Retrospective validation study demonstrates rapid functional reclassification of uncertain splicing variants in hereditary cancer genetics, highlighting a scalable diagnostic tool.
Key Points
To establish a fast, patient-RNA-free minigene workflow for functionally evaluating and reclassifying pre-mRNA splicing variants of uncertain significance in cancer diagnostics.
Analyzed next-generation sequencing data from a customized 77-gene cancer panel across 2,142 individuals.
Engineered a minigene assay using synthetic DNA and recombination-based cloning integrated with AlphaGenome computational predictions to test splicing outcomes without patient-derived RNA.
Identified 384 pathogenic or likely pathogenic variants across 54 genes (18% diagnostic yield) alongside a 17% rate of variants of uncertain significance in the initial cohort.
Successfully resolved variants with discordant computational predictions and reclassified previously uncertain splicing variants within clinically compatible reporting timeframes.