Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 251-272 |
| Number of pages | 22 |
| Journal | American Journal of Human Genetics |
| Volume | 110 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - 2 Feb 2023 |
Bibliographical note
Funding Information:The authors would like to thank the individuals and their family members for their collaboration. We thank Nicole van Koetsveld, Annemieke Trebitsch, Ayse Sener, Sally den Boer, Chantal Elling, Cindy Becht, and Jamilieh Hosseini for culturing the fibroblasts. We thank Gideon Huigen, Roy Lamping, Martine van Amelsvoort, Lida Prins, and Chérise Jurriens for performing the RNA isolations. Furthermore, we would like to thank Stefan Barakat for critically reading the manuscript. T.J.v.H. was funded by an Erasmus University Rotterdam (EUR) fellowship. Conceptualization: J.D. R.S. G.M.S.M. T.J.v.H.; methodology: J.D. R.S. M.B. W.G.d.V. M.M.v.V.-P. K.M. H.D. P.E. E.K. L.M.A.v.U. G.G. J.J.J. F.W.V. M.L.T.v.d.S. F.S.N. M.W. L.H.H. M.H.-W. M.N. T.J.v.H.; formal analysis and investigation: J.D. R.S. M.B. W.G.d.V. M.M.v.V.-P. K.M. H.D. P.E. E.K. L.M.A.v.U. G.G. Y.v.I. F.W.V. M.L.T.v.d.S. F.S.N. H.H.H. M.W. V.J.M.V. M.J. A.J.A.K. I.M.B.H.v.d.L. M.H.-W. M.N. G.M.S.M.; writing - original draft preparation: J.D. R.S. G.M.S.M. T.J.v.H.; writing - review and editing: J.D. R.S. G.M.S.M. D.J.S. M.N. T.J.v.H.; supervision: G.M.S.M. T.J.v.H. All authors read, commented, and approved the final manuscript.
Funding Information:
The authors would like to thank the individuals and their family members for their collaboration. We thank Nicole van Koetsveld, Annemieke Trebitsch, Ayse Sener, Sally den Boer, Chantal Elling, Cindy Becht, and Jamilieh Hosseini for culturing the fibroblasts. We thank Gideon Huigen, Roy Lamping, Martine van Amelsvoort, Lida Prins, and Chérise Jurriens for performing the RNA isolations. Furthermore, we would like to thank Stefan Barakat for critically reading the manuscript. T.J.v.H. was funded by an Erasmus University Rotterdam (EUR) fellowship.
Publisher Copyright:
© 2022 American Society of Human Genetics