Quantifying the potential of functional evidence to reclassify variants of uncertain significance in the categorical and Bayesian interpretation frameworks

Abstract

Additional variant interpretation tools are required to effectively harness genomic sequencing for clinical applications. The American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP) published guidelines for clinical sequence variant interpretation, incorporating different types of data that lend varying levels of support towards a benign or pathogenic interpretation. Variants of uncertain significance (VUS) are those with either contradictory or insufficient evidence, and their uncertainty complicates patient counseling and management. Functional assays may provide a solution to evidence gaps relegating variants to the VUS category, but the impact of functional evidence in this framework has not been assessed. We employ an algorithmic analysis of the ACMG/AMP combining rules to assess how the availability of strong functional evidence could theoretically improve the ability to make a benign or pathogenic assertion. We follow this with analysis of actual evidence combinations met by variants through expert curations as part of the Clinical Genome Resource (ClinGen). We also examine the impact of functional evidence in a Bayesian adaptation of the ACMG/AMP framework. This lays the groundwork for an evidence-based prioritization of assay development and variant assessment by identifying genes and variants that may benefit the most from functional data.

Keywords: ACMG/AMP guidelines; Bayesian; VUS; functional assay; variant interpretation.

Figure 1. ACMG/AMP categorical combining rules for variant classification.

Possible evidence combinations are represented alongside the corresponding variant classification according to Richards et al. 2015 combining rules. The first letter of each evidence code indicates support towards a pathogenic (P) or benign (B) classification and the second letter indicates the assigned evidence strength: supporting (P), moderate (M), strong (S), very strong (VS), or stand-alone (A). Evidence code boxes are colored by evidence strength. Final classifications are determined by combinations of evidence codes resulting in the following: pathogenic, likely pathogenic, variant of uncertain significance, likely benign, or benign.

Figure 2. ACMG/AMP evidence codes for classifying sequence variants as applicable to variant molecular consequence.

The first letter of each evidence code indicates support towards a pathogenic (P) or benign (B) variant classification, and is followed by an indicated weight of evidence: supporting (P), moderate (M), strong (S), very strong (VS) or stand-alone (A). See Richards et al. 2015 for a complete description of each code. Variant type refers to the molecular consequence (truncating, missense, in-frame indel, synonymous). If evidence applies to the specific variant type, it is indicated with a “/”, while evidence that does not apply is indicated with an “X”.

Figure 3. Reclassifying missense VUS evidence combinations in the categorical ACMG/AMP framework and the adapted Bayesian framework.

(A) Modeling the addition of well-validated functional evidence showing a damaging effect (PS3) or no effect (BS3) to non-conflicting missense variant evidence combinations in the categorical ACMG/AMP framework described by Richards et al. (2015) finds 97% of combinations would be reclassified with functional data. (B) Theoretical conflicting and non-conflicting missense evidence combinations resulting in a VUS classification in the categorical ACMG/AMP combining rules (Prior class categorical) were entered into the Bayesian adaptation described by Tavtigian et al. (2018) and the corresponding variant classifications are shown (New class Bayesian). (C) From the theoretical missense evidence combinations resulting in a VUS classification in (B), we examined those that did not include PS3 or BS3 criteria (Prior Class Bayesian). Modeling the addition of well-validated functional evidence (BS3 or PS3) reclassified all combinations (New class Bayesian) by posterior probability calculations in the Bayesian framework described by Tavtigian et al. (2018). ACMG/AMP, American College of Medical Genetics and Genomics/Association for Molecular Pathology; B, benign; BS3, well-established functional studies show no deleterious effect; LB, likely benign; LP, likely pathogenic; P, pathogenic; PS3, well established functional studies show a deleterious effect; VUS, variant of uncertain significance

Figure 4. Non-conflicting missense variants classified as VUS by expert panels.

^aEvidence Satisfied, as determined by expert panels in Gelb et al. (2018) and Kelly et al. (2018), where abbreviations indicate specific criteria described in Richards et al. (2015). Boxes are colored according to evidence strength. ^bVariant listed by gene, cDNA, and protein. Variants were curated by expert panels using the following reference transcripts: HRAS (NM_005343.2), MAP2K2 (NM_030662.3), RAF1 (NM_002880.3), SHOC2 (NM_007373.3), and MYH7 (NM_000257.3). ^c Variant class assessed according to categorical ACMG/AMP combining rules (Table 5 in Richards et al., 2015), with Expert Panel (EP) variant classifications considering only evidence satisfied in (a). ^dVariant class with the addition of either PS3 or BS3 criteria in the categorical interpretation framework (Table 5 in Richards et al.). ^eVariant class in Bayesian-adapted framework was determined by posterior probability of pathogenicity (Post_P). Post_P was calculated by entering evidence totals by strength in Supplemental Table 1 from Tavtigian et al. (2018), using the default settings: Prior Probability (Prior_P) = 0.100; Odds Path Very Strong = 350; Exponential progression (X) = 2.000. ^fThese column shows the Post_P value if a strong pathogenic criteria (PS3) or strong benign criteria (BS3) were to be added within the quantitative framework as described in (e). Box colors in (c-f) are coded by variant classification. BS3, well-established functional studies show no deleterious effect; EP, expert panel; LBEN, likely benign; LPATH, likely pathogenic; N/A, none applied; PS3, well established functional studies show a deleterious effect; VUS, variant of uncertain significance

Figure 5. Expert-curated missense variants with conflicting evidence.

^aEvidence Satisfied, as determined by expert panels in Gelb et al. (2018) and Kelly et al. (2018), where abbreviations indicate specific criteria described in Richards et al. (2015). Boxes are colored according to evidence strength. ^bVariant listed by gene, cDNA, and protein. Variants were curated by expert panels using the following reference transcripts: SOS1 (NM_005633.3), PTPN11 (NM_002834.3), and MYH7 (NM_000257.3). ^cExpert Panel (EP) Class as determined by CMP-EP or RAS-EP using modified categorical ACMG/AMP combining rules. ^dVariant class in Bayesian-adapted framework was determined by posterior probability of pathogenicity (Post_P). Post_P was calculated by entering evidence totals by strength in Supplemental Table 1 from Tavtigian et al. (2018), using the default settings: Prior Probability (Prior_P) = 0.100; Odds Path Very Strong = 350; Exponential progression (X) = 2.000. ^e,fThese column shows the Post_P value if a strong pathogenic criteria (e) or strong benign criteria (f) were to be added within the quantitative framework as described in (d). Box colors in (c-f) are coded by variant classification. BS3, well-established functional studies show no deleterious effect; EP, expert panel; LBEN, likely benign; LPATH, likely pathogenic; N/A, none applied; PATH, pathogenic; PS3, well established functional studies show a deleterious effect; VUS, variant of uncertain significance