{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,29]],"date-time":"2026-06-29T18:00:20Z","timestamp":1782756020528,"version":"3.54.5"},"reference-count":38,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2021,5,13]],"date-time":"2021-05-13T00:00:00Z","timestamp":1620864000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"European Union\u2019s Horizon 2020 research and innovation programme","award":["grant agreement No. 739593"],"award-info":[{"award-number":["grant agreement No. 739593"]}]},{"name":"National Research, Development and Innovation Office","award":["GINOP-2.3.2\u201315-2016\u201300001, GINOP-2.3.2\u201315-2016\u201300024, and GINOP-2.3.2\u201315-2016\u201300026"],"award-info":[{"award-number":["GINOP-2.3.2\u201315-2016\u201300001, GINOP-2.3.2\u201315-2016\u201300024, and GINOP-2.3.2\u201315-2016\u201300026"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cancers"],"abstract":"<jats:p>Germline mutations in the BRCA1 and BRCA2 genes are responsible for hereditary breast and ovarian cancer syndrome. Germline and somatic BRCA1\/2 mutations may define therapeutic targets and refine cancer treatment options. However, routine BRCA diagnostic approaches cannot reveal the exact time and origin of BRCA1\/2 mutation formation, and thus, the fine details of their contribution to tumor progression remain less clear. Here, we establish a diagnostic pipeline using high-resolution microscopy and laser microcapture microscopy to test for BRCA1\/2 mutations in the tumor at the single-cell level, followed by deep next-generation sequencing of various tissues from the patient. To demonstrate the power of our approach, here, we describe a detailed single-cell-level analysis of an ovarian cancer patient we found to exhibit constitutional somatic mosaicism of a pathogenic BRCA2 mutation. Employing next-generation sequencing, BRCA2 c.7795G&gt;T, p.(Glu2599Ter) was detected in 78% of reads in DNA extracted from ovarian cancer tissue and 25% of reads in DNA derived from peripheral blood, which differs significantly from the expected 50% of a hereditary mutation. The BRCA2 mutation was subsequently observed at 17\u201320% levels in the normal ovarian and buccal tissue of the patient. Together, our findings suggest that this mutation occurred early in embryonic development. Characterization of the mosaic mutation at the single-cell level contributes to a better understanding of BRCA mutation formation and supports the concept that the combination of single-cell and next-generation sequencing methods is advantageous over traditional mutational analysis methods. This study is the first to characterize constitutional mosaicism down to the single-cell level, and it demonstrates that BRCA2 mosaicism occurring early during embryogenesis can drive tumorigenesis in ovarian cancer.<\/jats:p>","DOI":"10.3390\/cancers13102354","type":"journal-article","created":{"date-parts":[[2021,5,13]],"date-time":"2021-05-13T11:10:06Z","timestamp":1620904206000},"page":"2354","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["The Combination of Single-Cell and Next-Generation Sequencing Can Reveal Mosaicism for BRCA2 Mutations and the Fine Molecular Details of Tumorigenesis"],"prefix":"10.3390","volume":"13","author":[{"given":"Alexandra","family":"Gr\u00e1f","sequence":"first","affiliation":[{"name":"HCEMM-BRC Mutagenesis and Carcinogenesis Research Group, Institute of Genetics, Biological Research Centre, 6726 Szeged, Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M\u00e1rton Zsolt","family":"Enyedi","sequence":"additional","affiliation":[{"name":"Delta Bio 2000 Ltd., 6726 Szeged, Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lajos","family":"Pint\u00e9r","sequence":"additional","affiliation":[{"name":"Delta Bio 2000 Ltd., 6726 Szeged, Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"\u00c9va","family":"Kriston-P\u00e1l","sequence":"additional","affiliation":[{"name":"Delta Bio 2000 Ltd., 6726 Szeged, Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"G\u00e1bor","family":"Jaksa","sequence":"additional","affiliation":[{"name":"Delta Bio 2000 Ltd., 6726 Szeged, Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"\u00c1rp\u00e1d","family":"B\u00e1lind","sequence":"additional","affiliation":[{"name":"Lend\u00fclet Laboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, Biological Research Centre, 6726 Szeged, Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"\u00c9va","family":"Ezer","sequence":"additional","affiliation":[{"name":"Department of Clinical Oncology, Teaching Hospital M\u00f3r Kaposi, 7400 Kaposv\u00e1r, Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"P\u00e9ter","family":"Horv\u00e1th","sequence":"additional","affiliation":[{"name":"Lend\u00fclet Laboratory of Microscopic Image Analysis and Machine Learning, Institute of Biochemistry, Biological Research Centre, 6726 Szeged, Hungary"},{"name":"Institute for Molecular Medicine Finland (FIMM), University of Helsinki, 00014 Helsinki, Finland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Farkas","family":"S\u00fck\u00f6sd","sequence":"additional","affiliation":[{"name":"Department of Pathology, Faculty of Medicine, University of Szeged, 6720 Szeged, Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7344-1750","authenticated-orcid":false,"given":"Ern\u0151","family":"Kiss","sequence":"additional","affiliation":[{"name":"HCEMM-BRC Mutagenesis and Carcinogenesis Research Group, Institute of