Spliceosome malfunction causes neurodevelopmental disorders with overlapping features
Dong Li, … , Yuanquan Song, Hakon Hakonarson
Dong Li, … , Yuanquan Song, Hakon Hakonarson
Published November 14, 2023
Citation Information: J Clin Invest. 2024;134(1):e171235. https://doi.org/10.1172/JCI171235.
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Spliceosome malfunction causes neurodevelopmental disorders with overlapping features

Abstract

Pre-mRNA splicing is a highly coordinated process. While its dysregulation has been linked to neurological deficits, our understanding of the underlying molecular and cellular mechanisms remains limited. We implicated pathogenic variants in U2AF2 and PRPF19, encoding spliceosome subunits in neurodevelopmental disorders (NDDs), by identifying 46 unrelated individuals with 23 de novo U2AF2 missense variants (including 7 recurrent variants in 30 individuals) and 6 individuals with de novo PRPF19 variants. Eight U2AF2 variants dysregulated splicing of a model substrate. Neuritogenesis was reduced in human neurons differentiated from human pluripotent stem cells carrying two U2AF2 hyper-recurrent variants. Neural loss of function (LoF) of the Drosophila orthologs U2af50 and Prp19 led to lethality, abnormal mushroom body (MB) patterning, and social deficits, which were differentially rescued by wild-type and mutant U2AF2 or PRPF19. Transcriptome profiling revealed splicing substrates or effectors (including Rbfox1, a third splicing factor), which rescued MB defects in U2af50-deficient flies. Upon reanalysis of negative clinical exomes followed by data sharing, we further identified 6 patients with NDD who carried RBFOX1 missense variants which, by in vitro testing, showed LoF. Our study implicates 3 splicing factors as NDD-causative genes and establishes a genetic network with hierarchy underlying human brain development and function.

Authors

Dong Li, Qin Wang, Allan Bayat, Mark R. Battig, Yijing Zhou, Daniëlle G.M. Bosch, Gijs van Haaften, Leslie Granger, Andrea K. Petersen, Luis A. Pérez-Jurado, Gemma Aznar-Laín, Anushree Aneja, Miroslava Hancarova, Sarka Bendova, Martin Schwarz, Radka Kremlikova Pourova, Zdenek Sedlacek, Beth A. Keena, Michael E. March, Cuiping Hou, Nora O’Connor, Elizabeth J. Bhoj, Margaret H. Harr, Gabrielle Lemire, Kym M. Boycott, Meghan Towne, Megan Li, Mark Tarnopolsky, Lauren Brady, Michael J. Parker, Hanna Faghfoury, Lea Kristin Parsley, Emanuele Agolini, Maria Lisa Dentici, Antonio Novelli, Meredith Wright, Rachel Palmquist, Khanh Lai, Marcello Scala, Pasquale Striano, Michele Iacomino, Federico Zara, Annina Cooper, Timothy J. Maarup, Melissa Byler, Robert Roger Lebel, Tugce B. Balci, Raymond Louie, Michael Lyons, Jessica Douglas, Catherine Nowak, Alexandra Afenjar, Juliane Hoyer, Boris Keren, Saskia M. Maas, Mahdi M. Motazacker, Julian A. Martinez-Agosto, Ahna M. Rabani, Elizabeth M. McCormick, Marni J. Falk, Sarah M. Ruggiero, Ingo Helbig, Rikke S. Møller, Lino Tessarollo, Francesco Tomassoni Ardori, Mary Ellen Palko, Tzung-Chien Hsieh, Peter M. Krawitz, Mythily Ganapathi, Bruce D. Gelb, Vaidehi Jobanputra, Ashley Wilson, John Greally, Sébastien Jacquemont, Khadijé Jizi, Ange-Line Bruel, Chloé Quelin, Vinod K. Misra, Erika Chick, Corrado Romano, Donatella Greco, Alessia Arena, Manuela Morleo, Vincenzo Nigro, Rie Seyama, Yuri Uchiyama, Naomichi Matsumoto, Ryoji Taira, Katsuya Tashiro, Yasunari Sakai, Gökhan Yigit, Bernd Wollnik, Michael Wagner, Barbara Kutsche, Anna C.E. Hurst, Michelle L. Thompson, Ryan Schmidt, Linda Randolph, Rebecca C. Spillmann, Vandana Shashi, Edward J. Higginbotham, Dawn Cordeiro, Amanda Carnevale, Gregory Costain, Tayyaba Khan, Benoît Funalot, Frederic Tran Mau-Them, Luis Fernandez Garcia Moya, Sixto García-Miñaúr, Matthew Osmond, Lauren Chad, Nada Quercia, Diana Carrasco, Chumei Li, Amarilis Sanchez-Valle, Meghan Kelley, Mathilde Nizon, Brynjar O. Jensson, Patrick Sulem, Kari Stefansson, Svetlana Gorokhova, Tiffany Busa, Marlène Rio, Hamza Hadj Habdallah, Marion Lesieur-Sebellin, Jeanne Amiel, Véronique Pingault, Sandra Mercier, Marie Vincent, Christophe Philippe, Clemence Fatus-Fauconnier, Kathryn Friend, Rebecca K. Halligan, Sunita Biswas, Jane Rosser, Cheryl Shoubridge, Mark Corbett, Christopher Barnett, Jozef Gecz, Kathleen Leppig, Anne Slavotinek, Carlo Marcelis, Rolph Pfundt, Bert B.A. de Vries, Marjon A. van Slegtenhorst, Alice S. Brooks, Benjamin Cogne, Thomas Rambaud, Zeynep Tümer, Elaine H. Zackai, Naiara Akizu, Yuanquan Song, Hakon Hakonarson

