A TALS-like 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 mutation 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.