Saturation Genome Editing reveals the functional impact of RAD51D and XRCC2 variants

Germline pathogenic variants in RAD51D and XRCC2, which encode RAD51 paralogs that form a heterodimer within the BCDX2 complex, confer increased cancer risk and homologous recombination deficiency. However, most RAD51D and XRCC2 variants in ClinVar are classified as variants of uncertain significance (VUS), limiting clinical utility. Here, we applied saturation genome editing (SGE) to measure the effects of 5,412 RAD51D and 3,743 XRCC2 variants on cellular fitness and additionally assessed 2,876 and 2,069 variants in these genes for effects on RNA expression. Fitness scores discriminated pathogenic from benign variants with near-perfect accuracy (AUC=0.994 for RAD51D; AUC=1.000 for XRCC2). Integration of RNA expression data revealed RAD51D, but not XRCC2, is exceptionally sensitive to splice-altering variation, with 24% of RAD51D loss-of-function missense variants acting through RNA-mediated mechanisms compared to only 5% in XRCC2. These SGE datasets provide strong, splice-resolved functional evidence to support variant classification across both genes.