Functional analysis of genetic variants in the high-risk breast cancer susceptibility gene PALB2

Rick A C M Boonen,Maaike P G Vreeswijk,Milan Sharma,Peter Devilee,Haico Van Attikum,Magdalena B Rother,Fergus Couch,Nandi Celosse,Amélie Rodrigue,Wouter W Wiegant,Jean-Yves Masson,Chantal Stoepker,Bas Vroling,Jacques Simard

doi:10.1038/s41467-019-13194-2

Abstract

Heterozygous carriers of germ-line loss-of-function variants in the DNA repair gene PALB2 are at a highly increased lifetime risk for developing breast cancer. While truncating variants in PALB2 are known to increase cancer risk, the interpretation of missense variants of uncertain significance (VUS) is in its infancy. Here we describe the development of a relatively fast and easy cDNA-based system for the semi high-throughput functional analysis of 48 VUS in human PALB2. By assessing the ability of PALB2 VUS to rescue the DNA repair and checkpoint defects in Palb2 knockout mouse embryonic stem (mES) cells, we identify various VUS in PALB2 that impair its function. Three VUS in the coiled-coil domain of PALB2 abrogate the interaction with BRCA1, whereas several VUS in the WD40 domain dramatically reduce protein stability. Thus, our functional assays identify damaging VUS in PALB2 that may increase cancer risk.

Highlights

Heterozygous carriers of germ-line loss-of-function variants in the DNA repair gene partner and localizer of BRCA2 (PALB2) are at a highly increased lifetime risk for developing breast cancer
This cell-based approach should combine efficient integration and equal expression of human PALB2 cDNA carrying these variants in a cellular background devoid of endogenous Palb[2] and with the ability to assess their effect on homologous recombination (HR)
Out of the 49 PALB2 missense variants tested in this study (Supplementary Data 1), we identified 15 variants (p.L24S, p.Y28C, p.L35P, p.W912G, p.G937R, p.I944N, p.L947S, p.L961P, p.L972Q, p.A1025R, p.T1030I, p.I1037T, p.G1043D, p.L1070P, and p.L1172P) as damaging, reducing HR by >60%

Summary

Introduction

Heterozygous carriers of germ-line loss-of-function variants in the DNA repair gene PALB2 are at a highly increased lifetime risk for developing breast cancer. 1234567890():,; Germline loss-of-function (LOF) variants in the breast cancer susceptibility genes BRCA1 and BRCA2 are known to result in an approximately tenfold increased lifetime risk of developing breast cancer[1]. The PALB2-BRCA1/2-RAD51 complex plays an essential role in homologous recombination (HR), which is a critical pathway for the repair of highly-deleterious DNA double-strand breaks (DSBs). We report on the development of a relatively rapid and easy functional assay that can determine the functional consequences of VUS in PALB2, thereby facilitating cancer risk assessment and predicting therapy response

Methods

Results

Conclusion