Integration of functional assay data results provides strong evidence for classification of hundreds of BRCA1 variants of uncertain significance

Abstract

Purpose: BRCA1 pathogenic variant heterozygotes are at a substantially increased risk for breast and ovarian cancer. The widespread uptake of testing has led to a significant increase in the detection of missense variants in BRCA1, the vast majority of which are variants of uncertain clinical significance (VUS), posing a challenge to genetic counseling. Here, we harness a wealth of functional data for thousands of variants to aid in variant classification.

Methods: We have collected, curated, and harmonized functional data for 2701 missense variants representing 24.5% of possible missense variants in BRCA1. Results were harmonized across studies by converting data into binary categorical variables (functional impact versus no functional impact). Using a panel of reference variants we identified a subset of assays with high sensitivity and specificity (≥80%) and apply the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) variant interpretation guidelines to assign evidence criteria for classification.

Results: Integration of data from validated assays provided ACMG/AMP evidence criteria in favor of pathogenicity for 297 variants or against pathogenicity for 2058 representing 96.2% of current VUS functionally assessed. We also explore discordant results and identify limitations in the approach.

Conclusion: High quality functional data are available for BRCA1 missense variants and provide evidence for classification of 2355 VUS according to their pathogenicity.

Keywords: ACMG/AMP guidelines; BRCA1; breast and ovarian cancer; functional assays; variants of uncertain significance.

Fig. 1. Overview of functional track assessment.

Subway chart illustrating the processes and stages of data collection, curation, and harmonization of functional data (green line); harmonization of reference variant classification (red line); and data analysis and evidence criteria assignment (yellow line). VUS variant of uncertain significance.

Fig. 2. Track validation.

(a) Specificity and sensitivity of 22 tracks. Side color bars indicate different classes of assays. Blue, green, and red bars represent sensitivity/specificity, lower and upper bound of 95% confidence interval (CI) respectively. Tracks in blue font were validated (specificity/sensitivity ≥80%) and tracks in black font were not. (b) Fraction of variants receiving different American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) evidence criteria.

Fig. 3. Overview of individual variant assessment using Hi Set.

Subway chart illustrating the stages of evidence criteria assignment to variants of uncertain significance (VUS) (yellow line) and estimation of sensitivity and specificity of the integrated analysis (red line). Dotted red line represents the estimation of sensitivity and specificity with prior knowledge of which variants affected splicing.

Fig. 4. Comparison of evidence criteria assignment for Hi Set and majority voting approaches.

Graphs show the fraction of each evidence criteria variants were assigned under the two alternative scenario, the percentage of VUS assigned and the estimated error rate.

Fig. 5. Location of pathogenic variants.

(a) Fraction of different evidence criteria assignments by 100–amino acid windows. The RING finger and the BRCT domains are indicated by red lines. The number of measurements (number of reported results from assays for all variants) in the 100–amino acid windows is shown in red font. (b) Fraction of different evidence criteria assignments by secondary structures and connecting loops in the BRCT domains.