The online version of this article (https://doi.org/10.1186/s13053-018-0086-0) contains supplementary material, which is available to authorized users.
In kindreds carrying path_BRCA1/2 variants, some women in these families will develop cancer despite testing negative for the family’s pathogenic variant. These families may have additional genetic variants, which not only may increase the susceptibility of the families’ path_BRCA1/2, but also be capable of causing cancer in the absence of the path_BRCA1/2 variants. We aimed to identify novel genetic variants in prospectively detected breast cancer (BC) or gynecological cancer cases tested negative for their families’ pathogenic BRCA1/2 variant (path_BRCA1 or path_BRCA2).
Women with BC or gynecological cancer who had tested negative for path_BRCA1 or path_BRCA2 variants were included. Forty-four cancer susceptibility genes were screened for genetic variation through a targeted amplicon-based sequencing assay. Protein- and RNA splicing-dedicated in silico analyses were performed for all variants of unknown significance (VUS). Variants predicted as the ones most likely affecting pre-mRNA splicing were experimentally analyzed in a minigene assay.
We identified 48 women who were tested negative for their family’s path_BRCA1 (n = 13) or path_BRCA2 (n = 35) variants. Pathogenic variants in the ATM, BRCA2, MSH6 and MUTYH genes were found in 10% (5/48) of the cases, of whom 15% (2/13) were from path_BRCA1 and 9% (3/35) from path_BRCA2 families. Out of the 26 unique VUS, 3 (12%) were predicted to affect RNA splicing (APC c.721G > A, MAP3K1 c.764A > G and MSH2 c.815C > T). However, by using a minigene, assay we here show that APC c.721G > A does not cause a splicing defect, similarly to what has been recently reported for the MAP3K1 c.764A > G. The MSH2 c.815C > T was previously described as causing partial exon skipping and it was identified in this work together with the path_BRCA2 c.9382C > T (p.R3128X).
All women in breast or breast/ovarian cancer kindreds would benefit from being offered genetic testing irrespective of which causative genetic variants have been demonstrated in their relatives.
Additional file 1: The concentration in a 10 ml PCR was 1xThermopol Reaction Buffer with 2 mM MgS04, 0.3 μM “reverse” primers, 0.15 μM “forward” primer, 0.1 μM, 6-Carboxyfluorescein-GC clamp primer, 600 μM dNTP, 100 μg Bovine Serum Albumine (Sigma-Aldrich, Oslo, Norway) and 0.75 U Taq DNA polymerase. Plates were sealed with two strips of electrical tape (Clas Ohlson, Oslo, Norway). The temperature cycling was repeated 35 times; 94 °C for 30 s, annealing temperature held for 30 s and extension at 72 °C for 60 s (Eppendorf Mastercycler ep gradient S (Eppendorf, Hamburg, Germany)). Table S1. primers used to amplify PCR product to be analysed by cycling temperature capillary electrophoresis. (DOCX 16 kb)13053_2018_86_MOESM1_ESM.docx
Additional file 2: Primers used in the pCAS2 minigene splicing assay. (DOCX 14 kb)13053_2018_86_MOESM2_ESM.docx
Antoniou A, Pharoah PD, Narod S, Risch HA, Eyfjord JE, Hopper JL, Loman N, Olsson H, Johannsson O, Borg A, et al. Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case series unselected for family history: a combined analysis of 22 studies. Am J Hum Genet. 2003;72(5):1117–30. CrossRefPubMedPubMedCentral
Evans DGR, Ingham SL, Buchan I, Woodward ER, Byers H, Howell A, Maher ER, Newman WG, Lalloo F. Increased rate of Phenocopies in all age groups in BRCA1/BRCA2 mutation kindred, but increased prospective breast cancer risk is confined to BRCA2 mutation carriers. Cancer Epidem Biomar. 2013;22(12):2269–76. CrossRef
Moller P, Evans G, Haites N, Vasen H, Reis MM, Anderson E, Apold J, Hodgson S, Eccles D, Olsson H, et al. Guidelines for follow-up of women at high risk for inherited breast cancer: consensus statement from the biomed 2 demonstration Programme on inherited breast cancer. Dis Markers. 1999;15(1–3):207–11. CrossRefPubMedPubMedCentral
Dominguez-Valentin M, Nakken S, Tubeuf H, Vodak D, Ekstrom PO, Nissen AM, Morak M, Holinski-Feder E, Martins A, Moller P, et al. Potentially pathogenic germline CHEK2 c.319+2T>A among multiple early-onset cancer families. Fam Cancer. 2017. https://doi.org/10.1007/s10689-017-0011-0. [Epub ahead of print].
