The widespread use of next generation sequencing for clinical testing is detecting an escalating number of variants in noncoding regions of the genome. The clinical significance of the majority of these variants is currently unknown, which presents a significant clinical challenge. We have screened over 6,000 early-onset and/or familial breast cancer (BC) cases collected by the ENIGMA consortium for sequence variants in the 5' noncoding regions of BC susceptibility genes BRCA1 and BRCA2, and identified 141 rare variants with global minor allele frequency < 0.01, 76 of which have not been reported previously. Bioinformatic analysis identified a set of 21 variants most likely to impact transcriptional regulation, and luciferase reporter assays detected altered promoter activity for four of these variants. Electrophoretic mobility shift assays demonstrated that three of these altered the binding of proteins to the respective BRCA1 or BRCA2 promoter regions, including NFYA binding to BRCA1:c.-287C>T and PAX5 binding to BRCA2:c.-296C>T. Clinical classification of variants affecting promoter activity, using existing prediction models, found no evidence to suggest that these variants confer a high risk of disease. Further studies are required to determine if such variation may be associated with a moderate or low risk of BC.

A C Camargo Cancer Center São Paulo Brazil

Area of Clinical and Molecular Genetics University Hospital Vall d'Hebron Barcelona Spain

Center for Genomic Medicine Copenhagen University Hospital Rigshospitalet Copenhagen Denmark

Center for Medical Genetics Ghent University Hospital and Cancer Research Institute Ghent Ghent University Ghent Belgium

CytoGnomix Inc London Ontario Canada

Department of Clinical Genetics Maastricht University Medical Centre Maastricht The Netherlands

Department of Genetics and Computational Biology QIMR Berghofer Medical Research Institute Brisbane Australia

Department of oncology Center for Translational Oncology Cancer Institute of the State of São Paulo ICESP São Paulo Brazil

Division of Cancer Prevention and Genetics Istituto Europeo di Oncologia Milan Italy

Fundación Pública Galega de Medicina Xenómica SERGAS Grupo de Medicina Xenómica USC CIBERER IDIS Santiago de Compostela Spain

Gustave Roussy Villejuif France

Huntsman Cancer Institute University of Utah Salt Lake City Utah

IFOM Fondazione Istituto FIRC di Oncologia Molecolare Milan Italy

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

Oncogenetics Group Vall d'Hebron Institute of Oncology Barcelona Spain

School of Chemistry and Molecular Biosciences University of Queensland Brisbane Australia

Section of Molecular Genetics Department of Laboratory Medicine University Hospital of Pisa Pisa Italy

Service de Génétique Department de Biologie des Tumeurs Institut Curie Paris France

Unit of Medical Genetics Department of Medical Oncology and Hematology Fondazione IRCCS Milan Italy

Unit of Molecular Bases of Genetic Risk and Genetic Testing Department of Research Fondazione IRCCS Istituto Nazionale dei Tumori di Milano Milan Italy

University of Western Ontario Department of Biochemistry Schulich School of Medicine and Dentistry London Ontario Canada

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