Assessment of the evidence yield for the calibrated PP3/BP4 computational recommendations

Purpose To investigate the number of rare missense variants observed in human genome sequences by ACMG/AMP PP3/BP4 evidence strength, following the calibrated PP3/BP4 computational recommendations. Methods Missense variants from the genome sequences of 300 probands from the Rare Genomes Project with suspected rare disease were analyzed using computational prediction tools able to reach PP3\_Strong and BP4\_Moderate evidence strengths (BayesDel, MutPred2, REVEL, and VEST4). The numbers of variants at each evidence strength were analyzed across disease-associated genes and genome-wide. Results From a median of 75.5 rare (≤1% allele frequency) missense variants in disease-associated genes per proband, a median of one reached PP3\_Strong, 3-5 PP3\_Moderate, and 3-5 PP3\_Supporting. Most were allocated BP4 evidence (median 41-49 per proband) or were indeterminate (median 17.5-19 per proband). Extending the analysis to all protein-coding genes genome-wide, the number of PP3\_Strong variants increased approximately 2.6-fold compared to disease-associated genes, with a median per proband of 1-3 PP3\_Strong, 8-16 PP3\_Moderate, and 10-17 PP3_Supporting. Conclusion A small number of variants per proband reached PP3\_Strong and PP3\_Moderate in 3,424 disease-associated genes, and though not the intended use of the recommendations, also genome-wide. Use of PP3/BP4 evidence as recommended from calibrated computational prediction tools in the clinical diagnostic laboratory is unlikely to inappropriately contribute to the classification of an excessive number of variants as Pathogenic or Likely Pathogenic by ACMG/AMP rules. ### Competing Interest Statement L.G.B. receives royalties from Wolters-Kluwer for authorship of UpToDate, is a member of the Illumina Medical Ethics Committee, and receives research support from Merck, Inc. V.P. and P.R. participated in the development of some of the tools assessed in this study. All other authors declare no conflict of interest. ### Funding Statement S.L.S. is supported by a fellowship from the Manton Center for Orphan Disease Research at Boston Children's Hospital. Data were provided by Broad Institute of MIT and Harvard Center for Mendelian Genomics with funding to A.O.D.L. by the National Human Genome Research Institute (NHGRI) grants UM1 HG008900 and U01 HG011755, and by the Chan Zuckerberg Initiative through an advised fund of the Silicon Valley Community Foundation grant 2020-224274. P.R. is supported by NHGRI grant U01 HG012022. E.N., M.S.G., and S.T. are supported by the NIH grant R01 CA264971. A.B.B. and S.M.H. are supported by NIH grant U24 HG006834. L.G.B. is supported by Z01 HG200328-18. ClinGen is primarily funded by the NHGRI with co-funding from the National Cancer Institute (NCI), through the following grants: U24 HG009649 (to Baylor/Stanford), U24 HG006834 (to Broad/Geisinger), and U24 HG009650 (to UNC/Kaiser). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All participants signed informed consent including the use of data for research purposes (Mass General Brigham IRB protocol 2016P001422). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript.