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Hum Mutat. 2018 May;39(5):729-741. doi: 10.1002/humu.23411. Epub 2018 Apr 6.

The BRCA2 c.68-7T > A variant is not pathogenic: A model for clinical calibration of spliceogenicity.

Colombo M1, Lòpez-Perolio I2, Meeks HD3, Caleca L1, Parsons MT4, Li H3, De Vecchi G1, Tudini E4, Foglia C1, Mondini P1, Manoukian S5, Behar R2, Garcia EBG6, Meindl A7, Montagna M8, Niederacher D9, Schmidt AY10, Varesco L11, Wappenschmidt B12,13, Bolla MK14, Dennis J14, Michailidou K14,15, Wang Q14, Aittomäki K16, Andrulis IL17,18, Anton-Culver H19, Arndt V20, Beckmann MW21, Beeghly-Fadel A22, Benitez J23,24, Boeckx B25,26, Bogdanova NV27,28,29, Bojesen SE30,31,32, Bonanni B33, Brauch H34,35,36, Brenner H20,36,37, Burwinkel B38,39, Chang-Claude J40,41, Conroy DM42, Couch FJ43, Cox A44, Cross SS45, Czene K46, Devilee P47,48, Dörk T28, Eriksson M46, Fasching PA21,49, Figueroa J50,51, Fletcher O52, Flyger H53, Gabrielson M46, García-Closas M51, Giles GG54,55, González-Neira A23, Guénel P56, Haiman CA57, Hall P46, Hamann U58, Hartman M59,60, Hauke J12,13,61, Hollestelle A62, Hopper JL55, Jakubowska A63, Jung A40, Kosma VM64,65,66, Lambrechts D25,26, Le Marchand L67, Lindblom A68, Lubinski J63, Mannermaa A64,65,66, Margolin S69, Miao H59, Milne RL54,55, Neuhausen SL70, Nevanlinna H71, Olson JE72, Peterlongo P73, Peto J74, Pylkäs K75,76, Sawyer EJ77, Schmidt MK78,79, Schmutzler RK12,13,61, Schneeweiss A38,80, Schoemaker MJ81, See MH82, Southey MC83, Swerdlow A81,84, Teo SH82,85, Toland AE86, Tomlinson I87, Truong T56, van Asperen CJ88, van den Ouweland AMW89, van der Kolk LE90, Winqvist R75,76, Yannoukakos D91, Zheng W22; kConFab/AOCS Investigators92, Dunning AM42, Easton DF14,42, Henderson A93, Hogervorst FBL90, Izatt L94, Offitt K95, Side LE96, van Rensburg EJ97, Embrace S98, Hebon S99, McGuffog L14, Antoniou AC14, Chenevix-Trench G4, Spurdle AB4, Goldgar DE3, Hoya M2, Radice P1.

