Recommendations for application of the functional evidence PS3/BS3 criterion using the ACMG/AMP sequence variant interpretation framework

Abstract

Background: The American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) clinical variant interpretation guidelines established criteria for different types of evidence. This includes the strong evidence codes PS3 and BS3 for "well-established" functional assays demonstrating a variant has abnormal or normal gene/protein function, respectively. However, they did not provide detailed guidance on how functional evidence should be evaluated, and differences in the application of the PS3/BS3 codes are a contributor to variant interpretation discordance between laboratories. This recommendation seeks to provide a more structured approach to the assessment of functional assays for variant interpretation and guidance on the use of various levels of strength based on assay validation.

Methods: The Clinical Genome Resource (ClinGen) Sequence Variant Interpretation (SVI) Working Group used curated functional evidence from ClinGen Variant Curation Expert Panel-developed rule specifications and expert opinions to refine the PS3/BS3 criteria over multiple in-person and virtual meetings. We estimated the odds of pathogenicity for assays using various numbers of variant controls to determine the minimum controls required to reach moderate level evidence. Feedback from the ClinGen Steering Committee and outside experts were incorporated into the recommendations at multiple stages of development.

Results: The SVI Working Group developed recommendations for evaluators regarding the assessment of the clinical validity of functional data and a four-step provisional framework to determine the appropriate strength of evidence that can be applied in clinical variant interpretation. These steps are as follows: (1) define the disease mechanism, (2) evaluate the applicability of general classes of assays used in the field, (3) evaluate the validity of specific instances of assays, and (4) apply evidence to individual variant interpretation. We found that a minimum of 11 total pathogenic and benign variant controls are required to reach moderate-level evidence in the absence of rigorous statistical analysis.

Conclusions: The recommendations and approach to functional evidence evaluation described here should help clarify the clinical variant interpretation process for functional assays. Further, we hope that these recommendations will help develop productive partnerships with basic scientists who have developed functional assays that are useful for interrogating the function of a variety of genes.

Keywords: Functional assays; Guidelines; Variant interpretation.

Fig. 1

Assembly of variant controls to set readout thresholds for normal and abnormal functions. Readout values across multiple specific instances of the same type can be plotted for any tested variant that reaches a likely benign/benign (LB/B) or likely pathogenic/pathogenic (LP/P) classification without PS3 or BS3 criteria. Each point on the plot represents the assay readout from a specific instance of an assay for the variant listed on the x-axis. Multiple points for the same variant indicate that the variant was tested in multiple specific instances of the same general class of assay. In this example, all LB/B variant controls (B1–B6) had readouts above 60%, with the exception of variant B6. When setting a readout threshold above which the readout is considered normal function, curators may draw this threshold at 60% and consider B6 to have an indeterminate readout. All LP/P variant controls (P1–P5) had readouts below 30%, with the exception of one specific instance for variant P1. With just 1 LB/B control variant with an indeterminate readout from a total of 11 variant controls (6 LB/B and 5 LP/P), PS3_moderate can be applied to variants with a readout indicating abnormal function and BS3_moderate can be applied to variants with a readout indicating normal function (see Additional file 1: Table S2). Variants of uncertain significance (VUS) tested on the same class of assay are plotted in the middle of the graph (indicated by light gray shading). VUS1 has an assay readout in the range of LB/B controls and would be above the threshold for normal function, so BS3_moderate could be applied. VUS3 has an assay readout consistent with LP/P control variants, below the threshold for abnormal function, so PS3_moderate could be applied. VUS2 has an indeterminate assay readout, so neither PS3_moderate nor BS3_moderate can be applied for this variant

Fig. 2

Decision tree for the evaluation of functional data for clinical variant interpretation. The SVI Working Group recommends that evaluators use a four-step process to determine the applicability and strength of evidence of functional assays for use in clinical variant interpretation (evidence codes PS3/BS3): (1) define the disease mechanism, (2) evaluate the applicability of general classes of assay used in the field, (3) evaluate the validity of specific instances of assays, and (4) apply evidence to individual variant interpretation