The clinical utility of genomic testing is constrained by variants of uncertain significance (VUS), which complicate diagnostic interpretation and patient management. The ACMG/AMP PS4 criterion, “prevalence in affected individuals statistically increased compared to controls,” offers strong evidence for pathogenicity but is often challenging to apply due to the limited availability of robust, matched case-control genomic and phenotypic data. Further, there are currently no options available to score evidence from case-control studies towards benignity.

We propose and validate a new code, RWE (real world evidence), by integrating de-identified, longitudinal clinical data with variant carriers and non-carriers identified from exome or genome sequence data across three large-scale clinicogenomic datasets: the Helix Research Network (HRN) dataset, UK Biobank (UKB) and All of US (AoU). Phenotypes for established gene-level disease associations were compiled from the longitudinal medical records of the individuals, enabling rigorous variant-specific case-control analyses from population data. This RWE approach was systematically applied to all variants, including previously identified VUS in clinically relevant genes, powering our VUS Early Surveillance Platform.

Across 20 hereditary cancer and cardiovascular genes, the application of RWE provided sufficient evidence to reclassify a VUS in 32% of VUS carriers–99.7% to B/LB and 0.3% to P/LP–directly resolving their ambiguous status. This reclassification rate varied by gene, ranging from 0.7% for BRCA2 up to 50% for LDLR.

The systematic integration of Real-World Evidence from large-scale clinicogenomic datasets into the ACMG/AMP scoring rubric through our newly developed and statistically robust RWE category is a significant improvement to variant interpretation that is projected to resolve over 50% of VUS carriers once longitudinal clinico-genomic databases are available for ~3M individuals. This approach markedly reduces the burden of Variants of Uncertain Significance, provides more definitive diagnoses for a substantial proportion of previously unresolved cases, and ultimately increases the clinical utility and adoption of genomic testing, representing a critical advancement for precision medicine.

KMSB, MJF, SC, IT, DI, NLW, CH, WL, AB, JL, and ETC are employees of Helix.

Funding was provided to the Healthy Nevada Project by the Renown Institute for Health Innovation and the Renown Health Foundation. The Healthy Nevada Project receives funding from Gilead Sciences, outside the scope of this research. Funding was provided to the myGenetics program by HealthPartners. This research also used data assets made available by National Safe Haven as part of the Data and Connectivity National Core Study, led by Health Data Research UK in partnership with the Office for National Statistics and funded by UK Research and Innovation(grant ref MC_PC_20058).

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:

The Helix cohorts were reviewed by Salus IRB (Reliance on Salus for all sites) and approved (approval number 21143), the WCG IRB (Western Institutional Review Board, WIRB-Copernicus Group) and approved (approval number 20224919), the MUSC Institutional Review Board for Human Research and approved (approval number Pro00129083), and the University of Nevada, Reno Institutional Review Board and approved (approval number 7701703417). The UKB study was approved by the North West Multicenter Research Ethics Committee, UK. The All of Us study is approved by the All of Us IRB. All data used for research were de-identified.

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).

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I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

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UKB data are available for download (https://www.ukbiobank.ac.uk/) to qualified researchers. The HRN data are available to qualified researchers upon reasonable request and with permission of the HRN Steering Committee and Helix. Researchers who would like to obtain the raw genotype data related to this study will be presented with a Data Use Agreement which requires that participants will not be reidentified and no data will be shared between individuals, third parties, or uploaded onto public domains. The HRN encourages collaboration with scientific researchers on an individual basis. Examples of restrictions that will be considered in requests to data access include but are not limited to: 1. Whether the request comes from an academic institution in good standing and will collaborate with our team to protect the privacy of the participants and the security of the data requested 2. Type and amount of data requested 3. Feasibility of the research suggested 4. Amount of resource allocation for Helix and HRN member institutions required to support a collaboration.