Functional analysis and clinical classification of 462 germline BRCA2 missense variants affecting the DNA binding domain

Abstract

Variants of uncertain significance (VUSs) in BRCA2 are a common result of hereditary cancer genetic testing. While more than 4,000 unique VUSs, comprised of missense or intronic variants, have been identified in BRCA2, the few missense variants now classified clinically as pathogenic or likely pathogenic are predominantly located in the region encoding the C-terminal DNA binding domain (DBD). We report on functional evaluation of the influence of 462 BRCA2 missense variants affecting the DBD on DNA repair activity of BRCA2 using a homology-directed DNA double-strand break repair assay. Of these, 137 were functionally abnormal, 313 were functionally normal, and 12 demonstrated intermediate function. Comparisons with other functional studies of BRCA2 missense variants yielded strong correlations. Sequence-based in silico prediction models had high sensitivity, but limited specificity, relative to the homology-directed repair assay. Combining the functional results with clinical and genetic data in an American College of Medical Genetics (ACMG)/Association for Molecular Pathology (AMP)-like variant classification framework from a clinical testing laboratory, after excluding known splicing variants and functionally intermediate variants, classified 431 of 442 (97.5%) missense variants (129 as pathogenic/likely pathogenic and 302 as benign/likely benign). Functionally abnormal variants classified as pathogenic by ACMG/AMP rules were associated with a slightly lower risk of breast cancer (odds ratio [OR] 5.15, 95% confidence interval [CI] 3.43-7.83) than BRCA2 DBD protein truncating variants (OR 8.56, 95% CI 6.03-12.36). Overall, functional studies of BRCA2 variants using validated assays substantially improved the variant classification yield from ACMG/AMP models and are expected to improve clinical management of many individuals found to harbor germline BRCA2 missense VUS.

Keywords: BRCA2; HDR; VUS; cancer; predisposition genes; variant classification.

Figure 1

HDR score of 462 BRCA2 missense variants Plot of 462 BRCA2 missense variants affecting the DBD ordered by amino acid position (x axis) against HDR score with 95% CIs. The thresholds for functionally abnormal (HDR < 1.49) and functionally normal (HDR > 2.5) were plotted as horizontal dotted lines. Variants reported in ClinVar were categorized by color.

Figure 2

Characteristics of 462 BRCA2 missense variants CIRCOS plot of BRCA2 missense variants. (1) Variant; (2) HDR class (blue: functionally normal, red: functionally abnormal); (3) histogram of HDR score with individual HDR 95% CI upper (red) and 95% CI lower (blue) (orange line indicates 1.49 cutoff point, gray line indicates 2.52 cutoff point); (4) splicing effect (blue: no splicing effect; red: known splicing effect; yellow: predicted splicing effect by SpliceAI); (5–8) MANO-B four drugs classification (olaparib, niraparib, rucaparib, and carboplatin [CBDCA]); (9) prime editing classification (blue: functional, red: nonfunctional, yellow: uncertain); (10–11) mESC functional assay result (Sharan^,, and Vreeswijk; blue: functional, red: nonfunctional, yellow: intermediate); (12) protein structure domain (HD, OB1, tower, OB2, OB3) with yellow dot indicating DSS1 contacting residue and red dot indicating single-stranded DNA (ssDNA) contacting residue.

Figure 3

Correlation of in silico prediction model scores with HDR Heatmaps indicating the correlation between HDR scores and the BayesDel in silico prediction model for BRCA2 effect. The heatmap contains 25 equal categories of BayesDel scores (y axis) and 25 categories for HDR score (x axis). Red dashed lines reflect thresholds for Ambry BRCA2-specific BayesDel functionally normal (0.056) and functionally abnormal (0.431). HDR score thresholds are <1.50 for functionally abnormal and >2.50 for functionally normal.

Figure 4

Reclassification of missense variants in ClinVar Changes in classification of 442 missense variants including 386 from ClinVar (left side) to the updated classification from all sources (updated; right side). Categories of variants are labeled by color.

Figure 5

Lifetime breast cancer absolute risks associated with BRCA2 variants Lifetime risks were estimated by combining case-control OR estimates for non-Hispanic White breast cancer subjects (breast cancer subjects subjected to cancer testing by Ambry Genetics versus gnomAD reference controls) with prevalence in the SEER registry. Lifetime risk curves include BRCA2 missense variants classified as benign (B) or likely benign (LB) (classified B or LB missense variants); BRCA2 missense variants classified as pathogenic (P) or likely pathogenic (LP) (classified as P or LP missense variants); pathogenic missense standards; BRCA2 protein truncating variants (PTVs) including frameshift and stop-gain variants affecting the DNA binding domain (DBD PTV); in exon 11 (exon 11 PTV); and general population risk of breast cancer (general population).