Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C

Sep 28, 2026

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Functional and clinical characterization identifies a key hotspot in RAD51C missense variants of uncertain significance (VUS) that become inactivating. Analysis of 173 missense variants using a homology-directed repair assay reveals 30 nonfunctional (deleterious) variants, including 18 within the ATP-binding region. Deleterious variants show sensitivity to cisplatin and olaparib, disrupt RAD51C/XRCC3 and RAD51B/RAD51C/RAD51D/XRCC2 complex formation, and match predicted structural effects on ATP binding. A subset recapitulates activity defects in RAD51C-depleted cancer cells, with case-control studies linking deleterious variants to moderate breast cancer risk and high ovarian cancer risk.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages15

Reading Time38 min

AccessFree to download

Introduction

RAD51C function and cancer predisposition

Methods and variant functional analysis

HDR assay and nonfunctional variant identification

ATP-binding hotspot effects

Clinical association and risk assessment

Breast and ovarian case-control results

Functional and Clinical Characterization of Variants of Uncertain Signiﬁcance Identiﬁes a Hotspot for Inactivating Missense Variants in RAD51C Chunling Hu1, Anil Belur Nagaraj1, Hermela Shimelis1, Gemma Montalban2, Kun Y. Lee1, Huaizhi Huang1, Carolyn A. Lumby1, Jie Na1, Lisa R. Susswein3, Maegan E. Roberts3, Megan L. Marshall3, Susan Hiraki3, Holly LaDuca4, Elizabeth Chao4, Amal Yussuf4, Tina Pesaran4, Susan L. Neuhausen5, Christopher A. Haiman6, Peter Kraft7, Sara Lindstrom8, Julie R. Palmer9, Lauren R. Teras10, Celine M. Vachon1, Song Yao11, Irene Ong12, Katherine L. Nathanson13, Jeffrey N. Weitzel14, Nicholas Boddicker1, Rohan Gnanaolivu1, Eric C. Polley15, Georges Mer1, Gaofeng Cui1, Rachid Karam4, Marcy E. Richardson4, Susan M. Domchek13, Siddhartha Yadav1, Kathleen S. Hruska3, Jill Dolinsky4, S. John Weroha1, Steven N. Hart1, Jacques Simard2, Jean Yves Masson2, Yuan-Ping Pang1, and Fergus J. Couch1 ◥ Pathogenic protein-truncating variants of RAD51C, which plays an integral role in promoting DNA damage repair, increase the risk of breast and ovarian cancer. A large number of RAD51C missense variants of uncertain signiﬁcance (VUS) have been identiﬁed, but the effects of the majority of these variantson RAD51C function and cancer predisposition have not been established. Here, analysis of 173 missense variants by a homology-directed repair (HDR) assay in reconstituted RAD51C􀀁/ 􀀁 cells identiﬁed 30 nonfunctional (deleterious) variants, including 18 in a hotspot within the ATPbinding region. The deleterious variants conferred sensitivity tocisplatin and olapariband disrupted formation of RAD51C/XRCC3 and RAD51B/RAD51C/RAD51D/XRCC2 complexes. Computational analysis indicated the deleterious variant effects were consistent with structural effects on ATP-binding to RAD51C. A subset of the variants displayed similar effects on RAD51C activity in reconstituted human RAD51C-depleted cancer cells. Case–control association studies of deleterious variants in women with breast and ovarian cancer and noncancer controls showed associations with moderate breast cancer risk [OR, 3.92; 95% conﬁdence interval (95% CI), 2.18–7.59] and high ovarian cancer risk (OR, 14.8; 95% CI, 7.71–30.36), similar to protein-truncating variants. This functional data supports the clinical classiﬁcation of inactivating RAD51C missense variants as pathogenic or likely pathogenic, which may improve the clinical management of variant carriers. Signiﬁcance: Functional analysis of the impact of a large number of missense variants on RAD51C function provides insight into RAD51C activity and information for classiﬁcation of the cancer relevance of RAD51C variants. 1Mayo Clinic, Rochester, Minnesota. 2CHU de Quebec-Universit Laval Research Center, Universit Laval, Quebec City, Quebec, Canada. 3GeneDx, Gaithersburg, Maryland. 4Ambry Genetics, Aliso Viejo, California. 5Beckman Research Institute of City of Hope, Duarte, California. 6Keck School of Medicine, University of Southern California, Los Angeles, California. 7T. H. Chan School of Public Health, Harvard University, Boston, Massachusetts. 8Department of Epidemiology, University of Washington, Seattle, Washington. 9Slone Epidemiology Center at Boston University, Boston, Massachusetts. 10Behavioral and Epidemiology Research Group, American Cancer Society, Atlanta, Georgia. 11Roswell Park Comprehensive Cancer Center, Buffalo, New York. 12University of WisconsinMadison, Madison, Wisconsin. 13University of Pennsylvania, Philadelphia, Pennsylvania. 14Latin American School of Oncology, Sierra Madre, California. 15University of Chicago, Chicago, Illinois. C. Hu, A. B. Nagaraj, H. Shimelis, and G. Montalban contributed equally to this article. Corresponding Author: Fergus J. Couch, Department of Laboratory Medicine and Pathology, Mayo Clinic, Stabile 2-42, 200 First Street SW, Rochester, MN 55905. E-mail: [couch.fergus@mayo.edu](couch.fergus@mayo.edu) [Cancer Res 2023](Cancer Res 2023);[83:2557](

FAQs

What was the goal of analyzing RAD51C VUS in this study?

To determine which missense variants of uncertain significance impair RAD51C function and influence cancer predisposition.

Which variants were identified as nonfunctional, and where was the key hotspot?

Thirty missense variants were nonfunctional, including 18 located in a hotspot within the RAD51C ATP-binding region.

How did deleterious RAD51C variants affect drug response and DNA repair complexes?

They conferred sensitivity to cisplatin and olaparib and disrupted formation of RAD51C/XRCC3 and RAD51B/RAD51C/RAD51D/XRCC2 complexes.

Sep 28, 2026

Uploaded by Angel