Closing the gap: Systematic integration of multiplexed functional data resolves variants of uncertain significance in BRCA1, TP53, and PTEN

Abstract

Clinical interpretation of missense variants is challenging because the majority identified by genetic testing are rare and their functional effects are unknown. Consequently, most variants are of uncertain significance and cannot be used for clinical diagnosis or management. Although not much can be done to ameliorate variant rarity, multiplexed assays of variant effect (MAVEs), where thousands of single-nucleotide variant effects are simultaneously measured experimentally, provide functional evidence that can help resolve variants of unknown significance (VUSs). However, a rigorous assessment of the clinical value of multiplexed functional data for variant interpretation is lacking. Thus, we systematically combined previously published BRCA1, TP53, and PTEN multiplexed functional data with phenotype and family history data for 324 VUSs identified by a single diagnostic testing laboratory. We curated 49,281 variant functional scores from MAVEs for these three genes and integrated four different TP53 multiplexed functional datasets into a single functional prediction for each variant by using machine learning. We then determined the strength of evidence provided by each multiplexed functional dataset and reevaluated 324 VUSs. Multiplexed functional data were effective in driving variant reclassification when combined with clinical data, eliminating 49% of VUSs for BRCA1, 69% for TP53, and 15% for PTEN. Thus, multiplexed functional data, which are being generated for numerous genes, are poised to have a major impact on clinical variant interpretation.

Keywords: BRCA1; MAVE; PTEN; TP53; functional data; variant interpretation.

Figure 1

Missense variants of uncertain significance are a large and growing problem (A) Single-nucleotide missense variants colored by ClinVar classifications (benign = 25,707; likely benign = 16,377; VUSs = 227,365; likely pathogenic = 14,716; pathogenic = 22,489; conflicting interpretations = 20,026). ClinVar data downloaded on 10/27/2020. (B) Missense variants in ClinVar from 2015 to 2020 shown by clinical significance.

Figure 2

Schematic for integration of multiplexed functional data into clinical variant interpretation Top: we first collected variant function scores and determined assay dynamic range and sensitivity and specificity for established pathogenic and benign variants. If a single assay had high sensitivity and specificity, we used the function scores directly to determine which variants were functionally normal and functionally abnormal. Where possible, we combined multiple MAVE datasets to increase predictive value of function scores and determine the functional class of variants. Finally, we computed the odds of pathogenicity for the assigned functional classes to determine the strength of evidence assigned to each dataset. Bottom: existing evidence for 324 VUSs were combined with the MAVE functional evidence to reinterpret variants as either likely pathogenic (orange), likely benign (blue), or VUSs (gray).

Figure 3

Function scores for BRCA1, TP53, and PTEN variants of known effect Histograms of function scores for variants colored by their ClinVar interpretations for each multiplexed functional assay in the left column and nonsense and synonymous variant distributions in the right column. (A and B) Function scores for BRCA1 derived from saturation genome editing in a BRCA1-deficient HAP1 cell line. (C–J) Function scores for TP53 derived from four different assays. From top to bottom: TP53-null A549 cell line with positive selection for loss-of-function variants with etoposide. TP53-null A549 cell line with negative selection for loss-of-function variants with nutlin-3. TP53-wild-type A549 cell line with positive selection for dominant negative variants with nutlin-3. TP53-wild-type AML reporter cell line with positive selection for dominant negative variants with nutlin-3. (K–N) Function scores for PTEN derived from two different assays. From top to bottom: PTEN variant abundance assayed in a HEK293 cell line and PTEN variant phosphatase activity in a humanized yeast system. Histogram color indicates known clinical effect as reported in the ClinVar database (dark blue, benign; light blue, likely benign; light red, likely pathogenic; dark red, pathogenic).

Figure 4

Reinterpretation of BRCA1, TP53, and PTEN VUSs with multiplexed functional data (A–C) Original variant classifications from Ambry Genetics. (D–F) Variant classifications after reinterpretation with existing evidence and multiplexed functional data. Dashed sections represent the proportion of VUSs reclassified to either likely pathogenic or likely benign.

Figure 5

Strength of evidence that could be assigned to variants of ACMG Secondary Findings v3.0 genes with hypothetical MAVEs that perfectly distinguish between pathogenic and benign controls