Study of the role of lncRNA PHOX2B-AS1 in the pathogenesis of Congenital Central Hypoventilation Syndrome

Paired-like homeobox 2B (PHOX2B) gene encodes for a transcription factor responsible for the development of the autonomic nervous system (ANS) and the neural structures involved in breathing control. Heterozygous mutation in PHOX2B cause Congenital Central Hypoventilation Syndrome (CCHS), a rare genetic neurodevelopmental disorder that affects the ANS and the central chemosensitivity. In vivo and in vitro studies suggest that a loss of function mechanism, combined with a dominant-negative effect and/or toxic gain of function of the mutated proteins, is responsible for the entire disease spectrum. No effective pharmacological ventilatory stimulant is currently available. PHOX2B-AS1, a natural antisense lncRNA mapping in the PHOX2B locus, was recently identified. To characterize and study the function of PHOX2B-AS1, we took advantage of neuroblastoma cell lines and of recently generated control and patient-derived (20/25 genotype) induced pluripotent stem cell (iPSC) lines (Previously published in: Cuadros Gamboa et al. (2022) Stem Cell Res 61, 102781). RNAscope experiments showed that both lncRNA PHOX2B-AS1 and PHOX2B mRNA are mainly cytoplasmic. Performing western blot analysis, we observed that a 50% reduction of PHOX2B-AS1, using a pool of three gapmers, does not affect the PHOX2B mRNA level, however there is a reduction in the protein level. Therefore, our data suggest that PHOX2B-AS1 acts at favouring PHOX2B translation. Furthermore, the qPCR analysis has shown an ectopic expression of PHOX2B and PHOX2B-AS1 in a patient-derived iPSC line, despite the expression of the pluripotency markers. Overall, these data suggest that dysregulated PHOX2B-AS1 and PHOX2B transcription at earlier developmental stages may be involved in CCHS pathogenesis and the possibility to modulate PHOX2B-AS1 expression as a new therapeutic strategy aimed at reducing the expression of mutant PHOX2B proteins.