Abstract
Clinical genetic testing readily detects germline genetic variants. Yet, the rarity of individual variants limits the evidence available for variant classification, leading to many variants of uncertain significance (VUS). VUS cannot guide clinical decisions, complicating counseling and management. In hereditary breast cancer gene PALB2, approximately 50% of clinically identified germline variants are VUS and approximately 90% of VUS are missense. Truncating PALB2 variants have homologous recombination (HR) defects and rely on error-prone nonhomologous end-joining for DNA damage repair (DDR). Recent reports show that some missense PALB2 variants may also be damaging, but most functional studies have lacked benchmarking controls required for sufficient predictive power for clinical use. Here, variant-level DDR capacity in hereditary breast cancer genes was assessed using the Traffic Light Reporter (TLR) to quantify cellular HR/nonhomologous end-joining with fluorescent markers. First, using BRCA2 missense variants of known significance as benchmarks, the TLR distinguished between normal/abnormal HR function. The TLR was then validated for PALB2 and used to test 37 PALB2 variants. Based on the TLR's ability to correctly classify PALB2 validation controls, these functional data where applied in subsequent germline variant interpretations at a moderate level of evidence toward a pathogenic interpretation (PS3_moderate) for 8 variants with abnormal DDR, or a supporting level of evidence toward a benign interpretation (BS3_supporting) for 13 variants with normal DDR.