Li L, Chen HC, Liu LX. Sequence alignment algorithm in similarity measurement. Int Forum Info Technol Appl Proc. 2009;1:453–456. https://doi.org/10.1109/Ifita.2009.119.
Houdayer C, Caux-Moncoutier V, Krieger S, Barrois M, Bonnet F, Bourdon V, Bronner M, Buisson M, Coulet F, Gaildrat P, et al. Guidelines for splicing analysis in molecular diagnosis derived from a set of 327 combined in silico/in vitro studies on BRCA1 and BRCA2 variants. Hum Mutat. 2012;33(8):1228–38. CrossRefPubMed
Antoniou AC, Kuchenbaecker KB, Soucy P, Beesley J, Chen XQ, McGuffog L, Lee A, Barrowdale D, Healey S, Sinilnikova OM, et al. Common variants at 12p11, 12q24, 9p21, 9q31.2 and in ZNF365 are associated with breast cancer risk for BRCA1 and/or BRCA2 mutation carriers. Breast Cancer Research. 2012;14(1):1–18.
Susswein LR, Marshall ML, Nusbaum R, Vogel Postula KJ, Weissman SM, Yackowski L, Vaccari EM, Bissonnette J, Booker JK, Cremona ML, et al. Pathogenic and likely pathogenic variant prevalence among the first 10,000 patients referred for next-generation cancer panel testing. Genet Med. 2016;18(8):823–32. CrossRefPubMed
Soukarieh O, Gaildrat P, Hamieh M, Drouet A, Baert-Desurmont S, Frebourg T, Tosi M, Martins A. Exonic Splicing Mutations Are More Prevalent than Currently Estimated and Can Be Predicted by Using In Silico Tools. Plos Genet. 2016;12(1):1–26. CrossRef
Sjursen W, Haukanes BI, Grindedal EM, Aarset H, Stormorken A, Engebretsen LF, Jonsrud C, Bjornevoll I, Andresen PA, Ariansen S, et al. Current clinical criteria for lynch syndrome are not sensitive enough to identify MSH6 mutation carriers. J Med Genet. 2010;47(9):579–85. CrossRefPubMedPubMedCentral
Hegde M, Ferber M, Mao R, Samowitz W, Ganguly A. Working Group of the American College of medical G, genomics laboratory quality assurance C: ACMG technical standards and guidelines for genetic testing for inherited colorectal cancer (lynch syndrome, familial adenomatous polyposis, and MYH-associated polyposis). Genet Med. 2014;16(1):101–16. CrossRefPubMed
Castera L, Krieger S, Rousselin A, Legros A, Baumann JJ, Bruet O, Brault B, Fouillet R, Goardon N, Letac O, et al. Next-generation sequencing for the diagnosis of hereditary breast and ovarian cancer using genomic capture targeting multiple candidate genes. Eur J Hum Genet. 2014;22(11):1305–13. CrossRefPubMedPubMedCentral
Kamps R, Brandao RD, Bosch BJ, Paulussen AD, Xanthoulea S, Blok MJ, Romano A. Next-Generation Sequencing in Oncology: Genetic Diagnosis, Risk Prediction and Cancer Classification. Int J Mol Sci. 2017;18(2):1–57. CrossRef
Smith MJ, Urquhart JE, Harkness EF, Miles EK, Bowers NL, Byers HJ, Bulman M, Gokhale C, Wallace AJ, Newman WG, et al. The contribution of whole gene deletions and large rearrangements to the mutation Spectrum in inherited tumor predisposing syndromes. Hum Mutat. 2015;
Lincoln SE, Kobayashi Y, Anderson MJ, Yang S, Desmond AJ, Mills MA, Nilsen GB, Jacobs KB, Monzon FA, Kurian AW, et al. A systematic comparison of traditional and multigene panel testing for hereditary breast and ovarian cancer genes in more than 1000 patients. J Mol Diagn. 2015;17(5):533–44. CrossRefPubMed
- Genetic variants of prospectively demonstrated phenocopies in BRCA1/2 kindreds
D. Gareth R. Evans
Per Olaf Ekstrøm
Anke M. Nissen
- BioMed Central
Neu im Fachgebiet Onkologie
e.Med Kampagnen-Visual, Mail Icon II