PURPOSE: Germline pathogenic variants in CHEK2 confer moderately elevated breast cancer risk (odds ratio, OR ∼ 2.5), qualifying carriers for enhanced breast cancer screening. Besides pathogenic variants, dozens of missense CHEK2 variants of uncertain significance (VUS) have been identified, hampering the clinical utility of germline genetic testing (GGT). EXPERIMENTAL DESIGN: We collected 460 CHEK2 missense VUS identified by the ENIGMA consortium in 15 countries. Their functional characterization was performed using CHEK2-complementation assays quantifying KAP1 phosphorylation and CHK2 autophosphorylation in human RPE1-CHEK2-knockout cells. Concordant results in both functional assays were used to categorize CHEK2 VUS from 12 ENIGMA case-control datasets, including 73,048 female patients with breast cancer and 88,658 ethnicity-matched controls. RESULTS: A total of 430/460 VUS were successfully analyzed, of which 340 (79.1%) were concordant in both functional assays and categorized as functionally impaired (N = 102), functionally intermediate (N = 12), or functionally wild-type (WT)-like (N = 226). We then examined their association with breast cancer risk in the case-control analysis. The OR and 95% CI (confidence intervals) for carriers of functionally impaired, intermediate, and WT-like variants were 2.83 (95% CI, 2.35-3.41), 1.57 (95% CI, 1.41-1.75), and 1.19 (95% CI, 1.08-1.31), respectively. The meta-analysis of population-specific datasets showed similar results. CONCLUSIONS: We determined the functional consequences for the majority of CHEK2 missense VUS found in patients with breast cancer (3,660/4,436; 82.5%). Carriers of functionally impaired missense variants accounted for 0.5% of patients with breast cancer and were associated with a moderate risk similar to that of truncating CHEK2 variants. In contrast, 2.2% of all patients with breast cancer carried functionally wild-type/intermediate missense variants with no clinically relevant breast cancer risk in heterozygous carriers.

A C Camargo Cancer Center and Oncology Center Hospital Sirio Libanes Sao Paulo Brazil

Ambry Genetics Aliso Viejo California

American Cancer Society Atlanta Georgia

Beckman Research Institute City of Hope Cancer Center Duarte California

Behavioral and Epidemiology Research Group American Cancer Society Atlanta Georgia

Biosciences Laboratory IRCCS Istituto Romagnolo per lo Studio dei Tumori Dino Amadori Meldola Italy

Biostatistics Unit The Cyprus Institute of Neurology and Genetics Nicosia Cyprus

Cancer Epidemiology Program Division of Population Sciences H Lee Moffitt Cancer Center and Research Institute Tampa Florida

Cancer Genetics Service National Cancer Centre Singapore Singapore

Cancer Genomics Laboratory Peter MacCallum Cancer Centre Melbourne Australia

CARRIERS USA

Center for Familial Breast and Ovarian Cancer Center for Integrated Oncology University of Cologne Faculty of Medicine and University Hospital Cologne Cologne Germany

Center for Genomic Medicine Copenhagen University Hospital Copenhagen Denmark

Center for Medical Genetics Ghent University and Ghent University Hospital Ghent Belgium

Center for Medical Genetics NorthShore University Health System Evanston Illinois

Center for Omics Sciences IRCCS San Raffaele Scientific Institute Milan Italy

Centro de Investigación Biomédica en Red de Cáncer Madrid Spain

Centro de Investigación en Red de Enfermedades Raras Santiago de Compostela Spain

Clinical Cancer Genetics and Family Consultants CLINICAGENE Athens Medical Center Athens Greece

CPS 2 USA

CPS3 Kennesaw Georgia

CTS USA

Department of Breast Medical Oncology University of Texas MD Anderson Cancer Center Houston Texas

Department of Cancer Biology and Genetics Comprehensive Cancer Center The Ohio State University Columbus Ohio

Department of Cancer Prevention and Control Roswell Park Cancer Center Buffalo New York

Department of Clinical and Experimental Sciences University of Brescia Brescia Italy

Department of Clinical and Molecular Medicine Sapienza University of Rome Rome Italy

Department of Clinical Genetics Copenhagen University Hospital Copenhagen Denmark

Department of Clinical Genetics Maastricht University Medical Centre Maastricht the Netherlands

Department of Clinical Medicine Faculty of Health and Medical Sciences Copenhagen University Copenhagen Denmark

