부산대학교 도서관

Keywords RNA-sequencing; transcriptomics; mRNA splicing; neurodevelopmental disorder; molecular diagnostics; web browser application Summary 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. Author Affiliation: (1) Department of Clinical Genetics, Erasmus MC, University Medical Center Rotterdam, PO Box 2040, 3000 Rotterdam, the Netherlands (2) Department of Pediatrics, Erasmus MC, University Medical Center Rotterdam, PO Box 2040, 3000 Rotterdam, the Netherlands * Corresponding author Article History: Received 8 July 2022; Accepted 21 December 2022 (miscellaneous) Published: January 19, 2023 (footnote)3 These authors contributed equally (footnote)4 Senior authors Byline: Jordy Dekker (1,3), Rachel Schot (1,3), Michiel Bongaerts (1), Walter G. de Valk (1), Monique M. van Veghel-Plandsoen (1), Kathryn Monfils (1), Hannie Douben (1), Peter Elfferich (1), Esmee Kasteleijn (1), Leontine M.A. van Unen (1), Geert Geeven (1), Jasper J. Saris (1), Yvette van Ierland (1), Frans W. Verheijen (1), Marianne L.T. van der Sterre (1), Farah Sadeghi Niaraki (1), Daphne J. Smits (1), Hidde H. Huidekoper (2), Monique Williams (2), Martina Wilke (1), Virginie J.M. Verhoeven (1), Marieke Joosten (1), Anneke J.A. Kievit (1), Ingrid M.B.H. van de Laar (1), Lies H. Hoefsloot (1), Marianne Hoogeveen-Westerveld (1), Mark Nellist (1), Grazia M.S. Mancini (1,4), Tjakko J. van Ham [t.vanham@erasmusmc.nl] (1,4,*)