From Uncertain to Actionable: Significant Reduction in Variants of Uncertain Significance in Hereditary Germline Testing via Multi-Institutional Real-World Evidence

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.