Clinical validation of RNA sequencing for Mendelian disorder diagnostics

Despite rapid advancements in clinical sequencing, over half of diagnostic evaluations still lack definitive results. RNA sequencing (RNA-seq) has shown promise in research settings for bridging this gap by providing essential functional data for accurate interpretation of diagnostic sequencing results. However, despite advanced research pipelines, clinical translation of diagnostic RNA-seq has not yet been realized. We have developed and validated a clinical diagnostic RNA-seq test for individuals with suspected genetic disorders who have existing or concurrent comprehensive DNA diagnostic testing. This diagnostic RNA-seq test processes RNA samples from fibroblasts or blood and derives clinical interpretations based on the analytical detection of outliers in gene expressions and splicing patterns. The clinical validation involves 130 samples, including 90 negative and 40 positive samples. We developed provisional expression and splicing benchmarks using short-read and long-read RNA-seq data from the GM24385 lymphoblastoid sample produced by the Genome in a Bottle Consortium. For clinical validation, we first established reference ranges for each gene and junction based on expression distributions from our control data. We then evaluated the clinical performance of our outlier-based pipeline using 40 positive samples with previously identified diagnostic findings from the Undiagnosed Diseases Network project. Our study provides a paradigm and necessary resources for independent laboratories to validate a clinical RNA-seq test.