Data from Functional Analysis Identifies Damaging <i>CHEK2</i> Missense Variants Associated with Increased Cancer Risk

Abstract

<div>Abstract<p>Heterozygous carriers of germline loss-of-function variants in the tumor suppressor gene checkpoint kinase 2 (<i>CHEK2)</i> are at an increased risk for developing breast and other cancers. While truncating variants in <i>CHEK2</i> are known to be pathogenic, the interpretation of missense variants of uncertain significance (VUS) is challenging. Consequently, many VUS remain unclassified both functionally and clinically. Here we describe a mouse embryonic stem (mES) cell–based system to quantitatively determine the functional impact of 50 missense VUS in human <i>CHEK2</i>. By assessing the activity of human CHK2 to phosphorylate one of its main targets, Kap1, in <i>Chek2</i> knockout mES cells, 31 missense VUS in <i>CHEK2</i> were found to impair protein function to a similar extent as truncating variants, while 9 <i>CHEK2</i> missense VUS resulted in intermediate functional defects. Mechanistically, most VUS impaired CHK2 kinase function by causing protein instability or by impairing activation through (auto)phosphorylation. Quantitative results showed that the degree of CHK2 kinase dysfunction correlates with an increased risk for breast cancer. Both damaging <i>CHEK2</i> variants as a group [OR 2.23; 95% confidence interval (CI), 1.62–3.07; <i>P</i> < 0.0001] and intermediate variants (OR 1.63; 95% CI, 1.21–2.20; <i>P</i> = 0.0014) were associated with an increased breast cancer risk, while functional variants did not show this association (OR 1.13; 95% CI, 0.87–1.46; <i>P</i> = 0.378). Finally, a damaging VUS in <i>CHEK2</i>, c.486A>G/p.D162G, was also identified, which cosegregated with familial prostate cancer. Altogether, these functional assays efficiently and reliably identified VUS in <i>CHEK2</i> that associate with cancer.</p>Significance:<p>Quantitative assessment of the functional consequences of <i>CHEK2</i> variants of uncertain significance identifies damaging variants associated with increased cancer risk, which may aid in the clinical management of patients and carriers.</p></div>