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Figure 5

The identified RBFOX1 missense variants alter the gene-splicing pattern.

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The identified RBFOX1 missense variants alter the gene-splicing pattern....
(A) Intolerance landscape plot generated by MetaDome for RBFOX1 (NM_018723.4) variant analysis (top panel) and a lollipop plot (middle panel) with a schematic outline of the RBFOX1 protein domains (lower panel) showing 1 recurrent variant, p.(Arg118Gln), and other conserved variants identified in individuals 5 and 6. (B) Schematic representation of the murine TrkB gene (top) and the TrkB-BAC minigene (bottom). The exons in black are commonly expressed in both the full-length (TrkB.FL) and the truncated T1 (TrkB.T1) isoforms. The TrkB.T1 isoform was generated by including the specific T1 exon (green), whereas the TrkB.FL isoform was generated by including exons in orange. The 164 kb TrkB-BAC minigene includes upstream, the transmembrane coding exon and, downstream, in addition to the TrkB.T1 coding exon, the 2 exons encoding the juxtamembrane region preceding the tyrosine kinase region. The cDNAs coding for the missing extracellular domain and the tyrosine kinase region of TrkB were fused inframe to an upstream exon (98 bp) and a downstream exon (131 bp) of the BAC region (dashed lines). A neomycin resistance cassette (NEO) is present in the minigene for selection, while a synthetic CAG promoter drives the minigene expression. (C) Representative immunoblot analysis of the clonal cell line expressing the TrkB-BAC minigene transiently transfected with plasmid vectors expressing the mouse Rbfox1 and the human RBFOX1 (as positive controls), the F158A mutant (as a negative control), and the de novo RBFOX1 variants. Ntrk2 (TrkB) protein levels were tested 48 hours after transfection with an antibody against the TrkB intracellular domain to specifically detect the full-length protein (TrkB.FL). (D) Immunoblot quantification analysis of TrkB.FL protein levels from 3 independent experiments, as in C. n = 3. Data indicate the mean ± SEM. ***P < 0.001, by ordinary 1-way ANOVA with Dunnett’s multiple-comparison test.