Saturation genome editing of BARD1 resolves VUS and provides insight into BRCA1-BARD1 tumor suppression

BARD1 variants are associated with hereditary breast cancer and neuroblastoma, yet, 98% of missense variants remain variants of uncertain significance (VUS). We applied Saturation Genome Editing (SGE) to assess 8,818 SNVs and 2,097 3-base pair deletions for their effects on cell survival and RNA abundance. We found that 13% of missense variants are loss-of-function (LoF), with 98% in BARD1's three folded domains. LoF missense variants in the ankyrin repeat and BRCT domains were enriched in breast cancer cohorts, linking their molecular functions to cancer risk. SGE discriminated known pathogenic from benign variants with exceptional accuracy (AUC = 0.99) and resolved 95.4% of VUS, demonstrating high clinical utility. The single nucleotide resolution allowed discrimination of variant effects on RNA abundance from those affecting protein function and provided further evidence linking BARD1's function in homology directed repair to tumor suppression. This comprehensive variant effect dataset informs inherited cancer risk and treatment decisions.