Loss-of-function variants in PALB2 give rise to defects in DNA damage repair by homologous recombination (HR), increasing the risk of breast cancer in female carriers. However, genetic testing frequently reveals missense variants of uncertain significance (VUS) for which the impact on protein function and cancer risk are unclear. Here we assay 84% of all possible missense variants in 11 out of 13 PALB2 exons using site-saturation functional screens with PARP inhibitor sensitivity as a readout for HR. These exons encode the coiled-coil and WD40 domains, which we identify as the minimal regions required for HR. Furthermore, we reveal the functional impact of 6718 missense variants, classifying 3904 variants as functional (58%), 2422 as intermediate (36%), and 392 as damaging (6%). A burden-type analysis shows that damaging missense variants in PALB2 are associated with a significantly increased risk of breast cancer, similar to that observed for truncating variants. These results will be valuable for the classification of PALB2 missense VUS and clinical management of carriers.

Ambry Genetics Aliso Viejo CA USA

Bio Prodict Nijmegen The Netherlands

Clinical Genetics Service Department of Medicine Memorial Sloan Kettering Cancer Center New York NY USA

Department of Biomedical Data Sciences Leiden University Medical Center Leiden The Netherlands

Department of Human Genetics Leiden University Medical Center Leiden The Netherlands

Department of Laboratory Medicine and Pathology Mayo Clinic Rochester MN USA

Department of Medicine Weill Cornell Medical Center Cornell University New York NY USA

Department of Pathology Leiden University Medical Center Leiden The Netherlands

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

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

Leiden Genome Technology Center Department of Human Genetics Leiden University Medical Center Leiden The Netherlands

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Yang, X. et al. Cancer risks associated with germline PALB2 pathogenic variants: an international study of 524 families. PubMed DOI PMC

Brnich, S. E. et al. Recommendations for application of the functional evidence PS3/BS3 criterion using the ACMG/AMP sequence variant interpretation framework. PubMed DOI PMC

Richards, S. et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. PubMed DOI PMC

Nepomuceno, T. C. et al. The role of PALB2 in the DNA damage response and cancer predisposition. PubMed PMC

Boonen, R., Vreeswijk, M. P. G. & van Attikum, H. Functional characterization of PALB2 variants of uncertain significance: toward cancer risk and therapy response prediction. PubMed DOI PMC

Nepomuceno, T. C. et al. PALB2 variants: protein domains and cancer susceptibility. PubMed DOI

Boonen, R. et al. Functional analysis of genetic variants in the high-risk breast cancer susceptibility gene PALB2. PubMed DOI PMC

Rodrigue, A. et al. A global functional analysis of missense mutations reveals two major hotspots in the PALB2 tumor suppressor. PubMed DOI PMC

Wiltshire, T. et al. Functional characterization of 84 PALB2 variants of uncertain significance. PubMed DOI PMC

Breast Cancer Association, C. et al. Breast cancer risk genes—association analysis in more than 113,000 women. PubMed DOI PMC

Hu, C. et al. A population-based study of genes previously implicated in breast cancer. PubMed DOI PMC

Bleuyard, J. Y., Buisson, R., Masson, J. Y. & Esashi, F. ChAM, a novel motif that mediates PALB2 intrinsic chromatin binding and facilitates DNA repair. PubMed DOI PMC

Rubin, A. F. et al. A statistical framework for analyzing deep mutational scanning data. PubMed DOI PMC

Rubin, A. F. et al. Correction to: A statistical framework for analyzing deep mutational scanning data. PubMed DOI PMC

Ng, P. S. et al. Characterisation of protein-truncating and missense variants in PALB2 in 15 768 women from Malaysia and Singapore. PubMed PMC

Starita, L. M. et al. Variant interpretation: functional assays to the rescue. PubMed DOI PMC

Blomen, V. A. et al. Gene essentiality and synthetic lethality in haploid human cells. PubMed DOI

Karczewski, K. J. et al. The mutational constraint spectrum quantified from variation in 141,456 humans. PubMed DOI PMC

Foo, T. K. et al. Compromised BRCA1-PALB2 interaction is associated with breast cancer risk. PubMed DOI PMC

Schymkowitz, J. et al. The FoldX web server: an online force field. PubMed DOI PMC

Blaabjerg, L. M. et al. Rapid protein stability prediction using deep learning representations. PubMed PMC

Adzhubei, I. A. et al. A method and server for predicting damaging missense mutations. PubMed DOI PMC

Sim, N. L. et al. SIFT web server: predicting effects of amino acid substitutions on proteins. PubMed DOI PMC

Tavtigian, S. V., Samollow, P. B., de Silva, D. & Thomas, A. An analysis of unclassified missense substitutions in human BRCA1. PubMed DOI

Ioannidis, N. M. et al. REVEL: an ensemble method for predicting the pathogenicity of rare missense variants. PubMed DOI PMC

Frazer, J. et al. Disease variant prediction with deep generative models of evolutionary data. PubMed DOI

Vroling, B. & Heijl, S. White paper: The Helix Pathogenicity Prediction Platform. https://arxiv.org/abs/2104.01033 (2021).

Feng, B. J. PERCH: a unified framework for disease gene prioritization. PubMed DOI PMC

Cheng, J. et al. Accurate proteome-wide missense variant effect prediction with AlphaMissense. PubMed DOI

Dorling, L. et al. Breast cancer risks associated with missense variants in breast cancer susceptibility genes. PubMed DOI PMC

Findlay, G. M. et al. Accurate classification of BRCA1 variants with saturation genome editing. PubMed DOI PMC

Starita, L. M. et al. A multiplex homology-directed DNA repair assay reveals the impact of more than 1,000 BRCA1 missense substitution variants on protein function. PubMed DOI PMC

Ikegami, M. et al. High-throughput functional evaluation of BRCA2 variants of unknown significance. PubMed DOI PMC

Li, H. et al. Functional annotation of variants of the BRCA2 gene via locally haploid human pluripotent stem cells. PubMed DOI PMC

Huang, H. et al. Functional evaluation and clinical classification of BRCA2 variants. PubMed DOI PMC

Sahu, S. et al. Saturation genome editing-based clinical classification of BRCA2 variants. PubMed DOI

Jaganathan, K. et al. Predicting splicing from primary sequence with deep learning. PubMed DOI

Ducy, M. et al. The tumor suppressor PALB2: inside out. PubMed DOI

Sidorova, J. A game of substrates: replication fork remodeling and its roles in genome stability and chemo-resistance. PubMed DOI PMC

Boonen, R. et al. Functional analysis identifies damaging CHEK2 missense variants associated with increased cancer risk. PubMed DOI PMC

Bouwman, P. et al. A high-throughput functional complementation assay for classification of BRCA1 missense variants. PubMed DOI

Kranz, A. et al. An improved Flp deleter mouse in C57Bl/6 based on Flpo recombinase. PubMed DOI

DeLong, E. R., DeLong, D. M. & Clarke-Pearson, D. L. Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach. PubMed DOI

Oliver, A. W., Swift, S., Lord, C. J., Ashworth, A. & Pearl, L. H. Structural basis for recruitment of BRCA2 by PALB2. PubMed DOI PMC

Soukupova, J. et al. Validation of CZECANCA (CZEch CAncer paNel for Clinical Application) for targeted NGS-based analysis of hereditary cancer syndromes. PubMed DOI PMC