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.
Highlights
Specialty section: This article was submitted to Cellular Biochemistry, a section of the journal Frontiers in Molecular Biosciences
We reported a similar impact on homologous recombination (HR) in DR-GFP assays for this variant (55% reduction in HR when compared to WT PALB2) (Boonen et al, 2019), we observed a partial loss of the PALB2-BRCA1 interaction in coimmunoprecipitation experiments, as well as the recruitment of PALB2 to sites of DNA damage induced by laser microirradiation (Boonen et al, 2019)
We have provided head-to-head comparisons of the different assays that were used for the functional characterization of variants in PALB2
Summary
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). Four PALB2 missense variants (p.L24S, p.L35P, p.I944N, and p.L1070P) were identified that strongly disrupted HR (>65% reduction in HR compared to WT PALB2) To corroborate their findings, a CRISPR-LMNA HR assay (Ducy et al, 2019) was performed in U2OS cells with endogenous PALB2 depletion by siRNA treatment, followed by transient expression of siRNA-resistant PALB2 cDNA with or without variant. The same four variants disrupted HR-mediated mRuby integration into the LMNA locus In this assay, the variants exhibited a >90% reduction in HR compared to cells that were complemented with WT PALB2 cDNA.
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