Department of Epidemiology University of Washington Seattle Washington

Department of Experimental Medicine Sapienza University of Rome Rome Italy

Department of Experimental Oncology Molecular Bases of Genetic Risk and Genetic Testing Unit Fondazione IRCCS Istituto Nazionale dei Tumori Milan Italy

Department of Family Medicine and Public Health University of California San Diego San Diego California

Department of Laboratory Medicine and Pathology Mayo Clinic Rochester Minnesota

Department of Medical Oncology Mayo Clinic Rochester Minnesota

Department of Medical Sciences University of Turin Turin Italy

Department of Medicine Perelman School of Medicine University of Pennsylvania Philadelphia Pennsylvania

Department of Obstetrics and Gynecology Helsinki University Hospital and University of Helsinki Helsinki Finland

Department of Obstetrics and Gynecology Ulm University Ulm Germany

Department of Oncology and Haematology Modena University Hospital Modena Italy

Department of Oncology Mayo Clinic Rochester Minnesota

Department of Pathophysiology 1st Faculty of Medicine Charles University Prague Czech Republic

Department of Population Science American Cancer Society Atlanta Georgia

Department of Population Sciences Beckman Research Institute of City of Hope Duarte California

Department of Precision and Regenerative Medicine and Ionian Area Medical Genetics Unit University of Bari Bari Italy

Department of Quantitative Health Sciences Mayo Clinic Rochester Minnesota

Department Quantitative Sciences Mayo Clinic Rochester Minnesota

Division of Biostatistics Institute for Health and Equity and Cancer Center Medical College of Wisconsin Milwaukee Wisconsin

Division of Cancer Prevention and Genetics IEO European Institute of Oncology IRCCS Milan Italy

Duke NUS Medical School Singapore Singapore

Endocrine and Metabolic Disease Unit ASST Spedali Civili of Brescia Brescia Italia

Fundacion Publica Galega de Medicina Xenomica Santiago de Compostela Spain

Hereditary Cancer Program Catalan Institute of Oncology IDIBELL IGTP IDIBGI L'Hospitalet Barcelona Spain

Human Molecular Genetics Laboratory INRaSTES National Center for Scientific Research Demokritos Athens Greece

Immunology and Molecular Oncology Unit Veneto Institute of Oncology Padua Italy

Inserm UMR1245 UNIROUEN Normandy Centre for Genomic and Personalized Medicine Normandie University Rouen France

Institute for Medical Informatics Statistics and Epidemiology University of Leipzig Leipzig Germany

Institute of Biology and Medical Genetics 1st Faculty of Medicine Charles University and General University Hospital Prague Prague Czech Republic

Institute of Human Genetics University Medical Center Hamburg Eppendorf Hamburg Germany

Institute of Medical Biochemistry and Laboratory Diagnostics 1st Faculty of Medicine Charles University and General University Hospital Prague Prague Czech Republic

Institute of Medical Science The University of Tokyo Tokyo Japan

Instituto de Investigación Sanitaria de Santiago de Compostela Santiago de Compostela Spain

Keck School of Medicine University of Southern California Los Angeles California

Laboratory for Genotyping Development RIKEN Center for Integrative Medical Sciences Yokohama Japan

Laboratory of Cancer Cell Biology Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

Lee Kong Chian School of Medicine Nanyang Technological University Singapore Singapore

Lifepool Peter MacCallum Cancer Centre Melbourne Australia

Manchester Centre for Genomic Medicine Division of Evolution and Genomic Sciences University of Manchester Manchester United Kingdom

Mayo Clinic Rochester Minnesota

MCBCS USA

MEC USA

Medical genetics University of Siena Siena Italy

MMHS USA

Molecular Diagnostics Laboratory INRaSTES National Center for Scientific Research Demokritos Athens Greece

Natera Inc Duarte California

NHS Reston Virginia

Parkville Familial Cancer Centre Peter MacCallum Cancer Centre and Royal Melbourne Hospital Melbourne Australia

Population Health Program QIMR Berghofer Medical Research Institute Brisbane Australia

Roswell Park Comprehensive Cancer Center Buffalo New York

School of Medicine and Public Health University of Wisconsin Madison Wisconsin

Sir Peter MacCallum Department of Oncology University of Melbourne Melbourne Australia

Slone Epidemiology Center Boston University Boston Massachusetts

T H Chan School of Public Health Harvard University Cambridge Massachusetts

University of Wisconsin Madison Wisconsin

WCHS Inc Baltimore Maryland

WHI USA

WWHS Charlotte North Carolina

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