Clinical, splicing, and functional analysis to classify BRCA2 exon 3 variants: Application of a points‐based ACMG/AMP approach
Human Mutation, vol. 43, pp. 1921–1944
Abstract
Skipping of BRCA2 exon 3 (∆E3) is a naturally occurring splicing event, complicating clinical classification of variants that may alter ∆E3 expression. This study used multiple evidence types to assess pathogenicity of 85 variants in/near BRCA2 exon 3. Bioinformatically predicted spliceogenic variants underwent mRNA splicing analysis using minigenes and/or patient samples. ∆E3 was measured using quantitative analysis. A mouse embryonic stem cell (mESC) based assay was used to determine the impact of 18 variants on mRNA splicing and protein function. For each variant, population frequency, bioinformatic predictions, clinical data, and existing mRNA splicing and functional results were collated. Variant class was assigned using a gene-specific adaptation of ACMG/AMP guidelines, following a recently proposed points-based system. mRNA and mESC analysis combined identified six variants with transcript and/or functional profiles interpreted as loss of function. Cryptic splice site use for acceptor site variants generated a transcript encoding a shorter protein that retains activity. Overall, 69/85 (81%) variants were classified using the points-based approach. Our analysis shows the value of applying gene-specific ACMG/AMP guidelines using a points-based approach and highlights the consideration of cryptic splice site usage to appropriately assign PVS1 code strength.
Authors 49
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Mads Thomassen corresponding
Affiliation as printed
Department of Clinical Genetics Odense University Hospital Odence C Denmark
Department of Clinical Genetics, Odense University Hospital, Odence C, Denmark
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Romy L.S. Mesman Aachen Department of Human Genetics Leiden University Medical Center Leiden The Netherlands Department of Human Genetics
Leiden University Medical Center
Affiliation as printed
Department of Human Genetics Leiden University Medical Center Leiden the Netherlands
Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands
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Copenhagen University Hospital · Rigshospitalet
Affiliation as printed
Department of Clinical Genetics, Rigshospitalet Copenhagen University Hospital Copenhagen Denmark
Department of Clinical Genetics, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark
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Institut d'Investigació Biomédica de Bellvitge · Institut Català d'Oncologia · Centro de Investigación Biomédica en Red de Cáncer
Affiliation as printed
Hereditary Cancer Program Catalan Institute of Oncology, ONCOBELL‐IDIBELL‐IDTP, CIBERONC Hospitalet de Llobregat Spain
Hereditary Cancer Program, Catalan Institute of Oncology, ONCOBELL-IDIBELL-IDTP, CIBERONC, Hospitalet de Llobregat, Spain
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Copenhagen University Hospital · Rigshospitalet
Affiliation as printed
Center for Genomic Medicine, Rigshospitalet Copenhagen University Hospital Copenhagen Denmark
Center for Genomic Medicine, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark
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Hospital Clínico San Carlos · Instituto de Investigación Sanitaria del Hospital Clínico San Carlos · Centro de Investigación Biomédica en Red de Cáncer
Affiliation as printed
Molecular Oncology Laboratory, CIBERONC, Hospital Clinico San Carlos, IdISSC (Instituto de Investigación Sanitaria del Hospital Clínico San Carlos) Madrid Spain
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QIMR Berghofer Medical Research Institute
Affiliation as printed
Department of Genetics and Computational Biology QIMR Berghofer Medical Research Institute Brisbane Queensland Australia
Department of Genetics and Computational Biology, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia
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Affiliation as printed
Division of Oncology, Department of Clinical Sciences Lund Lund University Lund Sweden
Division of Oncology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden
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QIMR Berghofer Medical Research Institute
Affiliation as printed
Department of Genetics and Computational Biology QIMR Berghofer Medical Research Institute Brisbane Queensland Australia
Department of Genetics and Computational Biology, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia
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Aalborg University Hospital · Aalborg University
Affiliation as printed
Clinical Cancer Research Center Aalborg University Hospital Aalborg Denmark
Department of Clinical Medicine Aalborg University Aalborg Denmark
Molecular Diagnostics, Aalborg University Hospital Aalborg Denmark
Clinical Cancer Research Center, Aalborg University Hospital, Aalborg, Denmark
Department of Clinical Medicine, Aalborg University, Aalborg, Denmark
Molecular Diagnostics, Aalborg University Hospital, Aalborg, Denmark
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University of Malaya · Cancer Research Malaysia
Affiliation as printed
Breast Cancer Research Programme Cancer Research Malaysia Subang Jaya Selangor Malaysia
Department of Surgery, Faculty of Medicine University of Malaya Kuala Lumpur Malaysia
Breast Cancer Research Programme, Cancer Research Malaysia, Subang Jaya, Selangor, Malaysia
Department of Surgery, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
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Affiliation as printed
Department of Clinical Genetics Odense University Hospital Odence C Denmark
Department of Clinical Genetics, Odense University Hospital, Odence C, Denmark
