Tecleab, Yohannes A.

Abstract Germline BRCA2 loss-of function (LOF) variants identified by clinical genetic testing predispose to breast, ovarian, prostate and pancreatic cancer. However, variants of uncertain significance (VUS) (n>4000) limit the clinical use of testing results. Thus, there is an urgent need for functional characterization and clinical classification of all BRCA2 variants. Here we report on comprehensive saturation genome editing-based functional characterization of 97% of all possible single nucleotide variants (SNVs) in the BRCA2 DNA Binding Domain hotspot for pathogenic missense variants that is encoded by exons 15 to 26. The assay was based on deep sequence analysis of surviving endogenously targeted haploid cells. A total of 7013 SNVs were characterized as functionally abnormal (n=955), intermediate/uncertain, or functionally normal (n=5224) based on 95% agreement with ClinVar known pathogenic and benign standards. Results were validated relative to batches of nonsense and synonymous variants and variants evaluated using a homology directed repair (HDR) functional assay. Breast cancer case-control association studies showed that pooled SNVs encoding functionally abnormal missense variants were associated with increased risks of breast and ovarian cancer (odds ratio (OR) 3.89, 95%CI: 2.77-5.51). In addition, 86% of tumors associated with abnormal missense SNVs displayed loss of heterozygosity (LOH), whereas 26% of tumors with normal variants had LOH. The functional data were added to other sources of information in a ClinGen/ACMG/AMP-like model and 700 functionally abnormal SNVs, including 220 missense SNVs, were classified as pathogenic or likely pathogenic, while 4862 functionally normal SNVs, including 3084 missense SNVs, were classified as benign or likely benign. These classified variants can now be used for risk assessment and clinical care of variant carriers and the remaining functional scores can be used directly for clinical classification and interpretation of many additional variants.

10501 Background: Greater than 90% of missense variants in BRCA2 are classified as variants of uncertain significance (VUS). In addition, among the few known missense pathogenic variants (PVs) in BRCA2, breast cancer risk has been reported to be lower compared to pathogenic truncating variants (PTVs) (Li et al., 2022) . Herein, we aim to reclassify several missense variants in BRCA2 and investigate the associated breast cancer risk. Methods: A total of 450 BRCA2 missense variants in the C-terminal DNA binding domain (DBD) were evaluated for functional effect utilizing a previously validated homology-directed DNA double-strand repair (HDR) assay. The results of the functional evaluation along with clinical, structural, and in-silico data were applied to the rules-based ACMG/AMP criteria for variant classification to identify pathogenic/likely pathogenic (P/LP) variants and benign/likely benign (B/LB) variants. A pooled case-control analysis was performed comparing the frequency of reclassified P/LP missense variants among 82,372 women with breast cancer within the Ambry Genetics database as cases and 174,167 unaffected women within gnomAD, CARRIERS, and BRIDGES datasets as controls to evaluate associated breast cancer risk. Separate case-control analysis and comparisons with BRCA2 DBD PTVs, exon 11 PTVs, and known missense PV standards in ENIGMA were also performed. Results: From the 450 missense variants in the DBD, 137 (30.4%) were noted to be functionally deleterious and 313 (69.6%) were functionally neutral by the HDR assay. Incorporation of the functional and other data into an ACMG/AMP model led to the reclassification of 439 (97.6%) variants, with 131 as P/LP and 308 as B/LB variants. In a pooled case-control analysis, reclassified BRCA2 missense P/LP variants were associated with clinically significant increased risks of breast cancer (Odds Ratio (OR):5.6, 95%CI:3.9–8.0), whereas B/LB missense variants were not (OR:1.1, 95%CI:1.1–1.2). Similarly elevated risks of breast cancer were observed for BRCA2 PTVs in the DBD (OR:6.8, 95%CI:5.1–9.0) and missense PV standards in ENIGMA (OR:7.8, 95%CI:4.1–14.7). However, PTVs in exon 11 of BRCA2 were associated with a lower risk of breast cancer (OR:4.4, 95%CI:3.8–4.9) compared to PTVs in DBD, confirming the ovarian cancer cluster region effect in BRCA2. Conclusions: The study demonstrates that the HDR functional assay in conjunction with other clinical and genomic data can be a powerful tool that can aid in the reclassification of > 95% of missense variants in BRCA2. In addition, missense PVs in DBD were noted to have a similarly elevated risk of breast cancer as PTVs. The reclassification of the BRCA2 missense VUS and identification of associated breast cancer risk in this study will significantly influence breast cancer risk assessment and management strategies among carriers of these missense variants.