRCA1 and BRCA2 is associated with aberrant splicing in breast/ovarian cancer patients. Clin Cancer Res 16(6):1957–1967. doi: 10.1158/1078-0432.ccr-09-2564 PubMedCrossRefGoogle Scholar
  47. 47.
    Fackenthal JD, Cartegni L, Krainer AR, Olopade OI (2002) BRCA2 T2722R is a deleterious allele that causes exon skipping. Am J Hum Genet 71(3):625–631PubMedCrossRefGoogle Scholar

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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