Dvacet let molekulární analýzy genů BRCA1 a BRCA2 v MOÚ – aktuální vývoj v klasifikaci nálezů

Download PDF English version

Authors: Eva Machackova ¹; Kathleen Claes ²; Miroslava Mikova ¹; Jana Házová ¹; Eva Hrabincová Sťahlová ¹; Petra Vasickova ¹; Martin Trbusek ³; Marie Navrátilová ¹; Marek Svoboda ¹; Lenka Foretová ¹
Authors‘ workplace: Department of Cancer Epidemiology and Genetics, Masaryk Memorial Cancer Institute, Brno ¹; Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium ²; Department of Internal Medicine Haematology and Oncology, University Hospital Brno ³
Published in: Klin Onkol 2019; 32(Supplementum2): 51-71
Category: Original Articles
doi: https://doi.org/10.14735/amko2019S51

Overview

Východiska: Patogenní mutace v genech BRCA1 a BRCA2 jsou majoritní příčinou dědičné dominantní predispozice ke vzniku nádoru prsu a vaječníku. Interpretace molekulárně-genetických nálezů vždy závisí na dostupných informacích v době uzavření laboratorní zprávy. Cílem této studie byla revize klasifikace všech výsledků testování BRCA genů v Masarykově onkologickém ústavu (MOÚ).

Soubor pacientů a metody: Pacienti ze 7 400 rodin s podezřením na dědičnou predispozici ke vzniku nádorů prsu a/nebo vaječníků byli v MOÚ vyšetřeni v období let 1999 až první poloviny 2018. Vyšetření genů BRCA bylo vždy indikováno klinickým genetikem. V průběhu 20 let laboratorní praxe byly použity různé metody – počínaje vyšetřením cíleným na detekci zkrácené délky proteinu a heteroduplexní analýzu přes vysokorozlišovací analýzu křivek tání a Sangerovo sekvenování až po masivní paralelní sekvenování.

Výsledky: Mutační analýza vedla k odhalení dědičné predispozice k nádoru prsu/ovaria u 20,5 % rodin. Vysoce riziková zárodečná mutace byla detekována u 1 021 rodin v genu BRCA1 a u 497 rodin v genu BRCA2. Bylo zachyceno široké spektrum patogenních a pravděpodobně patogenních unikátních mutací v obou genech – 124 různých mutací v genu BRCA1 a 123 různých mutací v genu BRCA2. Jako benigní nebo pravděpodobně benigní bylo klasifikováno 96 unikátních variant v genu BRCA1 a 126 variant v genu BRCA2. Zbývajících 82 vzácných unikátních variant zůstalo klasifikováno jako „nejasného významu“, především z důvodu ojedinělého výskytu a nedostatku podkladů pro jejich zařazení do ostatních skupin. Výsledky jsou shrnuty v tabulkách dle typu mutace/varianty vč. podkladů pro jejich klasifikaci.

Závěr: Co nejpřesnější klinická klasifikace variant identifikovaných v BRCA genech má dopad na genetické poradenství a následnou klinickou péči. V této studii uvádíme přehled frekvencí BRCA mutací detekovaných v našem regionu, retrospektivní hodnocení a případně reklasifikaci u některých dříve reportovaných variant ve světle nedávných zjištění.

Děkujeme laborantkám MOÚ, které se podílely na laboratorních analýzách: Hana Pavlů, Jitka Kuklová, Veronika Kosinová, Zuzana Jurášková, Marcela Macků. Děkujeme lékařům ostatních genetických pracovišť v České republice, kteří se podíleli na indikaci pacientů vyšetřovaných v MOÚ: Ústav biologie a lékařské genetiky 2. LF UK a FN Motol v Praze; Klinika lékařské genetiky Thomayerovy nemocnice v Praze; Klinika lékařské genetiky FN Hradec Králové; Klinika lékařské genetiky FN Olomouc; Klinika lékařské genetiky FN Ostrava; Oddělení lékařské genetiky FN Brno; Klinika klinické genetiky Nemocnice České Budějovice, Klinika klinické genetiky Masarykovy nemocnice Ústí nad Labem; a další genetičtí poradci z různých oblastí České republiky.

