The increasing application of gene panels for familial cancer susceptibility disorders will probably lead to an increased proposal of susceptibility gene candidates. Using ERCC2 DNA repair gene as an example, we show that proof of a possible role in cancer susceptibility requires a detailed dissection and characterization of the underlying mutations for genes with diverse cellular functions (in this case mainly DNA repair and basic cellular transcription). In case of ERCC2, panel sequencing of 1345 index cases from 587 German, 405 Lithuanian and 353 Czech families with breast and ovarian cancer (BC/OC) predisposition revealed 25 mutations (3 frameshift, 2 splice-affecting, 20 missense), all absent or very rare in the ExAC database. While 16 mutations were unique, 9 mutations showed up repeatedly with population-specific appearance. Ten out of eleven mutations that were tested exemplarily in cell-based functional assays exert diminished excision repair efficiency and/or decreased transcriptional activation capability. In order to provide evidence for BC/OC predisposition, we performed familial segregation analyses and screened ethnically matching controls. However, unlike the recently published RECQL example, none of our recurrent ERCC2 mutations showed convincing co-segregation with BC/OC or significant overrepresentation in the BC/OC cohort. Interestingly, we detected that some deleterious founder mutations had an unexpectedly high frequency of > 1% in the corresponding populations, suggesting that either homozygous carriers are not clinically recognized or homozygosity for these mutations is embryonically lethal. In conclusion, we provide a useful resource on the mutational landscape of ERCC2 mutations in hereditary BC/OC patients and, as our key finding, we demonstrate the complexity of correct interpretation for the discovery of "bonafide" breast cancer susceptibility genes.

Clinic for Dermatology Venerology and Allergology Göttingen Germany

Clinic of Dermatology Rostock Germany

Cologne Center for Genomics Cologne Germany

Department of Gynecology and Obstetrics Medical Faculty and University Hospital Carl Gustav Carus Technische Universität Dresden Germany

German Cancer Consortium Dresden Germany

German Cancer Research Center Heidelberg Germany

Institute for Clinical Genetics Faculty of Medicine Carl Gustav Carus Technische Universität Dresden Dresden Germany

Institute of Biochemistry and Experimental Oncology 1st Faculty of Medicine Charles University Prague Prague Czech Republic

Masaryk Memorial Cancer Institute Brno Czech Republic

MGZ Medical Genetics Center Munich Germany

National Center for Tumor Diseases Partner Site Dresden Germany

State Research Institute Innovative Medicine Center Vilnius Lithuania

Vilnius University Hospital Santariskiu Clinics Hematology Oncology and Transfusion Medicine Center Vilnius Lithuania

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Couch FJ, Nathanson KL, Offit K (2014) Two decades after BRCA: setting paradigms in personalized cancer care and prevention. Science 343: 1466–1470. 10.1126/science.1251827 PubMed DOI PMC

Turnbull C, Rahman N (2008) Genetic predisposition to breast cancer: past, present, and future. Annu Rev Genomics Hum Genet 9: 321–345. 10.1146/annurev.genom.9.081307.164339 PubMed DOI

Antoniou AC, Easton DF (2006) Models of genetic susceptibility to breast cancer. Oncogene 25: 5898–5905. PubMed

Easton DF, Pharoah PD, Antoniou AC, Tischkowitz M, Tavtigian SV, et al. (2015) Gene-panel sequencing and the prediction of breast-cancer risk. N Engl J Med 372: 2243–2257. 10.1056/NEJMsr1501341 PubMed DOI PMC

Evans BJ, Burke W, Jarvik GP (2015) The FDA and genomic tests—getting regulation right. N Engl J Med 372: 2258–2264. 10.1056/NEJMsr1501194 PubMed DOI PMC

Zhang G, Zeng Y, Liu Z, Wei W (2013) Significant association between Nijmegen breakage syndrome 1 657del5 polymorphism and breast cancer risk. Tumour Biol 34: 2753–2757. 10.1007/s13277-013-0830-z PubMed DOI

