Abstract
Richieri-Costa-Pereira syndrome (RCPS) is a congenital craniofacial malformation originated from disturbances in embryonic craniofacial development. RCPS is caused by loss-of-function mutations in EIF4A3, which acts in basic cellular functions such as mRNA splicing and nonsense-mediated decay. Development of the craniofacial complex is dependent upon morphogenetic stages orchestrated by cellular proliferation, migration, apoptosis and differentiation within the neuroepithelium and in neuroepithelium-derived neural crest cells, which form the structures affected in RCPS and in many other craniofacial syndromes. Many of these syndromes are caused by mutations in genes associated with presumably basic and ubiquitous functions (such as EIF4A3 itself and other genes related to mRNA splicing and ribosome biogenesis). This fact contradicts the observed tissue-specific phenotypes largely confined to the craniofacial complex, bringing bout an apparent paradox that represents an important unsolved issue in relation to RCPS and other syndromes. Ergo, this project proposes the investigation of RCPS to dissect its pathogenetic mechanism (which still needs clarification), as a means to contribute to the aforementioned discussion, ultimately promoting a better understanding of other craniofacial syndromes and of human craniofacial development. The following hypotheses will be tested: A) RCPS can be caused by disturbances in neuroepithelial proliferation and apoptosis and/or alterations in neural crest cell proliferation, apoptosis, migration and/or differentiation; B) Splicing-related disturbances may result in alterations in these cellular mechanisms in a cell-specific fashion. The project will be carried out via disease modelling with the use of cell types representative of craniofacial development: induced pluripotent stem cells (iPSCs) already derived from 3 patients and 4 controls will be differentiated into neuroepithelial cells, in which proliferation and apoptosis will be evaluated. Further, neural crest cells previously differentiated from these iPSC samples will be subjected to proliferation, apoptosis and migration assays. These cellular models are being validated in Eif4a3-knockout mice (in collaboration), not detailed herein. We will also carry out RNAseq analyses in neural crest cells to identify splicing-related alterations, which will complement results from the cellular models and may generate new hypotheses. This project is innovative in that (1) the mechanism through which mutations in genes exerting basic and ubiquitous functions cause tissue-specific defects is largely unknown, and (2) there is no published research making use of such cellular models to investigate craniofacial dysmorphologies. Finally, the establishment of an in vitro model that recapitulates human craniofacial development opens new possibilities for studying other diseases, pharmacological screening aimed at developing preventive strategies, and functional validation of candidate variants detected via next-generation sequencing. (AU)
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