Author information

1
Unit of Molecular Bases of Genetic Risk and Genetic Testing, Department of Research, Fondazione IRCCS (Istituto di Ricovero e Cura a Carattere Scientifico) Istituto Nazionale dei Tumori (INT), Milan, Italy.
2
Molecular Oncology Laboratory CIBERONC, Hospital Clinico San Carlos, IdISSC (Instituto de Investigación Sanitaria del Hospital Clínico San Carlos), Madrid, Spain.
3
Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah.
4
Department of Genetics and Computational Biology, QIMR Berghofer Medical Research Institute, Brisbane, QLD 4006, Australia.
5
Unit of Medical Genetics, Department of Medical Oncology and Hematology, Fondazione IRCCS (Istituto di Ricovero e Cura a Carattere Scientifico) Istituto Nazionale dei Tumori (INT), Milan, Italy.
6
Department of Clinical Genetics and GROW, School for Oncology and Developmental Biology, MUMC, Maastricht, The Netherlands.
7
Department of Obstetrics and Gynecology, University Hospital, LMU Munich, Germany.
8
Immunology and Molecular Oncology Unit, Veneto Institute of Oncology IOV - IRCCS, Padua, Italy.
9
Department of Gynaecology and Obstetrics, University Hospital Düsseldorf, Heinrich-Heine University, Duesseldorf, Germany.
10
Center for Genomic Medicine, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark.
11
Hereditary Cancer Unit, IRCCS AOU San Martino -IST, Genova, Italy.
12
Center for Hereditary Breast and Ovarian Cancer, University Hospital of Cologne, Cologne, Germany.
13
Center for Integrated Oncology (CIO), Medical Faculty, University Hospital of Cologne, Cologne, Germany.
14
Centre for Cancer Genetic Epidemiology, Department of Public Health and Primary Care, University of Cambridge, Cambridge, UK.
15
Department of Electron Microscopy/Molecular Pathology, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus.
16
Department of Clinical Genetics, Helsinki University Hospital, University of Helsinki, Helsinki, Finland.
17
Fred A. Litwin Center for Cancer Genetics, Lunenfeld-Tanenbaum Research Institute of Mount Sinai Hospital, Toronto, Ontario.
18
Department of Molecular Genetics, University of Toronto, Toronto, Canada.
19
Department of Epidemiology, University of California Irvine, Irvine, California.
20
Division of Clinical Epidemiology and Aging Research, German Cancer Research Center (DKFZ), Heidelberg, Germany.
21
Department of Gynaecology and Obstetrics, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nuremberg, Comprehensive Cancer Center Erlangen-EMN, Erlangen, Germany.
22
Division of Epidemiology, Department of Medicine, Vanderbilt Epidemiology Center, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee.
23
Human Cancer Genetics Program, Spanish National Cancer Research Centre, Madrid, Spain.
24
Centro de Investigación en Red de Enfermedades Raras (CIBERER), Valencia, Spain.
25
VIB Center for Cancer Biology, VIB, Leuven, Belgium.
26
Laboratory for Translational Genetics, Department of Human Genetics, University of Leuven, Leuven, Belgium.
27
Department of Radiation Oncology, Hannover Medical School, Hannover, Germany.
28
Gynaecology Research Unit, Hannover Medical School, Hannover, Germany.
29
N.N. Alexandrov Research Institute of Oncology and Medical Radiology, Minsk, Belarus.
30
Copenhagen General Population Study, Herlevand Gentofte Hospital, Copenhagen University Hospital, Herlev, Denmark.
31
Department of Clinical Biochemistry, Herlev and Gentofte Hospital, Copenhagen University Hospital, Herlev, Denmark.
32
Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
33
Division of Cancer Prevention and Genetics, Istituto Europeo di Oncologia, Milan, Italy.
34
Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany.
35
University of Tübingen, Tübingen, Germany.
36
German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany.
37
Division of Preventive Oncology, German Cancer Research Center (DKFZ) and National Center for Tumor Diseases (NCT), Heidelberg, Germany.
38
Department of Obstetrics and Gynecology, University of Heidelberg, Heidelberg, Germany.
39
Molecular Epidemiology Group, C080, German Cancer Research Center (DKFZ), Heidelberg, Germany.
40
Division of Cancer Epidemiology, German Cancer Research Center (DKFZ), Heidelberg, Germany.
41
Research Group Genetic Cancer Epidemiology, University Cancer Center Hamburg (UCCH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
42
Centre for Cancer Genetic Epidemiology, Department of Oncology, University of Cambridge, Cambridge, UK.
43
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, New York.
44
Sheffield Institute for Nucleic Acids (SInFoNiA), Department of Oncology and Metabolism, University of Sheffield, Sheffield, UK.
45
Academic Unit of Pathology, Department of Neuroscience, University of Sheffield, Sheffield, UK.
46
Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.
47
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
48
Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.
49
David Geffen School of Medicine, Department of Medicine Division of Hematology and Oncology, University of California at Los Angeles, Los Angeles, California.
50
Usher Institute of Population Health Sciences and Informatics, The University of Edinburgh Medical School, Edinburgh, UK.
51
Division of Cancer Epidemiology and Genetics, National Cancer Institute, Rockville, Maryland.
52
The Breast Cancer Now Toby Robins Research Centre, The Institute of Cancer Research, London, UK.
53
Department of Breast Surgery, Herlev and Gentofte Hospital, Copenhagen University Hospital, Herlev, Denmark.
54
Cancer Epidemiology & Intelligence Division, Cancer Council Victoria, Melbourne, Australia.
55
Centre for Epidemiology and Biostatistics, Melbourne School of Population and Global health, The University of Melbourne, Melbourne, Australia.
56
Cancer & Environment Group, Center for Research in Epidemiology and Population Health (CESP), INSERM, University Paris-Sud, University Paris-Saclay, Villejuif, France.
57
Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California.
58
Molecular Genetics of Breast Cancer, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany.
59
Saw Swee Hock School of Public Health, National University of Singapore, Singapore, Singapore.
60
Department of Surgery, National University Health System, Singapore, Singapore.
61
Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.
62
Department of Medical Oncology, Family Cancer Clinic, Erasmus MC Cancer Institute, Rotterdam, The Netherlands.
63
Department of Genetics and Pathology, Pomeranian Medical University, Szczecin, Poland.
64
Translational Cancer Research Area, University of Eastern Finland, Kuopio, Finland.
65
Institute of Clinical Medicine, Pathology and Forensic Medicine, University of Eastern Finland, Kuopio, Finland.
66
Imaging Center, Department of Clinical Pathology, Kuopio University Hospital, Kuopio, Finland.
67
Epidemiology Program, University of Hawaii Cancer Center, Honolulu, Hawaii.
68
Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden.
69
Department of Clinical Science and Education Södersjukhuset, Karolinska Institutet, Stockholm, Sweden.
70
Department of Population Sciences, Beckman Research Institute of City of Hope, Duarte, California.
71
Department of Obstetrics and Gynecology, Helsinki University Hospital, University of Helsinki, Helsinki, Finland.
72
Department of Health Sciences Research, Mayo Clinic, Rochester, New York.
73
IFOM, The FIRC (Italian Foundation for Cancer Research) Institute of Molecular Oncology, Milan, Italy.
74
Department of Non-Communicable Disease Epidemiology, London School of Hygiene and Tropical Medicine, London, UK.
75
Laboratory of Cancer Genetics and Tumor Biology, Cancer and Translational Medicine Research Unit, Biocenter Oulu, University of Oulu, Oulu, Finland.
76
Laboratory of Cancer Genetics and Tumor Biology, Northern Finland Laboratory Centre Oulu, Oulu, Finland.
77
Research Oncology, Guy's Hospital, King's College London, London, UK.
78
Division of Molecular Pathology, The Netherlands Cancer Institute - Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands.
79
Division of Psychosocial Research and Epidemiology, The Netherlands Cancer Institute - Antoni van Leeuwenhoek hospital, Amsterdam, The Netherlands.
80
National Center for Tumor Diseases, University of Heidelberg, Heidelberg, Germany.
81
Division of Genetics and Epidemiology, The Institute of Cancer Research, London, UK.
82
Breast Cancer Research Unit, Cancer Research Institute, University Malaya Medical Centre, Kuala Lumpur, Malaysia.
83
Department of Pathology, The University of Melbourne, Melbourne, Australia.
84
Division of Breast Cancer Research, The Institute of Cancer Research, London, UK.
85
Cancer Research Malaysia, Subang Jaya, Selangor, Malaysia.
86
Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio.
87
Wellcome Trust Centre for Human Genetics and Oxford NIHR Biomedical Research Centre, University of Oxford, Oxford, UK.
88
Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands.
89
Department of Clinical Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands.
90
Family Cancer Clinic, The Netherlands Cancer Institute - Antoni van Leeuwenhoek hospital, Amsterdam, The Netherlands.
91
Molecular Diagnostics Laboratory, INRASTES, National Centre for Scientific Research "Demokritos", Athens, Greece.
92
Peter MacCallum Cancer Center, Melbourne, Australia.
93
Institute of Genetic Medicine, Centre for Life, Newcastle Upon Tyne Hospitals NHS Trust, Newcastle upon Tyne, UK.
94
Clinical Genetics, Guy's and St. Thomas' NHS Foundation Trust, London, UK.
95
Clinical Genetics Research Laboratory, Dept. of Medicine, Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, New York.
96
Wessex Clinical Genetics Service, Mailpoint 627, Princess Anne Hospital, Coxford Road, Southampton, SO16 5YA.
97
Cancer Genetics Laboratory, Department of Genetics, University of Pretoria, Pretoria, South Africa.
98
Centre for Cancer Genetic Epidemiology, Department of Public Health and Primary Care, University of Cambridge, Strangeways Research Laboratory, Worts Causeway, Cambridge, UK.
99
The Hereditary Breast and Ovarian Cancer Research Group Netherlands (HEBON), Coordinating center: Netherlands Cancer Institute, Amsterdam, The Netherlands.

