Assessment of the Clinical Relevance of BRCA2 Missense Variants by Functional and Computational Approaches

Abstract

Many variants of uncertain significance (VUS) have been identified in BRCA2 through clinical genetic testing. VUS pose a significant clinical challenge because the contribution of these variants to cancer risk has not been determined. We conducted a comprehensive assessment of VUS in the BRCA2 C-terminal DNA binding domain (DBD) by using a validated functional assay of BRCA2 homologous recombination (HR) DNA-repair activity and defined a classifier of variant pathogenicity. Among 139 variants evaluated, 54 had ?99% probability of pathogenicity, and 73 had ?95% probability of neutrality. Functional assay results were compared with predictions of variant pathogenicity from the Align-GVGD protein-sequence-based prediction algorithm, which has been used for variant classification. Relative to the HR assay, Align-GVGD significantly (p < 0.05) over-predicted pathogenic variants. We subsequently combined functional and Align-GVGD prediction results in a Bayesian hierarchical model (VarCall) to estimate the overall probability of pathogenicity for each VUS. In addition, to predict the effects of all other BRCA2 DBD variants and to prioritize variants for functional studies, we used the endoPhenotype-Optimized Sequence Ensemble (ePOSE) algorithm to train classifiers for BRCA2 variants by using data from the HR functional assay. Together, the results show that systematic functional assays in combination with in silico predictors of pathogenicity provide robust tools for clinical annotation of BRCA2 VUS.

Keywords: BRCA2; VUS; cancer predisposition; functional assay.

Figure 1

Influence of BRCA2-DBD Missense Variants on HDR Activity in Relation to Known Pathogenic and Neutral Variants Defined as Class 1, 4, or 5 in a Multifactorial Likelihood Model The model-based HDR fold change with SE is displayed on a logarithmic scale for the class 1, unclassified, class 4, and class 5 variants. Solid lines represent 99% probability of pathogenicity and 99% probability of neutrality (fold increase in GFP(+) cells < 1.66 for deleterious variants and > 2.41 for neutral variants). Dotted lines represent 95% probability of pathogenicity and 95% probability of neutrality (fold increase in GFP(+) cells < 1.78 for deleterious variants and > 2.25 for neutral variants).

Figure 2

Comparison of HDR Activity and Align-GVGD Prediction for 139 BRCA2 Variants (A) HDR normalized fold changes for 139 BRCA2-DBD missense changes are displayed for each of the seven Align-GVGD classification grades (C0–C65). (B and C) 73 BRCA2-DBD missense changes with >95% probability in favor of neutrality (B) and 58 BRCA2-DBD missense changes with >95% probability in favor of pathogenicity (C) are displayed for each of the seven Align-GVGD classification grades (C0–C65).

Figure 3

Plot of VarCall-Estimated Variant Effects Shows Individual Variants Plotted against HDR Activity Variants are shown as the natural log of the normalized fold change in HDR activity. Error bars represent SE within individual measurements for each variant. The probability of pathogenicity (Pr(eta\data)) based on the lower pathogenic component and upper neutral component is plotted in the right-hand margin. The wild-type (WT), p.Tyr3098His positive controls, p.Asp2723His negative control, known pathogenic variants, known neutral variants, and VUS are color coded as indicated. Variants in green (IARC < 3) are classes 1 and 2. Variants shown in pink (IARC > 3) are classes 4 and 5.

Figure 4

ROC Curves for the Continuous-Valued ePOSE Score and Variants Defined as Pathogenic or Neutral For each of five variant groupings, P is the upper-bound HDR fold-change threshold for pathogenicity, and N is the lower-bound threshold for defining a variant as neutral. The color-coded inset shows the AUC at each of the five groupings.