To study the potential contribution of genes other than BRCA1/2, PTEN, and TP53 to the biological and clinical characteristics of multiple early-onset cancers in Norwegian families, including early-onset breast cancer, Cowden-like and Li-Fraumeni-like syndromes (BC, CSL and LFL, respectively). The Hereditary Cancer Biobank from the Norwegian Radium Hospital was used to identify early-onset BC, CSL or LFL for whom no pathogenic variants in BRCA1/2, PTEN, or TP53 had been found in routine diagnostic DNA sequencing. Forty-four cancer susceptibility genes were selected and analyzed by our in-house designed TruSeq amplicon-based assay for targeted sequencing. Protein- and RNA splicing-dedicated in silico analyses were performed for all variants of unknown significance (VUS). Variants predicted as the more likely to affect splicing were experimentally analyzed by minigene assay. We identified a CSL individual carrying a variant in CHEK2 (c.319+2T>A, IVS2), here considered as likely pathogenic. Out of the five VUS (BRCA2, CDH1, CHEK2, MAP3K1, NOTCH3) tested in the minigene splicing assay, only NOTCH3 c.14090C>T (p.Ser497Leu) showed a significant effect on RNA splicing, notably by inducing partial skipping of exon 9. Among 13 early-onset BC, CSL and LFL patients, gene panel sequencing identified a potentially pathogenic variant in CHEK2 that affects a canonical RNA splicing signal. Our study provides new information on genetic loci that may affect the risk of developing cancer in these patients and their families, demonstrating that genes presently not routinely tested in molecular diagnostic settings may be important for capturing cancer predisposition in these families.
Early-onset breast cancer Cowden-like syndrome Li-Fraumeni-like syndrome Gene panel testing CHEK2RNA splicing mutations
American College of Medical Genetics and Genomics
Cowden syndrome like
Li-Fraumeni like syndrome
Mismatch repair genes
Next generation sequencing
Single nucleotide polymorphism
Variants of unclassified significance
We thank the included families for their participation and contribution to this study.
This work was supported by the Radium Hospital Foundation (Oslo, Norway), Helse Sør-Øst (Norway), the French Association Recherche contre le Cancer (ARC), the Groupement des Entreprises Françaises dans la Lutte contre le Cancer (Gefluc), the Association Nationale de la Recherche et de la Technologie (ANRT, CIFRE PhD fellowship to H.T.) and by the OpenHealth Institute.
Compliance with ethical standards
Conflict of interests
The authors declare that they have no competing interests.
Hegde M, Ferber M, Mao R et al (2014) ACMG technical standards and guidelines for genetic testing for inherited colorectal cancer (lynch syndrome, familial adenomatous polyposis, and MYH-associated polyposis). Genet Med 16(1):101–116. doi:10.1038/gim.2013.166CrossRefPubMedGoogle Scholar
Richards S, Aziz N, Bale S et al (2015) Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 17(5):405–424. doi:10.1038/gim.2015.30CrossRefPubMedPubMedCentralGoogle Scholar
Park KS, Cho EY, Nam SJ, Ki CS, Kim JW (2016) Comparative analysis of BRCA1 and BRCA2 variants of uncertain significance in patients with breast cancer: a multifactorial probability-based model versus ACMG standards and guidelines for interpreting sequence variants. Genet Med 18(12):1250–1257. doi:10.1038/gim.2016.39CrossRefPubMedGoogle Scholar
Malone KE, Daling JR, Thompson JD, O’Brien CA, Francisco LV, Ostrander EA (1998) BRCA1 mutations and breast cancer in the general population - Analyses in women before age 35 years and in women before age 45 years with first-degree family history. Jama 279(12):922–929 doi:10.1001/jama.279.12.922CrossRefPubMedGoogle Scholar
