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

Abstract

In recent years it has become clear that pathogenic variants in PALB2 are associated with a high risk for breast, ovarian and pancreatic cancer. However, the clinical relevance of variants of uncertain significance (VUS) in PALB2, which are increasingly identified through clinical genetic testing, is unclear. Here we review recent advances in the functional characterization of VUS in PALB2. A combination of assays has been used to assess the impact of PALB2 VUS on its function in DNA repair by homologous recombination, cell cycle regulation and the control of cellular levels of reactive oxygen species (ROS). We discuss the outcome of this comprehensive analysis of PALB2 VUS, which showed that VUS in PALB2's Coiled-Coil (CC) domain can impair the interaction with BRCA1, whereas VUS in its WD40 domain affect PALB2 protein stability. Accordingly, the CC and WD40 domains of PALB2 represent hotspots for variants that impair PALB2 protein function. We also provide a future perspective on the high-throughput analysis of VUS in PALB2, as well as the functional characterization of variants that affect PALB2 RNA splicing. Finally, we discuss how results from these functional assays can be valuable for predicting cancer risk and responsiveness to cancer therapy, such as treatment with PARP inhibitor- or platinum-based chemotherapy.

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

FIGURE 1

Schematic representation of PALB2 variants, functional domains, interacting proteins, and exons. The nucleotide numbers refer to the last nucleotide of each exon in PALB2 cDNA (NM_024675.3). The amino acid numbers are shown to specify the evolutionarily conserved functional domains of PALB2; Coiled-coil (CC) (Tischkowitz et al., 2007; Sy et al., 2009b; Zhang et al., 2009a,b), Chromatin-Association Motif (ChAM) (Bleuyard et al., 2012), MORF-Related Gene on chromosome 15 (MRG15) binding domain (Sy et al., 2009a) and WD40 domain (Xia et al., 2006; Tischkowitz et al., 2007). 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. The two damaging synthetic MVs are highlighted in purple.

FIGURE 2

Overview of the functional assays used for the functional characterization of PALB2 genetic variants. Either Palb2 KO mouse cells or PALB2 siRNA-depleted human cells were complemented by expressing human PALB2 (siRNA-resistant) cDNA, without or with a variant. PALB2 deficiency is indicated with a red cross, whereas a red arrow marks the position of a variant in the PALB2 cDNA. Complementation was either by transient (B400 mouse cells or human cell lines) or stable expression (mES cells) (top section). PALB2 complemented cells were subjected to multiple cell-based functional assays (bottom section). The functional assays determine in a quantitative manner: (1) homology-directed repair of an I-SceI–induced DSB in DR-GFP, which results in the restoration of a functional GFP gene whose expression can be monitored by fluorescence-activated cell sorting (FACS), (2) HR-mediated integration of mRuby into the LMNA A/C locus (LMNA) at a break site induced by CRISPR/Cas9, (3) the formation of IR-induced RAD51 foci, which is PALB2 dependent and provides a measure for the HR efficiency, (4) sensitivity to PARPi or cisplatin treatment, which leads to cell killing when HR is impaired, (5) G2/M checkpoint maintenance after extensive DNA damage, which is dependent on PALB2-mediated HR. Deficiency in PALB2 results in the progression of cells from G2-phase into M-phase. Consequently, the mitotic fraction represents a measure for the functional impact of PALB2 variants.

FIGURE 3

Comparison and correlation between DR-GFP- and PARPi-based HR assays from three different studies. (A) Bar graph comparing results from DR-GFP-based functional assays for 26 overlapping PALB2 variants from studies by us and Wiltshire et al. (2019; Boonen et al., 2019). Mean percentages of GFP-positive cells relative to wild type PALB2 (WT) are shown, with cells expressing WT PALB2 being set to 100%. (B) Bar graph comparing results from PARPi-based functional assays for 14 overlapping PALB2 variants from studies by us and Rodrigue et al. (2019; Boonen et al., 2019). Mean percentages of viability relative to WT PALB2 are shown, with cells expressing WT PALB2 being set to 100%. (C) Scatter plot showing the correlation between the results from our study and Wiltshire et al. (2019) as shown in (A) (Boonen et al., 2019). The color of the datapoints corresponds to the different variants/conditions: wild type (black), benign based on ClinVar (green), truncating (red), VUS (blue). (D) Scatter plot showing the correlation between the results from our study and Rodrigue et al. (2019) as shown in (B) (Boonen et al., 2019). The color of the datapoints corresponds to the different variants/conditions: wild type (black), benign based on ClinVar (green), VUS (orange). (E) Bar graph comparing results from DR-GFP- and CRISPR-LMNA-based HR assays for 9 overlapping PALB2 variants from studies by us and Rodrigue et al. (2019; Boonen et al., 2019). Mean percentages of GFP- or mRuby-positive cells relative to WT PALB2 are shown as in (A). (F) Scatter plot showing the correlation between the results from our study and Rodrigue et al. (2019) as shown in (E) (Boonen et al., 2019). The color of the datapoints is as shown in (D).