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

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.

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.