Tato práce byla podpořena MZ ČR – DRO (MOÚ, 00209805) a granty NV15-28830A, NV15-27695A.

Autoři deklarují, že v souvislosti s předmětem studie nemají žádné komerční zájmy.

Redakční rada potvrzuje, že rukopis práce splnil ICMJE kritéria pro publikace zasílané do biomedicínských časopisů.

Obdrženo: 27. 2. 2019

Přijato: 18. 4. 2019

Klíčová slova:

nádor prsu – nádor ovaria – gen BRCA1 – gen BRCA2 – zárodečné mutace

Sources

1. Maxwell KN, Domchek SM, Nathanson KL et al. Population frequency of germline BRCA1/2 mutations. J Clin Oncol 2016; 34 (34): 4183–4185. doi: 10.1200/JCO.2016.67.0554.

2. Manickam K, Buchanan AH, Schwartz MLB et al. Exome sequencing–based screening for BRCA1/2 expected pathogenic variants among adult biobank participants. JAMA Netw Open 2018; 1 (5): e182140. doi: 10.1001/jamanetworkopen.2018.2140.

3. Kuchenbaecker KB, Hopper JL, Barnes DR et al. Risks of breast, ovarian, and contralateral breast cancer for BRCA1 and BRCA2 mutation carriers. JAMA 2017; 317 (23): 2402–2416. doi: 10.1001/jama.2017.7112.

4. Plon SE, Eccles DM, Easton D et al. Sequence variant classification and reporting: recommendations for improving the interpretation of cancer susceptibility genetic test results. Hum Mutat 2008; 29 (11): 1282–1291. doi: 10.1002/humu.20880.

5. Richards CS, Bale S, Bellissimo DB et al. ACMG recommendations for standards for interpretation and reporting of sequence variations: Revisions 2007. Genet Med 2008; 10 (4): 294–300. doi: 10.1097/GIM.0b013e31816b5cae.

6. Enigma consortium.org. Evidence-based Network for the Interpretation of Germline Mutant Alleles: ENIGMA criteria. [online]. Available from: https: //enigmaconsortium.org/library/general-documents/enigma-classification-criteria.

7. Foretova L, Machackova E, Palacova M et al. Recommended extension of indication criteria for genetic testing of BRCA1 and BRCA2 mutations in hereditary breast and ovarian cancer syndrome. Klin Onkol 2016; 29 (Suppl 1): 9–13. doi: 10.14735/amko2016S9.

8. Machackova E, Foretova L, Lukesova M et al. Spectrum and characterisation of BRCA1 and BRCA2 deleterious mutations in high-risk Czech patients with breast and/or ovarian cancer. BMC Cancer 2008; 8: 140. doi: 10.1186/1471-2407-8-140.

9. Vasickova P, Machackova E, Lukesova M et al. High occurrence of BRCA1 intragenic rearrangements in hereditary breast and ovarian cancer syndrome in the Czech Republic. BMC Med Genet 2007; 8: 32. doi: 10.1186/1471-2350-8-32.

10. Machackova E, Hazova J, Stahlova Hrabincova E at el. Retrospective NGS study in high-risk hereditary cancer patients at Masaryk Memorial Cancer Institute. Klin Onkol 2016; 29 (Suppl 1): 35–45. doi: 10.14735/amko2016S35.

11. Soukupova J, Zemankova P, Lhotova K et al. Validation of CZECANCA (CZEch CAncer paNel for Clinical Application) for targeted NGS-based analysis of hereditary cancer syndromes. PLoS One 2018; 13 (4): e0195761. doi: 10.1371/journal.pone.0195761.

