Germline BRCA2 loss-of function variants, which can be identified through clinical genetic testing, predispose to several cancers^1,2,3,4,5. However, variants of uncertain significance limit the clinical utility of test results. Thus, there is a need for functional characterization and clinical classification of all BRCA2 variants to facilitate the clinical management of individuals with these variants. Here we analysed all possible single-nucleotide variants from exons 15 to 26 that encode the BRCA2 DNA-binding domain hotspot for pathogenic missense variants. To enable this, we used saturation genome editing CRISPR–Cas9-based knock-in endogenous targeting of human haploid HAP1 cells⁶. The assay was calibrated relative to nonsense and silent variants and was validated using pathogenic and benign standards from ClinVar and results from a homology-directed repair functional assay⁷. Variants (6,959 out of 6,960 evaluated) were assigned to seven categories of pathogenicity based on a VarCall Bayesian model⁸. Single-nucleotide variants that encode loss-of-function missense variants were associated with increased risks of breast cancer and ovarian cancer. The functional assay results were integrated into models from ClinGen, the American College of Medical Genetics and Genomics, and the Association for Molecular Pathology⁹ for clinical classification of BRCA2 variants. Using this approach, 91% were classified as pathogenic or likely pathogenic or as benign or likely benign. These classified variants can be used to improve clinical management of individuals with a BRCA2 variant.

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.

Prior studies have suggested the existence of reduced penetrance pathogenic variants (RPPVs) in BRCA1 and BRCA2 (BRCA) which pose challenges for patient counseling and care. Here, we sought to establish RPPVs as a new category of variants. Candidate BRCA RPPVs provided by two large clinical diagnostic laboratories were compiled to identify those with the highest likelihood of being a RPPV, based on concordant interpretations. Sixteen concordant candidate BRCA RPPVs across both laboratories were systematically assessed. RPPVs included missense, splice site, and frameshift variants. Our study establishes RPPVs as a new class of variants imparting a moderately increased risk of breast cancer, which impacts risk-informed cancer prevention strategies, and provides a framework to standardize interpretation and reporting of BRCA RPPVs. Further work to define clinically meaningful risk thresholds and categories for reporting BRCA RPPVs is needed to personalize cancer risks in conjunction with other risk factors.

Purpose Gross duplications are ambiguous in terms of clinical interpretation due to the limitations of the detection methods that cannot infer their context, namely, whether they occur in tandem or are duplicated and inserted elsewhere in the genome. We investigated the proportion of gross duplications occurring in tandem in breast cancer predisposition genes with the intent of informing their classifications. Methods The DNA breakpoint assay (DBA) is a custom, paired-end, next-generation sequencing (NGS) method designed to capture and detect deep-intronic DNA breakpoints in gross duplications in BRCA1, BRCA2, ATM, CDH1, PALB2, and CHEK2. Results DBA allowed us to ascertain breakpoints for 44 unique gross duplications from 147 probands. We determined that the duplications occurred in tandem in 114 (78%) carriers from this cohort, while the remainder have unknown tandem status. Among the tandem gross duplications that were eligible for reclassification, 95% of them were upgraded to pathogenic. Conclusion DBA is a novel, high-throughput, NGS-based method that informs the tandem status, and thereby the classification of, gross duplications. This method revealed that most gross duplications in the investigated genes occurred in tandem and resulted in a pathogenic classification, which helps to secure the necessary treatment options for their carriers.

Loss of function variants in the NF1 gene cause neurofibromatosis type 1, a genetic disorder characterized by complete penetrance, characteristic physical exam findings, and a substantially increased risk for malignancy. However, our understanding of the disorder is based on patients ascertained through phenotype-first approaches, which estimate prevalence at 1 in 3000. Leveraging a genotype-first approach in multiple large patient cohorts including over one million individuals, we demonstrate an unexpectedly high prevalence (1 in 1,286) of NF1 pathogenic variants. Half are identified in individuals lacking clinical features of NF1, with many appearing to have post-zygotic mosaicism for the identified variant. Incidentally discovered variants are not associated with classic neurofibromatosis features but are associated with an increased incidence of malignancy compared to control populations. Our findings suggest that NF1 pathogenic variants are substantially more common than previously thought, often characterized by somatic mosaicism and reduced penetrance, and are important contributors to cancer risk in the general population.