Strong functional data for pathogenicity or neutrality classify BRCA2 DNA-binding-domain variants of uncertain significance

Abstract

Determination of the clinical relevance of rare germline variants of uncertain significance (VUSs) in the BRCA2 cancer predisposition gene remains a challenge as a result of limited availability of data for use in classification models. However, laboratory-based functional data derived from validated functional assays of known sensitivity and specificity may influence the interpretation of VUSs. We evaluated 252 missense VUSs from the BRCA2 DNA-binding domain by using a homology-directed DNA repair (HDR) assay and identified 90 as non-functional and 162 as functional. The functional assay results were integrated with other available data sources into an ACMG/AMP rules-based classification framework used by a hereditary cancer testing laboratory. Of the 186 missense variants observed by the testing laboratory, 154 were classified as VUSs without functional data. However, after applying protein functional data, 86% (132/154) of the VUSs were reclassified as either likely pathogenic/pathogenic (39/132) or likely benign/benign (93/132), which impacted testing results for 1,900 individuals. These results indicate that validated functional assay data can have a substantial impact on VUS classification and associated clinical management for many individuals with inherited alterations in BRCA2.

Keywords: ACMG/AMP; BRCA2; breast cancer; functional assay; predisposition gene; variant of uncertain significance.

Figure 1

Homology-directed repair (HDR) activity HDR activity of 252 BRCA2 DNA-binding-domain (DBD) missense variants based on an HDR DR-GFP assay. The HDR fold-change based on proportions of GFP positive cells resulting from HDR activity is displayed on a linear scale between 1 (non-functional, p.Asp2723His) and 5 (functional, wild-type). Functional variants are shown in light gray, and non-functional variants are shown in dark gray. The 95% confidence intervals (CIs) for the HDR scores are included as a measure of the reproducibility of the HDR assay for each variant. Horizontal dotted lines represent 99% probability of pathogenicity (fold increase in GFP [+] cells < 1.66) and 95% probability of neutrality (fold increase in GFP [+] cells > 2.25). BRCA2 DBDs are denoted above each section, with the tower domain in OB2 indicated by a bracket. Different amino acid substitutions at the same position are grouped by brackets.

Figure 2

Variant workflow Summary of the number of variants subjected to the HDR assay and to variant interpretation. The number of variants within each classification outcome (LP/P, VUS, and LB/B) (first bullet, lower panels) and classification rates for VUS in the absence of protein functional data (second bullet, lower panels) are shown. P, pathogenic; LP, likely pathogenic; VUS, variant of uncertain significance; LB, likely benign; B, benign.

Figure 3

Influence of HDR functional data on variant classification with an ACMG/AMP-like model Classification of variants before (black bars) and after (gray bars) the application of protein functional data. The number of variants is indicated above the bar. P, pathogenic; LP, likely pathogenic; VUS, variant of uncertain significance; LB, likely benign; B, benign.

Figure 4

Final variant classification compared to ClinVar assertions Variants are grouped according to final classification (LP/P, VUS, LB/B, and unobserved) and further subdivided into the general assertion provided by ClinVar (LP/P, VUS, and LB/B). Each bar is shaded to indicate a conflicting ClinVar assertion (dark gray) or a non-conflicting assertion (light gray). Categories without a conflicting or non-conflicting sub-category (VUS) are shown as ”0.” The total number of variants in each final classification category is represented in parentheses, and the number of variants designated as conflicting or non-conflicting is indicated within the bar. P, pathogenic; LP, likely pathogenic; VUS, variant of uncertain significance; LB, likely benign; B, benign.