12. HGVS.org. Human Genome Variation Society. [online]. Available from: http: //varnomen.hgvs.org/.

13. den Dunnen JT, Dalgleish R, Maglott DR et al. HGVS recommendations for the description of sequence variants: 2016 update. Hum Mutat 2016; 37 (6): 564–569. doi: 10.1002/humu.22981.

14. NNSplice. BDGP Splice Site Prediction by Neural Network, National Human Genome Research Institute. [online]. Available from: http: //www.fruitfly.org/seq_tools/splice.html.

15. NetGene2. Technical University of Denmark, DTU Bioinformatics. [online]. Available from: http: //www.cbs.dtu.dk/services/NetGene2/.

16. GeneSplicer. University of Maryland, CBCB: Centre for Bioinformatics and Computational Biology. [online]. Available from: http: //www.cbcb.umd.edu/software/GeneSplicer/gene_spl.shtml.

17. MaxEntScan. Massachusetts Institute of Technology, Burge Laboratory web server. [online]. Available from: http: //genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html.

18. SpliceSiteFinder-like. Aix-Marseille University, Bioinformatics and Genetics Team, Human Spicing Finder. [online]. Available from: http: //www.umd.be/HSF3/HSF.shtml.

19. Claes K, Poppe B, Machackova E et al. Differentiating pathogenic mutations from polymorphic alterations in the splice sites of BRCA1 and BRCA2. Genes Chromosomes Cancer 2003; 37 (3): 314–320. doi: 10.1002/gcc.10221.

20. Baert A, Machackova E, Coene I et al. Thorough in silico and in vitro cDNA analysis of 21 putative BRCA1 and BRCA2 splice variants and a complex tandem duplication in BRCA2 allowing the identification of activated cryptic splice donor sites in BRCA2 exon 11. Hum Mutat 2018; 39 (4): 515–526. doi: 10.1002/humu.23390.

21. Align-GVGD. Huntsman Cancer Institute University of Utah. [online]. Available from: http: //agvgd.hci.utah.edu.

22. Vallée MP, Di Sera TL, Nix DA et al. Adding in silico assessment of potential splice aberration to the integrated evaluation of BRCA gene unclassified variants. Hum Mutat 2016; 37 (7): 627–639. doi: 10.1002/humu.22973.

23. PROVEAN. Protein Variation Effect Analyser, J. Craig Venter Institute. [online]. Available from: http: //provean.jcvi.org/seq_submit.php.

24. Choi Y, Chan AP. PROVEAN web server: a tool to predict the functional effect of amino acid substitutions and indels. Bioinformatics 2015; 31 (16): 2745–2747. doi: 10.1093/bioinformatics/btv195.

25. Kopanos C, Tsiolkas V, Kouris A et al. VarSome: the human genomic variant search engine. Bioinformatics 2019; 35 (11): 1978–1980. doi: 10.1093/bioinformatics/bty897.

26. Meeks HD, Song H, Michailidou K et al. BRCA2 polymorphic stop codon K3326X and the risk of breast, prostate, and ovarian cancers. J Nat Cancer Inst 2016; 108 (2): djv315. doi: 10.1093/jnci/djv315.

27. Wappenschmidt B, Becker AA, Hauke J et al. Analysis of 30 putative BRCA1 splicing mutations in hereditary breast and ovarian cancer families identifies exonic splice site mutations that escape in silico prediction. PLoS One 2012; 7 (12): e50800. doi: 10.1371/journal.pone.0050800.

28. Byrjalsen A, Steffensen AY, Hansen TO et al. Classification of the spliceogenic BRCA1 c.4096+3A>G variant as likely benign based on cosegregation data and identification of a healthy homozygous carrier. Clin Case Rep 2017; 5 (6): 876–879. doi: 10.1002/ccr3.944.

