Investigating the pathogenic mechanism underlying Richieri-Costa-Pereira syndrome

• Ano de defesa: 2019
• Autor(a) principal: Musso, Camila Manso
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Biblioteca Digitais de Teses e Dissertações da USP
• Palavras-chave em Português: Células de crista neural; iPSCs; Modelagem in vitro; Modelo animal; RCPS
• Link de acesso: http://www.teses.usp.br/teses/disponiveis/41/41131/tde-27112019-160620/

Richieri-Costa-Pereira syndrome (RCPS) is a rare autosomal-recessive disorder characterized by craniofacial abnormalities, including mandible cleft, microstomia, Robin sequence and microcephaly, as well as limb defects and learning impairments. RCPS is mainly caused by an increased number of repeat motifs within the EIF4A3 5\'UTR, which in turn leads to decreased expression of the gene product. The DEAD-box RNA helicase eIF4A3 is a core component of the RNA-binding exon junction complex (EJC), which is involved in post-transcriptional events such as alternative splicing, nonsense-mediated mRNA decay (NMD), translation initiation and mRNA localization. The EIF4A3 5\'UTR varies in both number and organization of three types of motifs between individuals. However, the origin of the RCPS-associated allelic pattern, as well as the functional effects of these motifs on EIF4A3 expression, remain to be uncovered. Although a relationship between EIF4A3 hypomorphic biallelic mutations and RCPS has been established, the pathogenetic mechanisms by which decreased levels of EIF4A3 lead to craniofacial malformation are unknown. To address these gaps, we first characterized the variation in the EIF4A3 5\'UTR at a populational level. This analysis demonstrated that this noncoding region displays a polymorphic nature and structural complexity presenting multiple patterns. The RCPS-associated allele patterns may have arisen from independent unequal crossing-over events between ancient alleles and can potentially emerge in any population containing alleles with the CGCA-20nt motif. Furthermore, there is a direct association between the number of motifs and EIF4A3 expression, revealing a potential cis-acting regulatory mechanism for these motifs and suggesting that the 5\'UTR structure plays a central role in phenotypic modulation. Next, we unraveled the cellular and molecular mechanisms responsible for RCPS using two complementary models, patient-derived induced pluripotent stem cells (iPSCs) and EIF4A3 haploinsufficient mouse models. The craniofacial structures compromised in RCPS patients are suggestive of disturbances in neural crest cells (NCCs), a transient cell population that originates at the neural plate border (NPB) in the developing embryo and gives rise to multiple cell types, generating most of the cranioskeleton. Accordingly, we differentiated iPSCs from RCPS patients and control individuals into NCCs and demonstrated that EIF4A3 deficiency impairs NCC development, leading to defective migration, premature osteogenic and dysregulated chondrogenic differentiation of NCC mesenchymal derivatives. Besides pinpointing that impaired NCC migratory capacity is a key cellular dysfunction underlying RCPS pathogenesis, we provided evidence by transcriptome analyses that impairment to cell adhesion dynamics is implicated in this dysfunction and involves alteration of cell-extracellular matrix (ECM) interaction components. Additionally, we suggest that ribosome defects contribute to RCPS pathogenesis, since aberrant splicing as well as alterations at the proteomic level of ribosomal components were identified in patient-derived NCCs. Elucidating the pathogenetic mechanism underlying RCPS will also aid in clarifying the etiology of other craniofacial syndromes and how mutations in genes with basic and ubiquitous functions such as EIF4A3 lead to specific phenotypes. Finally, we provided a simple protocol for quickly deriving human NCCs in vitro in a manner that recapitulates multiple stages from the early NPB onward. This method represents a promising approach to better understand human craniofacial development