Functional analysis of patient-derived PALB2 missense variants of uncertain significance.

Abstract

1531 Background: Inherited PALB2 pathogenic variants are associated with an increased lifetime risk for breast cancer development. However, the interpretation of numerous PALB2 missense variants of uncertain significance (VUS) identified in germline genetic testing remains a challenge. Here, we assessed the impact of breast cancer patient-derived VUS on PALB2 function and identified pathogenic PALB2 missense variants that may increase cancer risk. Methods: A total of seven potentially pathogenic PALB2 VUS identified in 2,279 breast cancer patients were selected for functional analysis. All these selected VUS were assessed by SIFT, Align-GVGD, and PolyPhen2 in silico and were predicted to be deleterious by at least two in silico algorithms. The p.L35P [c.104T > C] variant was also included, for which pathogenicity has been recently confirmed. The effects of the VUS on the homologous recombination (HR) activity of PALB2 were tested by U2OS/DR-GFP reporting system. Functional characterization was further validated by protein co-immunoprecipitation and RAD51 recruitment assay. Results: PALB2 variants p.L24F [c.72G > C] and p.L35P [c.104T > C] showed the most significant disruption to the HR activity of PALB2 relative to the wild-type condition, retaining only 52.2% ( p = 0.0013) and 8.5% ( p < 0.0001) of HR activity respectively. Moderate but statistically significant HR deficiency was observed for four other variants (p.P405A [c.1213C > G], p.T1012I [c.3035C > T], p.E1018D [c.3054G > C], and p.T1099M [c.3296C > T]). We found no statistical differences for the p.K628N [c.1884G > T] and p.R663C [c.1987C > T] in the HR activity compared to wild-type PALB2. The p.L24F and p.L35P variants compromised the BRCA1-PALB2 interaction and reduced RAD51 foci formation in response to DNA damage. Conclusions: We have identified a novel patient-derived pathogenic PALB2 missense variant, p.L24F [c.72G > C], that compromises PALB2-mediated HR activity. We suggest the integration of the identified pathogenic variants into breast cancer genetic counseling and individualized treatment regimens for better clinical outcomes.