Genetics, Biological Research Centre, 6726 Szeged, Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lajos","family":"Haracska","sequence":"additional","affiliation":[{"name":"HCEMM-BRC Mutagenesis and Carcinogenesis Research Group, Institute of Genetics, Biological Research Centre, 6726 Szeged, Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,5,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"306","DOI":"10.1038\/nature10762","article-title":"Clonal evolution in cancer","volume":"481","author":"Greaves","year":"2012","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"40","DOI":"10.3109\/10408363.2015.1083944","article-title":"Clonal evolution and tumor-initiating cells: New dimensions in cancer patient treatment","volume":"53","author":"Apostoli","year":"2016","journal-title":"Crit. Rev. Clin. Lab. Sci."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Black, J.R.M., and McGranahan, N. (2021). Genetic and non-genetic clonal diversity in cancer evolution. Nat. Rev. Cancer.","DOI":"10.1038\/s41568-021-00336-2"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"429","DOI":"10.1038\/35044000","article-title":"In search of the tumour-suppressor functions of BRCA1 and BRCA2","volume":"408","author":"Scully","year":"2000","journal-title":"Nature"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"8981","DOI":"10.1038\/sj.onc.1206176","article-title":"Homologous repair of DNA damage and tumorigenesis: The BRCA connection","volume":"21","author":"Jasin","year":"2002","journal-title":"Oncogene"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1016\/S0092-8674(02)00615-3","article-title":"Cancer susceptibility and the functions of BRCA1 and BRCA2","volume":"108","author":"Venkitaraman","year":"2002","journal-title":"Cell"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"5085","DOI":"10.1038\/onc.2017.213","article-title":"Loss of BRCA1 or BRCA2 markedly increases the rate of base substitution mutagenesis and has distinct effects on genomic deletions","volume":"36","author":"Zamborszky","year":"2017","journal-title":"Oncogene"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1016\/j.molcel.2012.07.029","article-title":"Playing the end game: DNA double-strand break repair pathway choice","volume":"47","author":"Chapman","year":"2012","journal-title":"Mol. Cell"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1388","DOI":"10.3389\/fonc.2019.01388","article-title":"Harnessing DNA double-strand break repair for cancer treatment","volume":"9","author":"Trenner","year":"2019","journal-title":"Front. Oncol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1038\/s41594-020-0374-z","article-title":"BRCA1 and S phase DNA repair pathways restrict LINE-1 retrotransposition in human cells","volume":"27","author":"Mita","year":"2020","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1007\/s00018-019-03365-1","article-title":"Structural basis of homologous recombination","volume":"77","author":"Sun","year":"2020","journal-title":"Cell Mol. Life Sci."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1016\/j.maturitas.2008.11.010","article-title":"Oncogenic pathways in hereditary and sporadic breast cancer","volume":"61","author":"Kenemans","year":"2008","journal-title":"Maturitas"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"611","DOI":"10.1136\/jcp.2005.032151","article-title":"Distinction between hereditary and sporadic breast cancer on the basis of clinicopathological data","volume":"59","author":"Bouter","year":"2006","journal-title":"J. Clin. Pathol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"609","DOI":"10.1038\/nature10166","article-title":"Integrated genomic analyses of ovarian carcinoma","volume":"474","year":"2011","journal-title":"Nature"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"764","DOI":"10.1158\/1078-0432.CCR-13-2287","article-title":"Germline and somatic mutations in homologous recombination genes predict platinum response and survival in ovarian, fallopian tube, and peritoneal carcinomas","volume":"20","author":"Pennington","year":"2014","journal-title":"Clin. Cancer Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1076","DOI":"10.18632\/oncotarget.6834","article-title":"BRCA somatic and germline mutation detection in paraffin embedded ovarian cancers by next-generation sequencing","volume":"7","author":"Mafficini","year":"2016","journal-title":"Oncotarget"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1640","DOI":"10.1002\/cam4.748","article-title":"Detection of somatic BRCA1\/2 mutations in ovarian cancer-next-generation sequencing analysis of 100 cases","volume":"5","author":"Koczkowska","year":"2016","journal-title":"Cancer Med."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"893","DOI":"10.1111\/cas.13530","article-title":"Crosstalk of DNA double-strand break repair pathways in poly(ADP-ribose) polymerase inhibitor treatment of breast cancer susceptibility gene 1\/2-mutated cancer","volume":"109","author":"Sunada","year":"2018","journal-title":"Cancer Sci."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"820","DOI":"10.1016\/j.tcb.2019.07.008","article-title":"PARP inhibitor resistance: A Tug-of-war in BRCA-mutated cells","volume":"29","author":"Noordermeer","year":"2019","journal-title":"Trends Cell Biol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"924","DOI":"10.1136\/jcp.2010.086405","article-title":"Melphalan as a treatment for BRCA-related ovarian carcinoma: Can you teach an old drug new tricks?","volume":"64","author":"Osher","year":"2011","journal-title":"J. Clin. Pathol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"122","DOI":"10.1186\/s13256-018-1674-3","article-title":"Complete response to orally administered melphalan in malignant pleural effusion from an occult female genital organ primary neoplasm with BRCA1\/2 mutations: A case report","volume":"12","author":"Fan","year":"2018","journal-title":"J. Med. Case Rep."