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Table 1 Text of original ACMG/AMP recommendation for functional assays, reproduced with permission [1]

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

“Functional studies can be a powerful tool in support of pathogenicity; however, not all functional studies are effective in predicting an impact on a gene or protein function. For example, certain enzymatic assays offer well-established approaches to assess the impact of a missense variant on enzymatic function in a metabolic pathway (e.g., α-galactosidase enzyme function). On the other hand, some functional assays may be less consistent predictors of the effect of variants on protein function. To assess the validity of a functional assay, one must consider how closely the functional assay reflects the biological environment. For example, assaying enzymatic function directly from biopsied tissue from the patient or an animal model provides stronger evidence than expressing the protein in vitro. Likewise, evidence is stronger if the assay reflects the full biological function of the protein (e.g., substrate breakdown by an enzyme) compared with only one component of function (e.g., adenosine triphosphate hydrolysis for a protein with additional binding properties). Validation, reproducibility, and robustness data that assess the analytical performance of the assay and account for specimen integrity, which can be affected by the method and time of acquisition, as well as storage and transport, are important factors to consider. These factors are mitigated in the case of an assay in a Clinical Laboratory Improvement Amendments laboratory–developed test or commercially available kit. Assays that assess the impact of variants at the messenger RNA level can be highly informative when evaluating the effects of variants at splice junctions and within coding sequences and untranslated regions, as well as deeper intronic regions (e.g., messenger RNA stability, processing, or translation). Technical approaches include direct analysis of RNA and/or complementary DNA derivatives and in vitro minigene splicing assays.”