Functional characterization of 84 PALB2 variants of uncertain significance

Abstract

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 PALB2mediated 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.

Molecular Mechanisms of PALB2 Function and Its Role in Breast Cancer Management

“…In the PALB2 N-terminus, p.L35P (c.104T>C) disrupts BRCA1-PALB2 interaction and abolishes the HR activity of PALB2, resulting in sensitivity to the PARPi (106). Moreover, p.P8L (c.23C>T), p.K18R (c.53A>G), p.L24S (c.71T>C), p.Y28C (c.83A>G), and p.R37H (c.110G>A) compromise HR activity of PALB2 and are suggested to be pathogenic (106)(107)(108). In the PALB2 C-terminus, p.W912G, p.G937R, p.L939W, p.I944N (c.2831T>A), p.L947F (c.2841G>T), p.L947S (c.2840T>C), p.L961P, p.L972Q, p.A1025R, p.T1030I (c.3089C>T), p.I1037T, p.G1043D, p.L1070P (c.3209T>C), p.P1088S (c.3262C>T), p.W1140G (c.3418T>C), p.L1143P, and p.L1172P promoted a decrease in the HR activity of PALB2 (20,(107)(108)(109)112).…”

“…Moreover, p.P8L (c.23C>T), p.K18R (c.53A>G), p.L24S (c.71T>C), p.Y28C (c.83A>G), and p.R37H (c.110G>A) compromise HR activity of PALB2 and are suggested to be pathogenic (106)(107)(108). In the PALB2 C-terminus, p.W912G, p.G937R, p.L939W, p.I944N (c.2831T>A), p.L947F (c.2841G>T), p.L947S (c.2840T>C), p.L961P, p.L972Q, p.A1025R, p.T1030I (c.3089C>T), p.I1037T, p.G1043D, p.L1070P (c.3209T>C), p.P1088S (c.3262C>T), p.W1140G (c.3418T>C), p.L1143P, and p.L1172P promoted a decrease in the HR activity of PALB2 (20,(107)(108)(109)112). Among these mutations, p.L939W, p.A1025R, p.T1030I, p.P1088S, and p.L1143P disrupt BRCA2-PALB2 interaction; p.W912G, p.G937R, p.I944N, p.L961P, p.L972Q, p.T1030I, p.I1037T, p.G1043D, and p.L1172P are associated with PALB2 protein instability; and p.I944N, p.L947F, p.L947S, p.T1030I, p.L1070P, and p.W1140G result in the mislocalization of PALB2 to the cytoplasm.…”

Functional Characterization of PALB2 Variants of Uncertain Significance: Toward Cancer Risk and Therapy Response Prediction

“…Assays using HR as a read-out have emerged as the standard for the functional characterisation of VUS in BRCA1 and BRCA2 (Bouwman et al, 2013;Woods et al, 2016;Shimelis et al, 2017;Starita et al, 2018;Mesman et al, 2019). More recently, VUS in PALB2 have also been characterized in a similar manner (Figures 1, 2) (Park et al, 2014;Foo et al, 2017;Boonen et al, 2019;Rodrigue et al, 2019;Wiltshire et al, 2019). To identify variants that impact HR, the well described DR-GFP reporter, as well as the more recently introduced CRISPR-LMNA HR assay were used (Kass et al, 2013;Ducy et al, 2019).…”

“…PALB2-interacting proteins are depicted underneath their respective PALB2 interacting domain/regions. All PALB2 genetic variants from five functional studies (Park et al, 2014;Foo et al, 2017;Boonen et al, 2019;Rodrigue et al, 2019;Wiltshire et al, 2019) are shown and categorized per (functional) domain as benign (green framed sections), truncating (red framed sections), or VUS and synthetic missense variants (MVs) (blue framed sections) based on ClinVar. All functionally damaging PALB2 VUS with an HR efficiency < 50% compared to wild type PALB2 in at least one functional assay are highlighted in red.…”

“…This phenotype was used as a readout for the functional characterization of several PALB2 variants, revealing that two variants, p.L939W and p.L1143P, impaired PALB2 function (Park et al, 2014). Lastly, since PALB2 interacts with BRCA1 and BRCA2 to load RAD51 at sites of DSBs, co-immunoprecipitation, recruitment to laser micro-irradiation induced DNA damage, and DNA-damage-induced RAD51 foci formation were among the additional functional readouts to study the impact of PALB2 variants on HR ( Figure 2) (Park et al, 2014;Foo et al, 2017;Boonen et al, 2019;Rodrigue et al, 2019;Wiltshire et al, 2019). A complete overview of all functional assays that were performed by three recent studies is provided in Table 1 (Boonen et al, 2019;Rodrigue et al, 2019;Wiltshire et al, 2019).…”