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"559","DOI":"10.1016\/j.cell.2010.04.033","article-title":"Cellular heterogeneity: Do differences make a difference?","volume":"141","author":"Altschuler","year":"2010","journal-title":"Cell"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"425","DOI":"10.1126\/science.1222161","article-title":"Cell Biology. Using cell-to-cell variability--a new era in molecular biology","volume":"336","author":"Pelkmans","year":"2012","journal-title":"Science"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1146\/annurev-pathol-020712-163923","article-title":"Cellular heterogeneity and molecular evolution in cancer","volume":"8","author":"Almendro","year":"2013","journal-title":"Ann. Rev. Pathol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"17957","DOI":"10.1073\/pnas.1907904116","article-title":"Addressing cellular heterogeneity in tumor and circulation for refined prognostication","volume":"116","author":"Lim","year":"2019","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"647","DOI":"10.1586\/14737159.7.5.647","article-title":"Laser capture microdissection technology","volume":"7","author":"Espina","year":"2007","journal-title":"Exp. Rev. Mol. Diagn."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"226","DOI":"10.1038\/s41467-017-02628-4","article-title":"Intelligent image-based in situ single-cell isolation","volume":"9","author":"Brasko","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Liu, X., and Harada, S. (2013). DNA isolation from mammalian samples. Curr. Protoc. Mol. Biol., 102.","DOI":"10.1002\/0471142727.mb0214s102"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Cline, M.S., Liao, R.G., Parsons, M.T., Paten, B., Alquaddoomi, F., Antoniou, A., Baxter, S., Brody, L., Cook-Deegan, R., and Coffin, A. (2018). BRCA Challenge: BRCA Exchange as a global resource for variants in BRCA1 and BRCA2. PLoS Genet., 14.","DOI":"10.1371\/journal.pgen.1007752"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"573","DOI":"10.1038\/nmeth.3867","article-title":"Clonal genotype and population structure inference from single-cell tumor sequencing","volume":"13","author":"Roth","year":"2016","journal-title":"Nat. Methods"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Hansen, T.V., Bisgaard, M.L., Jonson, L., Albrechtsen, A., Filtenborg-Barnkob, B., Eiberg, H., Ejlertsen, B., and Nielsen, F.C. (2008). Novel de novo BRCA2 mutation in a patient with a family history of breast cancer. BMC Med. Genet., 9.","DOI":"10.1186\/1471-2350-9-58"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"479","DOI":"10.1007\/s10689-009-9270-8","article-title":"Identification of a de novo BRCA1 mutation in a woman with early onset bilateral breast cancer","volume":"8","author":"Edwards","year":"2009","journal-title":"Fam. Cancer"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1007\/s10689-011-9429-y","article-title":"A novel de novo BRCA1 mutation in a Chinese woman with early onset breast cancer","volume":"10","author":"Kwong","year":"2011","journal-title":"Fam. Cancer"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1007\/s10689-020-00186-1","article-title":"Constitutional mosaicism for a BRCA2 mutation as a cause of early-onset breast cancer","volume":"19","author":"Alhopuro","year":"2020","journal-title":"Fam. Cancer"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Azzollini, J., Pesenti, C., Ferrari, L., Fontana, L., Calvello, M., Peissel, B., Portera, G., Tabano, S., Carcangiu, M.L., and Riva, P. (2017). Revertant mosaicism for family mutations is not observed in BRCA1\/2 phenocopies. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0171663"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"140","DOI":"10.1016\/j.molmed.2010.11.003","article-title":"Revertant mosaicism in skin: Natural gene therapy","volume":"17","author":"McGrath","year":"2011","journal-title":"Trends Mol. Med."},{"key":"ref_37","first-page":"167","article-title":"Revertant mosaicism in heritable skin diseases: Mechanisms of natural gene therapy","volume":"14","author":"Pasmooij","year":"2012","journal-title":"Discov. Med."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"513","DOI":"10.1007\/s00438-015-1130-7","article-title":"Unrevealed mosaicism in the next-generation sequencing era","volume":"291","author":"Gajecka","year":"2016","journal-title":"Mol. Genet. Genom."}],"container-title":["Cancers"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6694\/13\/10\/2354\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:00:24Z","timestamp":1760162424000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6694\/13\/10\/2354"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,5,13]]},"references-count":38,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2021,5]]}},"alternative-id":["cancers13102354"],"URL":"https:\/\/doi.org\/10.3390\/cancers13102354","relation":{},"ISSN":["2072-6694"],"issn-type":[{"value":"2072-6694","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,5,13]]}}}