Evaluation of non-canonical p53 functions in DNA replication and recombination for variant classification

Pathogenic germline TP53 variants predispose to diverse Li-Fraumeni syndrome (LFS) phenotypes and a broad cancer spectrum, whereby carriers of hypomorphic variants cluster in a cohort with attenuated disease onset and an overrepresentation of breast cancer (BC). Recently, functional assays have gained importance among the criteria used to predict the pathogenicity of hereditary breast and ovarian cancer (HBOC) risk-gene variants. Experimental assays scoring p53 functions in transcription and growth control have contributed to variant classification, yet a significant fraction of TP53 variants remain of unknown significance (VUS). To understand whether non-canonical functions of p53 in the fidelity control of DNA replication may aid variant classification, we subjected 23 TP53 VUS and 20 control variants identified in the German Consortium for HBOC (GC-HBOC) to assays that monitor nascent DNA synthesis and recombination-mediated bypass of replication barriers. Our results reveal a clear functional separation between benign (B)/likely benign (LB) and pathogenic (P)/likely pathogenic (LP) variants in recombination measurements, with B/LB variants associated with high recombination frequencies and P/LP variants with low recombination frequencies. Importantly, 8/23 VUS exhibited activities within the B/LB or P/LP ranges and therefore emerge as candidates for revised classification. Variant-specific recombination activities showed significant correlations with functional scores from four earlier studies systematically analyzing canonical p53 functions. Differently, in DNA fiber spreading assays B/LB and P/LP variants showed a more heterogeneous pattern and thus did not consistently recapitulate replication slow-down and acceleration observed in the presence and absence of p53, respectively. Structural modeling of separation-of-function (SOF) variants in transcription and recombination indicates varying effects on protein stability and the conformation of surface-exposed regions, affecting for example, the flexibility of Loop 1 (L1). Intriguingly, individual SOF variants suggest that loss-of-function (LOF) in recombination may drive BC, underscoring the predictive power of this assay for low-penetrance TP53 variants.