A TALS-like associated RTTN mutation impedes neural rosette formation in human cortical organoids

The Taybi-Linder syndrome (TALS) is a rare genetic disorder characterized by a severe microcephaly with abnormal gyral pattern, severe growth retardation, bone abnormalities and a reduced life span for the most severe cases. It is caused by mutations in RNU4ATAC whose transcript, the small nuclear RNA U4atac, is a core component of the minor spliceosome involved in the excision of minor introns spread over ~750 genes. Here, we report a patient presenting with TALS features but no mutations in RNU4ATAC; instead, she carries the RTTN c.2953A>G variant at the homozygous state. This variant, already reported in patients with syndromic microcephaly, encodes the missense p.Arg985Gly amino acid change. It is also known to affect RTTN pre-mRNA splicing, with the expression of two forms lacking either exon 23 (in-frame) or exons 22-23 (out-of-frame). By using the engineered RTTN depleted RPE1 cellular model, we analysed independently the impact of the missense and in-frame deletion of exon 23 RTTN isoforms on the localisation and function of the protein at the centrosome, and showed that the pathogenicity of the c.2953A>G variant is mostly due to the latter. In patient fibroblasts, we observed a reduction of the centriole length and an alteration of ciliary function, while the analysis of neuronal stem cells (NSC) derived from CRISPR/Cas9-edited induced pluripotent stem cells revealed major cell cycle and mitotic abnormalities, leading to aneuploidy, cell cycle arrest and increased cell death. Finally, by generating cortical organoids, we discovered a new function of RTTN in the self-organisation of NSC into neural rosettes. We observed a delayed apico-basal polarization of NSC, accompanied with decreased cell division and increased apoptosis. Altogether, these defects lead to a marked decrease of rosette number and size in RTTN-mutated organoids, thus impeding their overall growth. To conclude, our study gives new insights on microcephaly-related pathophysiological mechanisms underlying the only recurrent RTTN mutation, that could also open a path to better understand those involved in RNU4ATAC-associated Taybi-Linder syndrome. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by CNRS, Inserm and Universite Lyon 1 through recurrent funding; the Agence Nationale de la Recherche (no. ANR-18CE12-0007; no. ANR-22CE12-0007); the Fondation Jerome Lejeune and the Fondation pour la recherche sur le Cerveau "Espoir en tete" (confocal microscope). J.G. was supported by the Ministere de l'Enseignement Superieur et de la Recherche and by the Fondation pour la Recherche Medicale. T-Y C. was supported by a postdoctoral fellowship from Academia Sinica, Taiwan and T.K.T by the National Science and Technology Council (NSTC 112-2326-B001-010) and Academia Sinica (AS-IA-109-L04), Taiwan. E.B. was supported by an EMBO long-term fellowship (ALTF-284-2019), and V.H. by the Swiss National Foundation (SNSF) 310030_205087. S.T. was supported by Agence Nationale de la Recherche (no. ANR-17-CE16-0003-01). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: French national ethical committee Comite de Protection des Personnes (number 2021-A01551-40) gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors