ENIGMACHEK2gether Project: A Comprehensive Study Identifies Functionally ImpairedCHEK2Germline Missense Variants Associated with Increased Breast Cancer Risk

Abstract

Purpose: Germline pathogenic variants in CHEK2 confer moderately elevated breast cancer risk (odds ratio, OR ∼ 2.5), qualifying carriers for enhanced breast cancer screening. Besides pathogenic variants, dozens of missense CHEK2 variants of uncertain significance (VUS) have been identified, hampering the clinical utility of germline genetic testing (GGT). Experimental Design: We collected 460 CHEK2 missense VUS identified by the ENIGMA consortium in 15 countries. Their functional characterization was performed using CHEK2-complementation assays quantifying KAP1 phosphorylation and CHK2 autophosphorylation in human RPE1–CHEK2-knockout cells. Concordant results in both functional assays were used to categorize CHEK2 VUS from 12 ENIGMA case–control datasets, including 73,048 female patients with breast cancer and 88,658 ethnicity-matched controls. Results: A total of 430/460 VUS were successfully analyzed, of which 340 (79.1%) were concordant in both functional assays and categorized as functionally impaired (N = 102), functionally intermediate (N = 12), or functionally wild-type (WT)–like (N = 226). We then examined their association with breast cancer risk in the case–control analysis. The OR and 95% CI (confidence intervals) for carriers of functionally impaired, intermediate, and WT-like variants were 2.83 (95% CI, 2.35–3.41), 1.57 (95% CI, 1.41–1.75), and 1.19 (95% CI, 1.08–1.31), respectively. The meta-analysis of population-specific datasets showed similar results. Conclusions: We determined the functional consequences for the majority of CHEK2 missense VUS found in patients with breast cancer (3,660/4,436; 82.5%). Carriers of functionally impaired missense variants accounted for 0.5% of patients with breast cancer and were associated with a moderate risk similar to that of truncating CHEK2 variants. In contrast, 2.2% of all patients with breast cancer carried functionally wild-type/intermediate missense variants with no clinically relevant breast cancer risk in heterozygous carriers.

Comprehensive analysis of the functional impact of single nucleotide variants of humanCHEK2

“…Such an approach is supported by the finding that concordant data from multiple assays has significantly more overlap with independent VEP predictions than data from a single assay. The value of a second, confirming assay is further supported by an analysis of ENIGMA patient data and two functional assays (KAP1 phosphorylation and CHK2 phosphorylation) carried out by Stolarova et al 22 , which shows that CHK2 variants confirmed to be damaging in more assays are associated with increasing risk for breast cancer. For example, the 140 variants that either of their functional assays classified as damaging, even if the two assays were discordant, showed a combined increased risk for breast cancer (OR 2.29; 95% CI 1.97 – 2.66), while the 91-variant subset of these that were confirmed to be damaging in our screen showed a marginally increased risk (OR 2.95; 95% CI 2.36 – 3.69), and the risk conferred by 74 variants confirmed to be damaging in all three assays was even higher (OR 3.05; 2.39 – 3.90) (Supplemental Table 3 and 22 ).…”

“…Another recent large study functionally characterized 427 CHEK2 VUSs selected by the international ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium for variants with an association to an increased risk of breast cancer 22 . That study used two parallel functional assays that quantified the phosphorylation of the KRAB-associated protein (KAP1) at S473 and autophosphorylation of CHK2 at S516.…”

“…When compared to our functionally characterized CHK2 variants, the phosphorylation of KAP1 and CHK2 autophosphorylation experiments agreed in 322/427 (75.4%) and 318/327 (91.2%) of the variant classifications, respectively. However, this analysis does not include those classified as “functionally intermediate” in the ENIGMA 22 assays; the KAP1 phosphorylation and CHEK2 phosphorylation assays classified as intermediate 46 and 28 variants, respectively, that were tolerated in our screen, and another 12 and 16 variants, respectively, that were damaging in our screen. Compared with the concordant experimental data, our variant classifications were consistent in 337/427 (78.9%) of the variants.…”

“…Several different functional assays have been used to test individual and small sets of CHK2 missense variants identified in patients, including tests of protein stability, kinase activity toward various substrates, and activation by DNA damaging agents 22,23,29–32 . Another method to assay the function of CHK2 variants is to test their ability to complement a yeast RAD53 loss-of-function mutant ( rad53 ).…”

Prevalence of Variants of Uncertain Significance in Patients Undergoing Genetic Testing for Hereditary Breast and Ovarian Cancer and Lynch Syndrome

“…These assays are designed to assess the impact of a particular variant on the structure and functionality of the corresponding protein, providing an estimated VUS pathogenicity [37]. Large investigator consortia, like ENIGMA or INSIGHT, have started to form, with the aim of collecting a large amount of data on VUSs and subsequently using machine learning prediction models or functional assays in determining the significance of variants [39][40][41].…”