Functional characterization of 84 PALB2 variants of uncertain significance

Abstract

Purpose: Inherited pathogenic variants in PALB2 are associated with increased risk of breast and pancreatic cancer. However, the functional and clinical relevance of many missense variants of uncertain significance (VUS) identified through clinical genetic testing is unclear. The ability of patient-derived germline missense VUS to disrupt PALB2 function was assessed to identify variants with potential clinical relevance.

Methods: The influence of 84 VUS on PALB2 function was evaluated using a cellular homology directed DNA repair (HDR) assay and VUS impacting activity were further characterized using secondary functional assays.

Results: Four (~5%) variants (p.L24S,c.71T>C; p.L35P,c.104T>C; pI944N,c.2831T>A; and p.L1070P,c.3209T>C) disrupted PALB2-mediated HDR activity. These variants conferred sensitivity to cisplatin and a poly(ADP-ribose) polymerase (PARP) inhibitor and reduced RAD51 foci formation in response to DNA damage. The p.L24S and p.L35P variants disrupted BRCA1-PALB2 protein complexes, p.I944N was associated with protein instability, and both p.I944N and p.L1070P mislocalized PALB2 to the cytoplasm.

Conclusion: These findings show that the HDR assay is an effective method for screening the influence of inherited variants on PALB2 function, that four missense variants impact PALB2 function and may influence cancer risk and response to therapy, and suggest that few inherited PALB2 missense variants disrupt PALB2 function in DNA repair.

Keywords: PALB2; PARP inhibitor; breast cancer; homologous recombination repair; variant of uncertain significance (VUS).

Fig. 1

Homology directed repair assay of PALB2 variants. (a) Plot of all variants assayed in homologous recombination (HR) repair assay. Results for each independent assay are scaled 1–5 relative to the p.Y551X negative control and wild-type PALB2 positive control. Error bars represent the standard error of the mean (SE) of independent replicates. (b) Illustration of the PALB2 protein (amino acids 11–1183) showing position of functional domains. Deleterious variants (red) and no functional impact variants (black) are shown as vertical lines above and below the protein. GFP green fluorescence protein.

Fig. 2

Influence of PALB2 variants on protein complex formation and protein half-life. (a) Western blot analysis of PALB2-interacting proteins after coimmunoprecipitation of FLAG-tagged PALB2 from HEK293T cells transiently transfected with PALB2 wild-type (WT) and variants. Whole cell lysate (WCL) shows levels of PALB2 expression. (b) Western blot analysis of PALB2 protein after indicated periods of incubation in the presence of cycloheximide (CHX) to define protein half-life. (c) Quantitation of PALB2 protein half-life using ImageJ at indicated time points.

Fig. 3

Influence of PALB2 variants on response to DNA damage. (a) Recruitment of full-length yellow fluorescence protein (YFP)-tagged PALB2 (PALB2-YFP) wild-type (WT) and variants to sites of laser-induced double-strand breaks (DSBs) at indicated time points. (b) Proportion of cells expressing PALB2 WT and variants with localization of PALB2-YFP to DSBs. (c) Nucleocytoplasmic distribution of wild-type PALB2-YFP and variants as indicated. (d) Immunofluorescence analysis of PALB2-YFP and RAD51 foci formation in cyclin A–positive HeLa cells after exposure to ionizing radiation (IR) (2 Gy). Cells were treated with PALB2 small interfering RNA (siRNA) cells to deplete endogenous PALB2. (e) Quantification of RAD51 foci in cyclin A–positive cells expressing PALB2-YFP WT and variants. Results are from three independent experiments.

Fig. 4

Sensitivity to DNA damaging agents. Survival of mouse mammary tumor B400 cells reconstituted with PALB2 wild-type (WT) and variants following exposure to varying doses of olaparib (a, c), and cisplatin (b, d).