Classification of BRCA2 Variants of Uncertain Significance (VUS) Using an ACMG/AMP Model Incorporating a Homology-Directed Repair (HDR) Functional Assay

Abstract

Purpose: The identification of variants of uncertain significance (VUS) in the BRCA1 and BRCA2 genes by hereditary cancer testing poses great challenges for the clinical management of variant carriers. The ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) variant classification framework, which incorporates multiple sources of evidence, has the potential to establish the clinical relevance of many VUS. We sought to classify the clinical relevance of 133 single-nucleotide substitution variants encoding missense variants in the DNA-binding domain (DBD) of BRCA2 by incorporating results from a validated functional assay into an ACMG/AMP-variant classification model from a hereditary cancer-testing laboratory.

Experimental design: The 133 selected VUS were evaluated using a validated homology-directed double-strand DNA break repair (HDR) functional assay. Results were combined with clinical and genetic data from variant carriers in a rules-based variant classification model for BRCA2.

Results: Of 133 missense variants, 44 were designated as non-functional and 89 were designated as functional in the HDR assay. When combined with genetic and clinical information from a single diagnostic laboratory in an ACMG/AMP-variant classification framework, 66 variants previously classified by the diagnostic laboratory were correctly classified, and 62 of 67 VUS (92.5%) were reclassified as likely pathogenic (n = 22) or likely benign (n = 40). In total, 44 variants were classified as pathogenic/likely pathogenic, 84 as benign/likely benign, and 5 remained as VUS.

Conclusions: Incorporation of HDR functional analysis into an ACMG/AMP framework model substantially improves BRCA2 VUS re-classification and provides an important tool for determining the clinical relevance of individual BRCA2 VUS.

Figure 1.

Homology-directed repair (HDR) assay evaluation of clinically observed BRCA2 DNA-binding domain (DBD) missense variants. HDR scores with 95% confidence intervals (CI) are presented on a 1 to 5 scale based on HDR activity of wild-type BRCA2 (HDR score = 5) and the p.Asp2723His non-functional pathogenic variant (HDR score = 1). HDR thresholds for functional (>2.50) and non-functional (<1.49) are indicated by horizontal dotted lines. Variants are ordered on the basis of amino acid position (x-axis).

Figure 2.

Incorporation of HDR functional data into a clinical ACMG/AMP framework for variant classification. BRCA2 DBD missense variants observed clinically by GeneDx were classified by an ACMG/AMP-like framework into five categories: PATH (pathogenic), LPATH (likely pathogenic), VUS (variants of uncertain significance), LBEN (likely benign), BEN (benign). The PATH and LPATH categories were combined, LBEN and BEN categories were combined. The number of variants in each classification category defined by ClinVar (ClinGen approved laboratories excluding GeneDx) and by GeneDx before and after applying HDR functional data are shown.