29. Hoffman JD, Hallam SE, Venne VL, Lyon E et al. Implications of a novel cryptic splice site in the BRCA1 gene. Am J Med Genet 1998; 2; 80 (2): 140–144.

30. Sanz DJ, Acedo A, Infante M et al. A high proportion of DNA variants of BRCA1 and BRCA2 is associated with aberrant splicing in breast/ovarian cancer patients. Clin Cancer Res 2010; 16 (6): 1957–1967. doi: 10.1158/1078-0432.CCR-09-2564.

31. Spurdle AB, Whiley PJ, Thompson B et al. BRCA1 R1699Q variant displaying ambiguous functional abrogation confers intermediate breast and ovarian cancer risk. J Med Genet 2012; 49 (8): 525–532. doi: 10.1136/jmedgenet-2012-101037.

32. Thompson ME. BRCA1 16 years later: nuclear import and export processes. FEBS J. 2010; 277 (15): 3072–3078. doi: 10.1111/j.1742-4658.2010.07733.x.

33. Guidugli L, Shimelis H, Masica DL et al. Assessment of the clinical relevance of BRCA2 missense variants by functional and computational approaches. Am J Hum Genet 2018; 102 (2): 233–248. doi: 10.1016/j.ajhg.2017.12.013.

34. Lindor MN, Guidugli L, Wang X et al. A review of a multifactorial probability based model for classification of BRCA1 and BRCA2 variants of uncertain significance (VUS) Hum Mutat 2012 Jan; 33 (1): 8–21. doi: 10.1002/humu.21627.

35. Morris JR, Pangon L, Boutell C, et at. Genetic analysis of BRCA1 ubiquitin ligase activity and its relationship to breast cancer susceptibility. Hum Mol Genet 2006; 15 (4): 599–606. doi: 10.1093/hmg/ddi476.

36. Sarkar M, Magliery TJ. Re-engineering a split-GFP reassembly screen to examine RING-domain interactions between BARD1 and BRCA1 mutants observed in cancer patients. Mol Biosyst 2008; 4 (6): 599–605. doi: 10.1039/b802481b.

37. Machackova E, Damborsky J, Valik D et al. Novel germline BRCA1 and BRCA2 mutations in breast and breast/ovarian cancer families from the Czech Republic. Hum Mutat 2001; 18 (6): 545. doi: 10.1002/humu. 1232.

38. Tavtigian SV, Deffenbaugh AM, Yin L et al. Comprehensive statistical study of 452 BRCA1 missense substitutions with classification of eight recurrent substitutions as neutral. J Med Genet 2006; 43 (4): 295–305. doi: 10.1136/jmg.2005.033878.

39. Shimelis H, Mesman RLS, Von Nicolai C et al. BRCA2 hypomorphic missense variants confer moderate risks of breast cancer. Cancer Res 2017; 77 (11): 2789–2799. doi: 10.1158/0008-5472.CAN-16-2568.

40. Thomassen M, Blanco A, Montagna M et al. Characterization of BRCA1 and BRCA2 splicing variants: a collaborative report by ENIGMA consortium members. Breast Cancer Res Treat; 132 (3): 1009–1023. doi: 10.1007/s10549-011-1674-0.

41. Chen CF, Li S, Chen Y et al. The nuclear localization sequences of the BRCA1 protein interact with the importin-alpha subunit of the nuclear transport signal receptor. J Biol Chem 1996; 271 (51): 32863–32868. doi: 10.1074/jbc.271.51.32863.

42. Spain BH, Larson CJ, Shihabuddin LS et al. Truncated BRCA2 is cytoplasmic: implications for cancer-linked mutations. Proc Natl Acad Sci USA 1999; 96 (24): 13920–13925. doi: 10.1073/pnas.96.24.13920.

43. Tram E, Savas S, Ozcelik H. Missense variants of uncertain significance (VUS) altering the phosphorylation patterns of BRCA1 and BRCA2. PLoS 2013; 8 (5): e62468. doi: 10.1371/journal.pone.0062468.

