Airi Hosokai

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.

Over 90% of missense variants across ∼4,000 disease-associated genes are variants of uncertain significance (VUS). Experimental variant effect measurements provide critical evidence about pathogenicity and inform disease biology, but most variants lack data and clinical translation has been limited. The Impact of Genomic Variation on Function Consortium generated experimental data for 62,215 variants across ten genes using multiplexed assays and 1,407 variants across 163 genes using arrayed assays, curated 193,139 additional community-generated variant effect measurements across 30 additional genes, and developed automated calibration methods for translating experimental data and variant effect predictions into clinical evidence. To reduce current VUS, we developed a scalable workflow using only experimental and predictive evidence, enabling reclassification of 75% of the 16,115 VUS in these genes as pathogenic or benign with 90,000 unobserved variants; 62% had enough evidence to be “preclassified” as pathogenic or benign. We validated our data, evidence and classifications using All of Us and created interactive resources to enable clinical use of the calibrated data. Thus, for 40 genes, representing 1% of the clinical genome, we resolve most existing VUS and future variants, illustrating how systematic use of scalable evidence can empower genomic medicine.

BARD1 encodes a dimeric partner of BRCA1 and is required for homology-directed double strand DNA break repair ^1–5 . Germline loss-of-function variants in BARD1 are linked to elevated risk for breast cancer and neuroblastoma ^6–8 and PARP inhibitors have shown effectiveness against BARD1-deficient tumors. However, the majority of BARD1 variants identified by genetic testing for cancer risk are variants of uncertain significance (VUS) ⁹ limiting clinical utility. We used saturation genome editing ¹⁰ to assess the impact of nearly 11,000 single-nucleotide variants and three base-pair deletions across all 11 coding exons of BARD1 on cellular fitness and gene expression. The cellular fitness data are nearly perfectly concordant with known pathogenic and benign BARD1 variants ⁹ (AUC > 0.99) and loss-of-function missense variants in all three functional domains were associated with elevated risk for breast cancer ^6,8 . When used for clinical variant classification, our data resolved 95.4% of existing BARD1 variants of uncertain significance. Comparison of cellular fitness data to known structures ^11–14 , further solidifies that BARD1’s role in homology-directed repair is required for tumor suppression in humans ¹⁵ . These results will immediately improve clinical genetic testing and decision-making for patients with BARD1 variants and deepen our understanding of BARD1’s role in maintaining genome integrity.