Mutagenesis of the PALB2 WD40 domain identifies variants defective in interaction with BRCA2 and DNA repair

PALB2 (Partner and Localizer of BRCA2) is a tumor suppressor gene in which germline pathogenic variants predispose to breast and pancreatic cancer. The PALB2 protein is critical for maintaining genome integrity and plays a significant role in homologous recombination DNA repair by recruiting BRCA2 to sites of damage via the PALB2 WD40 domain. However, the impact of most PALB2 WD40 missense variants on PALB2 activity, the BRCA2 interaction, and predisposition to cancer remains poorly explored. Here, we assessed the functional impact of 78 PALB2 missense variants reported as variants of uncertain clinical significance or with conflicting interpretation in ClinVar. We identified variants that disrupt binding to BRCA2, promote PALB2 subcellular mislocalization, and protein instability. We also identified nine variants with a severe impact on BRCA2 binding and homologous recombination repair. We propose that these variants, which consistently show loss of function in different functional assays, represent pathogenic cancer predisposing variants.