Peto J, Collins N, Barfoot R et al (1999) Prevalence of BRCA1 and BRCA2 gene mutations in patients with early-onset breast cancer. J Natl Cancer Inst 91(11):943–949CrossRefPubMedGoogle Scholar
Pharoah PDP, Guilford P, Caldas C, Consortiu IGCL (2001) Incidence of gastric cancer and breast cancer in CDH1 (E-cadherin) mutation carriers from hereditary diffuse gastric cancer families. Gastroenterology 121(6):1348–1353. doi:10.1053/gast.2001.29611CrossRefPubMedGoogle Scholar
Aloraifi F, McCartan D, McDevitt T, Green AJ, Bracken A, Geraghty J (2015) Protein-truncating variants in moderate-risk breast cancer susceptibility genes: a meta-analysis of high-risk case-control screening studies. Cancer Genet 208(9):455–463. doi:10.1016/j.cancergen.2015.06.001CrossRefPubMedGoogle Scholar
Birch JM, Hartley AL, Tricker KJ et al (1994) Prevalence and diversity of constitutional mutations in the P53 Gene among 21 Li-Fraumeni families. Cancer Res 54(5):1298–1304PubMedGoogle Scholar
Malkin D, Li FP, Strong LC et al (1990) Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 250(4985):1233–1238CrossRefPubMedGoogle Scholar
Srivastava S, Zou ZQ, Pirollo K, Blattner W, Chang EH (1990) Germ-line transmission of a mutated p53 gene in a cancer-prone family with Li-Fraumeni syndrome. Nature 348(6303):747–749. doi:10.1038/348747a0CrossRefPubMedGoogle Scholar
den Dunnen JT, Antonarakis SE (2000) Mutation nomenclature extensions and suggestions to describe complex mutations: A discussion. Hum Mutat 15(1):7–12CrossRefPubMedGoogle Scholar
Thompson BA, Spurdle AB, Plazzer JP et al (2014) Application of a 5-tiered scheme for standardized classification of 2,360 unique mismatch repair gene variants in the InSiGHT locus-specific database. Nat Genet 46(2):107–115. doi:10.1038/ng.2854CrossRefPubMedGoogle Scholar
Houdayer C, Caux-Moncoutier V, Krieger S et al (2012) 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 33(8):1228–1238. doi:10.1002/humu.22101CrossRefPubMedGoogle Scholar
Di Giacomo D, Gaildrat P, Abuli A et al (2013) Functional analysis of a large set of BRCA2 exon 7 variants highlights the predictive value of hexamer scores in detecting alterations of exonic splicing regulatory elements. Hum Mutat 34(11):1547–1557. doi:10.1002/humu.22428CrossRefPubMedGoogle Scholar
Tavtigian SV, Deffenbaugh AM, Yin L et al (2006) Comprehensive statistical study of 452 BRCA1 missense substitutions with classification of eight recurrent substitutions as neutral. J Med Genet 43(4):295–305. doi:10.1136/jmg.2005.033878CrossRefPubMedGoogle Scholar
Kalia SS, Adelman K, Bale SJ et al (2017) Recommendations for reporting of secondary findings in clinical exome and genome sequencing, 2016 update (ACMG SF v2.0): a policy statement of the American College of Medical Genetics and Genomics. Genet Med 19(2):249–255. doi:10.1038/gim.2016.190CrossRefPubMedGoogle Scholar
Pinto P, Paulo P, Santos C et al (2016) Implementation of next-generation sequencing for molecular diagnosis of hereditary breast and ovarian cancer highlights its genetic heterogeneity. Breast Cancer Res Treat 159(2):245–256. doi:10.1007/s10549-016-3948-zCrossRefPubMedGoogle Scholar
Yadav S, Fulbright J, Dreyfuss H et al (2015) Outcomes of retesting BRCA-negative patients using multigene panels. J Clin Oncol 33(Suppl 28):23Google Scholar
Villacis RA, Miranda PM, Gomy I et al (2016) Contribution of rare germline copy number variations and common susceptibility loci in Lynch syndrome patients negative for mutations in the mismatch repair genes. Int J Cancer 138(8):1928–1935. doi:10.1002/ijc.29948CrossRefPubMedGoogle Scholar