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Affiliation as printed
Department of Tumour Biology The Norwegian Radium Hospital, Oslo University Hospital Oslo Norway
Department of Tumour Biology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
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Affiliation as printed
Division of Oncology, Department of Clinical Sciences Lund Lund University Lund Sweden
Division of Oncology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden
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Affiliation as printed
Department of Clinical Genetics Aarhus University Hospital Aarhus N Denmark
Department of Clinical Genetics, Aarhus University Hospital, Aarhus N, Denmark
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Affiliation as printed
Department of Molecular Medicine Aarhus University Hospital Aarhus Denmark
Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark
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Comprehensive Cancer Center Vienna · Medical University of Vienna
Affiliation as printed
Department of OB/GYN and Comprehensive Cancer Center Medical University of Vienna Vienna Austria
Department of OB/GYN and Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria
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Comprehensive Cancer Center Vienna · Medical University of Vienna
Affiliation as printed
Department of OB/GYN and Comprehensive Cancer Center Medical University of Vienna Vienna Austria
Department of OB/GYN and Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria
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Complejo Hospitalario Universitario de Santiago · Centre for Biomedical Network Research on Rare Diseases · Fundación Pública Galega de Medicina Xenómica · Instituto de Investigación Sanitaria de Santiago · Servicio Gallego de Salud
Affiliation as printed
Centro de Investigación en Red de Enfermedades Raras (CIBERER) Madrid Spain
Fundación Pública Galega de Medicina Xenómica Santiago de Compostela Spain
Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS), Complejo Hospitalario Universitario de Santiago, SERGAS Santiago de Compostela Spain
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Maastricht University Medical Centre · Maastricht University
Affiliation as printed
Department of Clinical Genetics Maastricht University Medical Center Maastricht the Netherlands
Department of Clinical Genetics, Maastricht University Medical Center, Maastricht, the Netherlands
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University of Southern Denmark
Affiliation as printed
Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences University of Southern Denmark Odense Denmark
Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense, Denmark
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University of Utah · Huntsman Cancer Institute
Affiliation as printed
Department of Dermatology Huntsman Cancer Institute, University of Utah School of Medicine Salt Lake City Utah USA
Department of Dermatology, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, Utah, USA
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QIMR Berghofer Medical Research Institute
Affiliation as printed
Department of Genetics and Computational Biology QIMR Berghofer Medical Research Institute Brisbane Queensland Australia
Department of Genetics and Computational Biology, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia
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Ambry Genetics (United States)
Affiliation as printed
Ambry Genetics Aliso Viejo California USA
Ambry Genetics, Aliso Viejo, California, USA
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Ambry Genetics (United States)
Affiliation as printed
Ambry Genetics Aliso Viejo California USA
Ambry Genetics, Aliso Viejo, California, USA
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Ambry Genetics (United States)
Affiliation as printed
Ambry Genetics Aliso Viejo California USA
Ambry Genetics, Aliso Viejo, California, USA
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Ambry Genetics (United States)
Affiliation as printed
Ambry Genetics Aliso Viejo California USA
Ambry Genetics, Aliso Viejo, California, USA
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Ambry Genetics (United States)
Affiliation as printed
Ambry Genetics Aliso Viejo California USA
Ambry Genetics, Aliso Viejo, California, USA
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Ambry Genetics (United States)
Affiliation as printed
Ambry Genetics Aliso Viejo California USA
Ambry Genetics, Aliso Viejo, California, USA
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Ambry Genetics (United States)
Affiliation as printed
Ambry Genetics Aliso Viejo California USA
Ambry Genetics, Aliso Viejo, California, USA
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Fabienne M.G.R. Calléja Aachen Department of Human Genetics Leiden University Medical Center Leiden The Netherlands Department of Human Genetics
Leiden University Medical Center
Affiliation as printed
Department of Human Genetics Leiden University Medical Center Leiden the Netherlands
Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands
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Affiliation as printed
Hereditary Cancer Clinic, Nelune Comprehensive Cancer Care Centre Sydney New South Wales Australia
Hereditary Cancer Clinic, Nelune Comprehensive Cancer Care Centre, Sydney, New South Wales, Australia
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The University of Melbourne · Peter MacCallum Cancer Centre
Affiliation as printed
Parkville Familial Cancer Centre, Peter MacCallum Cancer Center Melbourne Victoria Australia
Sir Peter MacCallum Department of Oncology The University of Melbourne Melbourne Victoria Australia
Parkville Familial Cancer Centre, Peter MacCallum Cancer Center, Melbourne, Victoria, Australia
Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, Victoria, Australia
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Affiliation as printed
Human Development and Health, Faculty of Medicine University of Southampton Southampton UK
Human Development and Health, Faculty of Medicine, University of Southampton, Southampton, UK
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Affiliation as printed
Middlesex Health Shoreline Cancer Center Westbrook Connecticut USA
Middlesex Health Shoreline Cancer Center, Westbrook, Connecticut, USA
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Fondazione IRCCS Istituto Nazionale dei Tumori
Affiliation as printed
Unit of Molecular Bases of Genetic Risk and Genetic Testing, Department of Research Fondazione IRCCS Istituto Nazionale dei Tumori (INT) Milan Italy
Unit of Molecular Bases of Genetic Risk and Genetic Testing, Department of Research, Fondazione IRCCS Istituto Nazionale dei Tumori (INT), Milan, Italy
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University of Utah · Huntsman Cancer Institute
Affiliation as printed
Department of Dermatology Huntsman Cancer Institute, University of Utah School of Medicine Salt Lake City Utah USA
Department of Dermatology, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, Utah, USA
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Affiliation as printed
Department of Pathology and Biomedical Science University of Otago Christchurch New Zealand
Department of Pathology and Biomedical Science, University of Otago, Christchurch, New Zealand
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Affiliation as printed
Institute for Medical Informatics, Statistics and Epidemiology University of Leipzig Leipzig Germany
Institute for Medical Informatics, Statistics and Epidemiology, University of Leipzig, Leipzig, Germany
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Affiliation as printed
Centre for Medical Genetics Ghent University Gent Belgium
Centre for Medical Genetics, Ghent University, Gent, Belgium
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Masaryk Memorial Cancer Institute
Affiliation as printed
Department of Cancer Epidemiology and Genetics Masaryk Memorial Cancer Institute Brno Czech Republic
Department of Cancer Epidemiology and Genetics, Masaryk Memorial Cancer Institute, Brno, Czech Republic
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Affiliation as printed
Human Development and Health, Faculty of Medicine University of Southampton Southampton UK
Human Development and Health, Faculty of Medicine, University of Southampton, Southampton, UK
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Centro di Riferimento Oncologico
Affiliation as printed
Division of Functional Onco‐genomics and Genetics Centro di Riferimento Oncologico di Aviano (CRO), IRCCS Aviano Italy
Division of Functional Onco-genomics and Genetics, Centro di Riferimento Oncologico di Aviano (CRO), IRCCS, Aviano, Italy
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University of Cologne · University Hospital Cologne
Affiliation as printed
Center for Familial Breast and Ovarian Cancer, Faculty of Medicine and University Hospital Cologne University of Cologne Cologne Germany
Center for Integrated Oncology (CIO), Faculty of Medicine and University Hospital Cologne University of Cologne Cologne Germany
Center for Familial Breast and Ovarian Cancer, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
Center for Integrated Oncology (CIO), Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
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Institut d'Investigació Biomédica de Bellvitge · Institut Català d'Oncologia · Centro de Investigación Biomédica en Red de Cáncer
Affiliation as printed
Hereditary Cancer Program Catalan Institute of Oncology, ONCOBELL‐IDIBELL‐IDTP, CIBERONC Hospitalet de Llobregat Spain
Hereditary Cancer Program, Catalan Institute of Oncology, ONCOBELL-IDIBELL-IDTP, CIBERONC, Hospitalet de Llobregat, Spain
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Complejo Hospitalario Universitario de Santiago · Centre for Biomedical Network Research on Rare Diseases · Fundación Pública Galega de Medicina Xenómica · Instituto de Investigación Sanitaria de Santiago · Servicio Gallego de Salud
Affiliation as printed
Centro de Investigación en Red de Enfermedades Raras (CIBERER) Madrid Spain
Fundación Pública Galega de Medicina Xenómica Santiago de Compostela Spain
Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS), Complejo Hospitalario Universitario de Santiago, SERGAS Santiago de Compostela Spain
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Maaike P.G. Vreeswijk Aachen Department of Human Genetics Leiden University Medical Center Leiden The Netherlands Department of Human Genetics
Leiden University Medical Center
Affiliation as printed
Department of Human Genetics Leiden University Medical Center Leiden the Netherlands
Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands
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Hospital Clínico San Carlos · Instituto de Investigación Sanitaria del Hospital Clínico San Carlos · Centro de Investigación Biomédica en Red de Cáncer
Affiliation as printed
Molecular Oncology Laboratory, CIBERONC, Hospital Clinico San Carlos, IdISSC (Instituto de Investigación Sanitaria del Hospital Clínico San Carlos) Madrid Spain
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Amanda B. Spurdle corresponding
QIMR Berghofer Medical Research Institute
Affiliation as printed
Department of Genetics and Computational Biology QIMR Berghofer Medical Research Institute Brisbane Queensland Australia
Department of Genetics and Computational Biology, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia
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