Abstract

Although the spliceogenic nature of the BRCA2 c.68-7T > A variant has been demonstrated, its association with cancer risk remains controversial. In this study, we accurately quantified by real-time PCR and digital PCR (dPCR), the BRCA2 isoforms retaining or missing exon 3. In addition, the combined odds ratio for causality of the variant was estimated using genetic and clinical data, and its associated cancer risk was estimated by case-control analysis in 83,636 individuals. Co-occurrence in trans with pathogenic BRCA2 variants was assessed in 5,382 families. Exon 3 exclusion rate was 4.5-fold higher in variant carriers (13%) than controls (3%), indicating an exclusion rate for the c.68-7T > A allele of approximately 20%. The posterior probability of pathogenicity was 7.44 × 10-115 . There was neither evidence for increased risk of breast cancer (OR 1.03; 95% CI 0.86-1.24) nor for a deleterious effect of the variant when co-occurring with pathogenic variants. Our data provide for the first time robust evidence of the nonpathogenicity of the BRCA2 c.68-7T > A. Genetic and quantitative transcript analyses together inform the threshold for the ratio between functional and altered BRCA2 isoforms compatible with normal cell function. These findings might be exploited to assess the relevance for cancer risk of other BRCA2 spliceogenic variants.

KEYWORDS:

BRCA2; digital PCR; multifactorial likelihood analysis; quantitative real-time PCR; spliceogenic variants

PMID:
29460995
PMCID:
PMC5947288
DOI:
10.1002/humu.23411
[Indexed for MEDLINE]
Free PMC Article

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