44. Svojgr K, Sumerauer D, Puchmajerova A et al. Fanconi anemia with biallelic FANCD1/BRCA2 mutations – case report of a family with three affected children. Eur J Med Genet 2016; 59 (3): 152–157. doi: 10.1016/j.ejmg.2015.11.013.

45. Reid S, Renwick A, Seal S et al. Biallelic BRCA2 mutations are associated with multiple malignancies in childhood including familial Wilms tumour. J Med Genet 2005; 42 (2): 147–51. doi: 10.1136/jmg.2004.022673.

46. Offit K, Levran O, Mullaney B et al. Shared genetic susceptibility to breast cancer, brain tumors, and Fanconi anemia. J Natl Cancer Inst 2003; 95 (20): 1548–1551. doi: 10.1093/jnci/djg072.

47. Fackenthal JD, Yoshimatsu T, Zhang B et al. Naturally occurring BRCA2 alternative mRNA splicing events in clinically relevant samples. J Med Genet 2016; 53 (8): 548–558. doi: 10.1136/jmedgenet-2015-103570.

48. Thirthagiri E, Klarmann KD, Shukla AK et al. BRCA2 minor transcript lacking exons 4-7 supports viability in mice and may account for survival of humans with a pathogenic biallelic mutation. Hum Mol Genet 2016; 25 (10): 1934–1945. doi: 10.1093/hmg/ddw066.

49. Acedo A, Hernández-Moro C, Curiel-García A et al. Functional classification of BRCA2 DNA variants by splicing assays in a large minigene with 9 exons. Hum Mutat 2015; 36 (2): 210–221. doi: 10.1002/humu.22725.

50. Fraile-Bethencourt E, Díez-Gómez B, Velásquez-Zapata V et al. Functional classification of DNA variants by hybrid minigenes: identification of 30 spliceogenic variants of BRCA2 exons 17 and 18. PLoS Genet 2017; 13 (3): e1006691. doi: 10.1371/journal.pgen.1006691.

51. Fraile-Bethencourt E, Valenzuela-Palomo A, Díez-Gómez B et al. Identification of eight spliceogenic variants in BRCA2 exon 16 by minigene assays. Front Genet 2018; 9: 188. doi: 10.3389/fgene.2018.00188.

52. Colombo M, De Vecchi G, Caleca L et al. Comparative in vitro and in silico analyses of variants in splicing regions of BRCA1 and BRCA2 genes and characterization of novel pathogenic mutations. PLoS One 2013; 8 (2): e57173. doi: 10.1371/journal.pone.0057173.

53. Colombo M, Blok MJ, Whiley P et al. Comprehensive annotation of splice junctions supports pervasive alternative splicing at the BRCA1 locus: a report from the ENIGMA consortium. Hum Mol Genet 2014; 23 (14): 3666–3680. doi: 10.1093/hmg/ddu075.

54. Wong-Brown M, McPhillips M, Gleeson M et al. When is a mutation not a mutation: the case of the c.594-2A>C splice variant in a woman harbouring another BRCA1 mutation in trans. Hered Cancer Clin Pract 2016; 14: 6. doi: 10.1186/s13053-015-0045-y.

55. de la Hoya M, Soukarieh O, López-Perolio I et al. Combined genetic and splicing analysis of BRCA1 c.[594-2A>C; 641A>G] highlights the relevance of naturally occurring in-frame transcripts for developing disease gene variant classification algorithms. Hum Mol Genet 2016; 25 (11): 2256–2268. doi: 10.1093/hmg/ddw094.

56. Sawyer SL, Tian L, Kähkönen M et al. Biallelic mutations in BRCA1 cause a new Fanconi anemia subtype. Cancer Discov 2015; 5 (2): 135–142. doi: 10.1158/2159-8290.CD-14-1156.

