PhD defense thesis Silvestre Cuinat "Pathophysiology of microcephalic primordial dwarfisms: novel clinical and molecular insights into RNU4ATAC-opathies, and investigation of the causes of microcephaly using an iPSC-derived cell model"

Key words: RNU4ATAC, minor splicing, microcephalic primordial dwarfisms, neural stem cells, centrosome

Abstract: Microcephalic primordial dwarfisms (MPDs) are a heterogeneous group of rare genetic disorders characterized by severe growth restriction beginning in utero and congenital microcephaly. The cellular mechanisms commonly affected suggest that these conditions arise from an imbalance between cell proliferation and cell death during early embryogenesis, resulting in global “hypocellularity”. Among these disorders, Taybi-Linder syndrome is caused by pathogenic variants in RNU4ATAC, which encodes the small non-coding RNA U4atac, a core component of the minor spliceosome. The minor spliceosome is responsible for the splicing of a rare class of introns, known as U12-type introns, which account for less than 1% of all introns in the human genome. These pathogenic variants therefore result in defective splicing of U12-type introns. However, the link between minor splicing defects and developmental abnormalities remains poorly understood. This thesis is structured around two complementary axes aimed at better characterizing MPDs, and more specifically RNU4ATAC-opathies.

In an introductive work, a systematic review of MPDs enabled us to identify 130 genes involved in essential cell processes such as centrosome biogenesis, mitosis, DNA replication and repair, and translation. A revised definition of MPDs is proposed, along with a mechanistic classification that facilitates genotype–phenotype correlations.

The first aim focuses on refining the clinical and biological spectrum of RNU4ATAC-opathies through the establishment and analysis of a large international cohort of 69 patients. This work identifies novel pathogenic variants and previously unreported clinical features, attenuated and pauci-symptomatic forms, as well as a high prevalence of immune abnormalities, suggesting that RNU4ATAC-related disorders remain likely underdiagnosed.

The second aim investigates the functional consequences of the two main RNU4ATAC mutations in neural progenitor cells derived from iPSCs. The results suggest that mutant cell lines exhibit abnormalities in centrosome biogenesis, leading to defective chromosome congression during mitosis, altered division axis orientation, and premature neuronal differentiation. These alterations may contribute to microcephaly and the brain malformations that significantly impact neurological outcomes in these conditions.

Finally, in complementary works, we contributed to expanding clinical and molecular knowledge regarding other MPD syndromes associated with RTTN, XRCC4, and PHGDH.

Overall, this work contributes to redefining the clinico-biological spectrum of MPDs, particularly RNU4ATAC-opathies, and explores novel pathophysiological links between the minor spliceosome and human neurodevelopment.