Cybulski C, Carrot-Zhang J, Kluzniak W, Rivera B, Kashyap A, et al. (2015) Germline RECQL mutations are associated with breast cancer susceptibility. Nat Genet. PubMed

Singh A, Compe E, Le May N, Egly JM (2015) TFIIH Subunit Alterations Causing Xeroderma Pigmentosum and Trichothiodystrophy Specifically Disturb Several Steps during Transcription. Am J Hum Genet 96: 194–207. 10.1016/j.ajhg.2014.12.012 PubMed DOI PMC

Takayama K, Salazar EP, Lehmann A, Stefanini M, Thompson LH, et al. (1995) Defects in the DNA repair and transcription gene ERCC2 in the cancer-prone disorder xeroderma pigmentosum group D. Cancer Res 55: 5656–5663. PubMed

Lehmann J, Schubert S, Emmert S (2014) Xeroderma pigmentosum: diagnostic procedures, interdisciplinary patient care, and novel therapeutic approaches. J Dtsch Dermatol Ges 12: 867–872. 10.1111/ddg.12419 PubMed DOI

Pabalan N, Francisco-Pabalan O, Sung L, Jarjanazi H, Ozcelik H (2010) Meta-analysis of two ERCC2 (XPD) polymorphisms, Asp312Asn and Lys751Gln, in breast cancer. Breast Cancer Res Treat 124: 531–541. 10.1007/s10549-010-0863-6 PubMed DOI

Kircher M, Witten DM, Jain P, O'Roak BJ, Cooper GM, et al. (2014) A general framework for estimating the relative pathogenicity of human genetic variants. Nat Genet 46: 310–315. 10.1038/ng.2892 PubMed DOI PMC

Fan L, Fuss JO, Cheng QJ, Arvai AS, Hammel M, et al. (2008) XPD helicase structures and activities: insights into the cancer and aging phenotypes from XPD mutations. Cell 133: 789–800. 10.1016/j.cell.2008.04.030 PubMed DOI PMC

Kuper J, Braun C, Elias A, Michels G, Sauer F, et al. (2014) In TFIIH, XPD helicase is exclusively devoted to DNA repair. PLoS Biol 12: e1001954 10.1371/journal.pbio.1001954 PubMed DOI PMC

Liu H, Rudolf J, Johnson KA, McMahon SA, Oke M, et al. (2008) Structure of the DNA repair helicase XPD. Cell 133: 801–812. 10.1016/j.cell.2008.04.029 PubMed DOI PMC

Botta E, Nardo T, Broughton BC, Marinoni S, Lehmann AR, et al. (1998) Analysis of mutations in the XPD gene in Italian patients with trichothiodystrophy: site of mutation correlates with repair deficiency, but gene dosage appears to determine clinical severity. Am J Hum Genet 63: 1036–1048. PubMed PMC

Viprakasit V, Gibbons RJ, Broughton BC, Tolmie JL, Brown D, et al. (2001) Mutations in the general transcription factor TFIIH result in beta-thalassaemia in individuals with trichothiodystrophy. Hum Mol Genet 10: 2797–2802. PubMed

Zhou X, Khan SG, Tamura D, Patronas NJ, Zein WM, et al. (2010) Brittle hair, developmental delay, neurologic abnormalities, and photosensitivity in a 4-year-old girl. J Am Acad Dermatol 63: 323–328. 10.1016/j.jaad.2010.03.041 PubMed DOI PMC

Broughton BC, Berneburg M, Fawcett H, Taylor EM, Arlett CF, et al. (2001) Two individuals with features of both xeroderma pigmentosum and trichothiodystrophy highlight the complexity of the clinical outcomes of mutations in the XPD gene. Hum Mol Genet 10: 2539–2547. PubMed

Nishiwaki Y, Kobayashi N, Imoto K, Iwamoto TA, Yamamoto A, et al. (2004) Trichothiodystrophy fibroblasts are deficient in the repair of ultraviolet-induced cyclobutane pyrimidine dimers and (6–4)photoproducts. J Invest Dermatol 122: 526–532. PubMed

