Web-accessible application for identifying pathogenic transcripts with RNA-seq: Increased sensitivity in diagnosis of neurodevelopmental disorders

• Published in: American journal of human genetics Vol. 110; no. 2; pp. 251 - 272
• Main Authors: Dekker, Jordy, Schot, Rachel, Bongaerts, Michiel, de Valk, Walter G., van Veghel-Plandsoen, Monique M., Monfils, Kathryn, Douben, Hannie, Elfferich, Peter, Kasteleijn, Esmee, van Unen, Leontine M.A., Geeven, Geert, Saris, Jasper J., van Ierland, Yvette, Verheijen, Frans W., van der Sterre, Marianne L.T., Sadeghi Niaraki, Farah, Smits, Daphne J., Huidekoper, Hidde H., Williams, Monique, Wilke, Martina, Verhoeven, Virginie J.M., Joosten, Marieke, Kievit, Anneke J.A., van de Laar, Ingrid M.B.H., Hoefsloot, Lies H., Hoogeveen-Westerveld, Marianne, Nellist, Mark, Mancini, Grazia M.S., van Ham, Tjakko J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02.02.2023; Elsevier
• Subjects: Cycloheximide; Gene Expression Profiling; Humans; molecular diagnostics; mRNA splicing; neurodevelopmental disorder; Neurodevelopmental Disorders - diagnosis; Neurodevelopmental Disorders - genetics; RNA-Seq; RNA-sequencing; Sequence Analysis, RNA - methods; transcriptomics; web browser application; RNA-sequencing; transcriptomics; molecular diagnostics; mRNA splicing; neurodevelopmental disorder; web browser application
• ISSN: 0002-9297; 1537-6605
• DOI: 10.1016/j.ajhg.2022.12.015

For neurodevelopmental disorders (NDDs), a molecular diagnosis is key for management, predicting outcome, and counseling. Often, routine DNA-based tests fail to establish a genetic diagnosis in NDDs. Transcriptome analysis (RNA sequencing [RNA-seq]) promises to improve the diagnostic yield but has not been applied to NDDs in routine diagnostics. Here, we explored the diagnostic potential of RNA-seq in 96 individuals including 67 undiagnosed subjects with NDDs. We performed RNA-seq on single individuals’ cultured skin fibroblasts, with and without cycloheximide treatment, and used modified OUTRIDER Z scores to detect gene expression outliers and mis-splicing by exonic and intronic outliers. Analysis was performed by a user-friendly web application, and candidate pathogenic transcriptional events were confirmed by secondary assays. We identified intragenic deletions, monoallelic expression, and pseudoexonic insertions but also synonymous and non-synonymous variants with deleterious effects on transcription, increasing the diagnostic yield for NDDs by 13%. We found that cycloheximide treatment and exonic/intronic Z score analysis increased detection and resolution of aberrant splicing. Importantly, in one individual mis-splicing was found in a candidate gene nearly matching the individual’s specific phenotype. However, pathogenic splicing occurred in another neuronal-expressed gene and provided a molecular diagnosis, stressing the need to customize RNA-seq. Lastly, our web browser application allowed custom analysis settings that facilitate diagnostic application and ranked pathogenic transcripts as top candidates. Our results demonstrate that RNA-seq is a complementary method in the genomic diagnosis of NDDs and, by providing accessible analysis with improved sensitivity, our transcriptome analysis approach facilitates wider implementation of RNA-seq in routine genome diagnostics. We present a user-friendly web application to detect pathogenic outlier gene expression and mRNA splicing in transcriptome data. Application of this pipeline in a cohort of 67 undiagnosed individuals with neurodevelopmental disorders increased the diagnostic yield by 13%. This approach will facilitate wider implementation of RNA-seq in routine genome diagnostics.