Transcriptomic analysis of the effects of PHOX2B polyAla expansions in cellular models of Congenital Central Hypoventilation Syndrome

This study investigates the effects of polyalanine (polyAla) expansion mutations of the PHOX2B gene with a focus on clarifying pathogenetic mechanisms and searching for potential therapeutic interventions in the still orphan Congenital Central Hypoventilation Syndrome (CCHS). Consistent with previous findings, further confirmed in this study, the severe PHOX2B+13Ala mutation disrupts protein conformation, induces cytoplasmic mislocalisation, reduces target gene transactivation and causes cytotoxicity. Therapeutic compounds such as 17-(AllylAmino)-17-demethoxyGeldanamycin (17-AAG) have shown in vitro promising results by correcting PHOX2B misfolding, restoring its nuclear localisation and increasing its transcriptional activity on the DBH promoter. Through the use of transcriptomic analysis of SK-N-BE cells expressing wild-type or mutant PHOX2B, this study identified dysregulated pathways affected by the mutation, assessing the restorative effects of 17-AAG. The RNA-seq data were analysed through gene set enrichment analysis tools (GSEA and Cytoscape) followed by in vitro evaluation of induced cellular stress and dysregulated cell cycle. After searching for Genes Differentially Expressed (DEGs) between treated and untreated PHOX2B+13Ala transfected cells, the Connectivity Map (CMap) drove the identification of additional compounds with potential therapeutic effects, which were evaluated for their ability to restore PHOX2B activity in vitro and improve central respiratory function ex vivo in Phox2b27Ala/+ mouse models. Among the selected molecules, SuberoylAnilide Hydroxamic Acid (SAHA) emerged as the most promising one, and now requires further evaluations through in vivo studies. To overcome the limitation of using immortalized cell lines, this study also aimed to obtain a more physiological in vitro model for CCHS research. To this end, stem cells were extracted from patients decidous teeth (SHEDs), which can be obtained through non-invasive methods and offer the advantage to derive from the neural crest. Therefore these cells express neurological markers and differentiate into neurons, making them particularly suitable for studying CCHS. SHEDs were characterized for disease-specific pathways using bulk and single-cell RNA sequencing. These approaches aimed to provide a comprehensive multi-omic understanding of CCHS pathogenetic mechanisms and establish a platform for testing therapeutic compounds. The findings presented here improve our understanding of the pathogenesis of PHOX2B mutations, introduce new cellular models and pave the way for therapeutic strategies for CCHS.