Multi-modal investigation reveals pathogenic features of diverse DDX3X missense mutations
Federica Mosti, Mariah L. Hoye, Carla F. Escobar-Tomlienovich, Debra L. Silver
Abstract
De novo mutations in the RNA binding protein DDX3X cause neurodevelopmental disorders including DDX3X syndrome and autism spectrum disorder. Amongst ~200 mutations identified to date, half are missense. While DDX3X loss of function is known to impair neural cell fate, how the landscape of missense mutations impacts neurodevelopment is almost entirely unknown. Here, we integrate transcriptomics, proteomics, and live imaging to demonstrate clinically diverse DDX3X missense mutations perturb neural development via distinct cellular and molecular mechanisms.
Introduction
The cerebral cortex controls our abilities to process outside information and generate appropriate behavioral responses. The foundational basis for these complex and essential tasks are established during embryonic development. In the embryonic cortex, neurons and glia are generated by radial glial progenitors and basal progenitors [1–3]. Spatial and temporal regulation of progenitor behavior relies on precise coordination of gene expression. In particular, post-transcriptional regulation, including translation, is essential for proper brain development, and associated with diverse neurodevelopmental disorders [4–7].
Materials and method
Ethics statement
All animal procedures were approved by the Duke Institutional Animal Care and Use Committee (IACUC) and performed in agreement with the ethical guidelines of the Division of Laboratory Animal Resources (DLAR) from Duke University. We used the previously described mouse line: Dcx::DsRed [37]. The following mouse strains were obtained from Charles Rivers: CD1 (strain 022). For embryo staging, plug dates were defined as embryonic day (E) 0.5 on the morning the plug was identified.
Results
Expression of DDX3X missense mutations in mouse primary neural progenitors through lentiviral delivery
Individuals carrying DDX3X missense mutations present with a spectrum of clinical and molecular phenotypes, encompassing neuroanatomical disruption, developmental disability, and altered biochemical activity. To model DDX3X syndrome in primary neural cells, we focused on four de novo DDX3X missense mutations, which cause diverse clinical presentations and molecular features, and are recurrently found in 4–8 individuals each, all females.
Discussion
DDX3X has emerged as a central causal gene for neurodevelopmental pathologies including ASD and DDX3X syndrome. Yet, we lack a fundamental understanding of how clinically diverse mutations impact DDX3X molecular and cellular functions. Here, we employ a multi-modal investigation of cell fate, subcellular localization, binding partners and molecular targets to discover common and unique mechanisms of DDX3X syndrome (Fig 8). We demonstrate for the first time that missense mutations impair neural development and discover distinct molecular signatures associated with aberrant neurogenesis and neuronal survival.
Acknowledgments
We thank the DDX3X Foundation and members of the Silver lab and Kate Meyer, Stephen Floor, Cagla Eroglu for helpful discussions and reading the manuscript. We thank Jianhong Ou for assistance with bioinformatics analysis, Carly Newman for mouse husbandry, Kate Meyer for plasmids. We thank the Duke proteomics, Regeneromics, and Microscopy core facilities.
Citation: Mosti F, Hoye ML, Escobar-Tomlienovich CF, Silver DL (2025) Multi-modal investigation reveals pathogenic features of diverse DDX3X missense mutations. PLoS Genet 21(1): e1011555. https://doi.org/10.1371/journal.pgen.1011555
Editor: Frank L. Conlon, University of North Carolina, UNITED STATES OF AMERICA
Received: October 8, 2024; Accepted: December 27, 2024; Published: January 21, 2025
Copyright: © 2025 Mosti et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All transcriptomic data in Fig 3 and S2 Table have been deposited in GEO with accession number GSE279078. All proteomics data (raw and normalized) are included in Fig 5 and S3 and S4 Tables. All numerical data for graphs and summary statistics underlying the findings are included in Supporting Information.
Funding: This work was supported by the following grants: National Institutes of Health (www.nih.gov) R01NS083897, R01NS120667, R37NS110388, R01MH132089, R21HD104514, and Ruth K. Broad Foundation (https://sites.duke.edu/broadfoundation/) to D.L.S.; National Institutes of Health F32NS112566 and Regeneration Next grant (https://sites.duke.edu/dukeregenerationcenter/) to M.L.H. M.L.H. is currently employed at the National Institutes of Health. This work was completed while M.L.H. was employed at Duke University. The opinions expressed in this article are the author’s own and do not reflect the views of the National Institutes of Health, the Department of Health and Human Services, or the United States Government. Duke University Tricem Award to F.M. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
