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

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 n...

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Published in:American journal of human genetics Vol. 110; no. 2; p. 251
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 02.02.2023
Subjects:
Cycloheximide
Gene Expression Profiling
Humans
Neurodevelopmental Disorders - diagnosis
Neurodevelopmental Disorders - genetics
RNA-Seq
Sequence Analysis, RNA - methods
RNA-sequencing
transcriptomics
molecular diagnostics
mRNA splicing
neurodevelopmental disorder
web browser application
ISSN:1537-6605, 1537-6605
Online Access:Get more information
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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.
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ISSN:1537-6605
1537-6605
DOI:10.1016/j.ajhg.2022.12.015