Cleaver JE, Thompson LH, Richardson AS, States JC (1999) A summary of mutations in the UV-sensitive disorders: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. Hum Mutat 14: 9–22. PubMed

Taylor EM, Broughton BC, Botta E, Stefanini M, Sarasin A, et al. (1997) Xeroderma pigmentosum and trichothiodystrophy are associated with different mutations in the XPD (ERCC2) repair/transcription gene. Proc Natl Acad Sci U S A 94: 8658–8663. PubMed PMC

Novembre J, Johnson T, Bryc K, Kutalik Z, Boyko AR, et al. (2008) Genes mirror geography within Europe. Nature 456: 98–101. 10.1038/nature07331 PubMed DOI PMC

Pohlreich P, Zikan M, Stribrna J, Kleibl Z, Janatova M, et al. (2005) High proportion of recurrent germline mutations in the BRCA1 gene in breast and ovarian cancer patients from the Prague area. Breast Cancer Res 7: R728–736. PubMed PMC

Lhota F, Zemankova P, Kleiblova P, Soukupova J, Vocka M, et al. (2016) Hereditary truncating mutations of DNA repair and other genes in BRCA1/BRCA2/PALB2-negatively tested breast cancer patients. Clin Genet. PubMed

Kleibl Z, Havranek O, Novotny J, Kleiblova P, Soucek P, et al. (2008) Analysis of CHEK2 FHA domain in Czech patients with sporadic breast cancer revealed distinct rare genetic alterations. Breast Cancer Res Treat 112: 159–164. PubMed

Berneburg M, Clingen PH, Harcourt SA, Lowe JE, Taylor EM, et al. (2000) The cancer-free phenotype in trichothiodystrophy is unrelated to its repair defect. Cancer Res 60: 431–438. PubMed

Kleijer WJ, Laugel V, Berneburg M, Nardo T, Fawcett H, et al. (2008) Incidence of DNA repair deficiency disorders in western Europe: Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. DNA Repair (Amst) 7: 744–750. PubMed

de Boer J, Donker I, de Wit J, Hoeijmakers JH, Weeda G (1998) Disruption of the mouse xeroderma pigmentosum group D DNA repair/basal transcription gene results in preimplantation lethality. Cancer Res 58: 89–94. PubMed

Lehmann AR (2001) The xeroderma pigmentosum group D (XPD) gene: one gene, two functions, three diseases. Genes Dev 15: 15–23. PubMed

Wang K, Li M, Hakonarson H (2010) ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res 38: e164 10.1093/nar/gkq603 PubMed DOI PMC

Protic-Sabljic M, Kraemer KH (1985) One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells. Proc Natl Acad Sci U S A 82: 6622–6626. PubMed PMC

Schafer A, Schubert S, Gratchev A, Seebode C, Apel A, et al. (2013) Characterization of three XPG-defective patients identifies three missense mutations that impair repair and transcription. J Invest Dermatol 133: 1841–1849. 10.1038/jid.2013.54 PubMed DOI

Khobta A, Anderhub S, Kitsera N, Epe B (2010) Gene silencing induced by oxidative DNA base damage: association with local decrease of histone H4 acetylation in the promoter region. Nucleic Acids Res 38: 4285–4295. 10.1093/nar/gkq170 PubMed DOI PMC

Grantham R (1974) Amino acid difference formula to help explain protein evolution. Science 185: 862–864. PubMed

Arnold M, Raffler J, Pfeufer A, Suhre K, Kastenmuller G (2015) SNiPA: an interactive, genetic variant-centered annotation browser. Bioinformatics 31: 1334–1336. 10.1093/bioinformatics/btu779 PubMed DOI PMC

Prevalence of Germline Pathogenic Variants in Cancer Predisposing Genes in Czech and Belgian Pancreatic Cancer Patients