57. Steffensen AY, Dandanel M, Jønson L et al. Functional characterization of BRCA1 gene variants by mini-gene splicing assay. Eur J Hum Genet 2014; 22 (12): 1362–1368. doi: 10.1038/ejhg.2014.40.

58.Whiley PJ, Guidugli L, Walker LC et al. Splicing and multifactorial analysis of intronic BRCA1 and BRCA2 sequence variants identifies clinically significant splicing aberrations up to 12 nucleotides from the intron/exon boundary. Hum Mutat 2011; 32 (6): 678–687. doi: 10.1002/humu.21495.

59. Easton DF, Deffenbaugh AM, Pruss D et al. A systematic genetic assessment of 1,433 sequence variants of unknown clinical significance in the BRCA1 and BRCA2 breast cancer-predisposition genes. Am J Hum Genet 2007; 81 (5): 873–883. doi: 10.1086/521032.

60. Walker LC, Whiley PJ, Couch FJ et al. Detection of splicing aberrations caused by BRCA1 and BRCA2 sequence variants encoding missense substitutions: implications for prediction of pathogenicity. Hum Mutat 2010; 31 (6): E1484–E1505. doi: 10.1002/humu.21267.

61. Rebbeck TR, Friebel TM, Friedman E et al. Mutational spectrum in a worldwide study of 29,700 families with BRCA1 or BRCA2 mutations. Hum Mutat 2018; 39 (5): 593–620. doi: 10.1002/humu.23406.

62. Menéndez M, Castellsagué J, Mirete M et al. Assessing the RNA effect of 26 DNA variants in the BRCA1 and BRCA2 genes. Breat Cancer Res Treat 2012; 132 (3): 979–992. doi: 10.1007/s10549-011-1661-5.

63. Sweet K, Senter L, Pilarski R et al. Characterization of BRCA1 ring finger variants of uncertain significance. Breast Cancer Res Treat 2010; 119 (3): 737–743. doi: 10.1007/s10549-009-0438-6.

64. Thouvenot P, Ben Yamin B, Fourrière L et al. Functional assessment of genetic variants with outcomes adapted to clinical decision-making. PLoS Genet 2016; 12 (6): e1006096. doi: 10.1371/journal.pgen.1006096.

65. Lee MS, Green R, Marsillac SM et al. Comprehensive analysis of missense variations in the BRCT domain of BRCA1 by structural and functional assays. Cancer Res 2010; 70 (12): 4880–4890. doi: 10.1158/0008-5472. CAN-09-4563.

66.Vallon-Christersson J, Cayanan C, Haraldsson K et al. Functional analysis of BRCA1 C-terminal missense mutations identified in breast and ovarian cancer families. Hum Mol Genet 2001; 10 (4): 353–360. doi: 10.1093/hmg/10.4.353.

67. Houdayer C, Caux-Moncoutier V, Krieger S 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): 1228–1238. doi: 10.1002/humu.22101.

68. Tavtigian SV, Byrnes GB, Goldgar DE et al. Classification of rare missense substitutions, using risk surfaces, with genetic-and molecular-epidemiology applications. Hum Mutat 2008; 29 (11): 1342–1354. doi: 10.1002/humu.20896.

69. Preisler-Adams S, Schönbuchner I, Fiebig B et al. Gross rearrangements in BRCA1 but not BRCA2 play a notable role in predisposition to breast and ovarian cancer in high-risk families of German origin. Cancer Genet Cytogenet 2006; 168 (1): 44–49. doi: 10.1016/j.cancergencyto.2005.07.005.

70. Judkins T, Hendrickson BC, Deffenbaugh AM et al. Application of embryonic lethal or other obvious phenotypes to characterize the clinical significance of genetic variants found in trans with known deleterious mutations. Cancer Res 2005; 65 (21): 10096–10103. doi: 10.1158/0008-5472.CAN-05-1241.

