Integrative and systematic genomic approaches to improve diagnosis in rare and undiagnosed diseases: results from the RareBoost project

Abstract Rare and undiagnosed diseases pose diagnostic challenges due to phenotypic and genetic heterogeneity and the limitations of conventional molecular testing. As part of the RareBoost Project, we implemented an integrated, stepwise genomic strategy in a cohort of 134 patients from 120 families with previously inconclusive genetic testing. Our strategy combined systematic exome with subsequent genome sequencing when indicated, followed by targeted RNA-sequencing, and complemented with comprehensive phenotyping and follow-up segregation analysis. Definitive molecular diagnoses (pathogenic/likely pathogenic variants) were achieved in 25.0% of families, and an additional 25.8% harbored clinically relevant variants of uncertain significance with strong phenotypic correlation. This approach identified non-coding and structural variants while resolving cases through systematic reanalysis of previously negative data. Collectively, our findings demonstrate that integrated genomic and transcriptomic analyses with reanalysis of existing data enhance diagnostic yield in complex rare disease cohorts and provides a scalable framework for implementing genomic medicine in clinical practice.