71. Liu J, Pan Y, Ma B et al. “Similarity trap” in protein-protein interactions could be carcinogenic: simulations of p53 core domain complexed with 53BP1 and BRCA1 BRCT domains. Structure 2006; 14 (12): 1811–1821. doi: 10.1016/j.str.2006.10.009.

72. Théry JC, Krieger S, Gaildrat P et al. Contribution of bioinformatics predictions and functional splicing assays to the interpretation of unclassified variants of the BRCA genes. Eur J Hum Genet 2011; 19 (10): 1052–1058. doi: 10.1038/ejhg.2011.100.

73. Bonnet C, Krieger S, Vezain M et al. 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 2008; 45 (7): 438–446. doi: 10.1136/jmg.2007.056895.

74. Bonatti F, Pepe C, Tancredi M et al. RNA-based analysis of BRCA1 and BRCA2 gene alterations. Cancer Genet Cytogenet 2006; 170 (2): 93–101. doi: 10.1016/j.cancergencyto.2006.05.005.

75. Heramb C, Wangenstee T, Grindedal EM et al. BRCA1 and BRCA2 mutation spectrum – an update on mutation distribution in a large cancer genetics clinic in Norway. Hered Cancer Clin Pract 2018; 16: 3. doi: 10.1186/s13053-017-0085-6.

76. Farrugia DJ, Agarwal MK, Pankratz VS, at al. Functional assays for classification of BRCA2 variants of uncertain significance. Cancer Res 2008; 68 (9): 3523–3531. doi: 10.1158/0008-5472.CAN-07-1587.

77. Biswas K, Das R, Alter BP et al. A comprehensive functional characterization of BRCA2 variants associated with Fanconi anemia using mouse ES cell-based assay. Blood 2011; 118 (9): 2430–2442. doi: 10.1182/blood-2010-12-324541.

78. Pölsler L, Fiegl H, Wimmer K et al. High prevalence of BRCA1 stop mutation c.4183C>T in the Tyrolean population: implications for genetic testing. Eur J Hum Genet 2016; 24 (2): 258–262. doi: 10.1038/ejhg.2015.108.

79. Karchin R, Agarwal M, Andrej Sali A et al. Classifying variants of undetermined significance in BRCA2 with protein likelihood ratios. Cancer Inform 2008; 6: 203–216.

80. Guidugli L, Carreira A, Caputo SM et al. Functional assays for analysis of variants of uncertain significance in BRCA2. Hum Mutat 2014; 35 (2): 151–164. doi: 10.1002/humu.22478.

81. Mesman RL, Calléja FM, Hendriks G et al. The functional impact of variants of uncertain significance in BRCA2. Genet Med 2019; 21 (2): 293–302. doi: 10.1038/s41436-018-0052-2.

82. Biswas K, Das R, Eggington JM et al. Functional evaluation of BRCA2 variants mapping to the PALB2-binding and C-terminal DNA-binding domains using a mouse ES cell-based assay. Hum Mol Genet 2012; 21 (18): 3993–4006. doi: 10.1093/hmg/dds222.

83. Muller D, Rouleau E, Schultz I et al. An entire exon 3 germ-line rearrangement in the BRCA2 gene: pathogenic relevance of exon 3 deletion in breast cancer predisposition. BMC Med Genet 2011; 12: 121. doi: 10.1186/1471-2350-12-121.

84. Colombo M, Lòpez-Perolio I, Meeks HD et al. The BRCA2 c.687T > A variant is not pathogenic: a model for clinical calibration of spliceogenicity. Hum Mutat 2018; 39 (5): 729–741. doi: 10.1002/humu.23411.

85. Joosse SA, Brandwijk KI, Devilee P et al. Prediction of BRCA2-association in hereditary breast carcinomas using array-CGH. Breast Cancer Res Treat 2012; 132 (2): 379–389. doi: 10.1007/s10549-010-1016-7.