Transcriptional dysregulation and impairment of PHOX2B auto-regulatory mechanism in the pathogenesis of Congenital Central Hypoventilation Syndrome

The PHOX2B transcription factor plays a crucial role in autonomic nervous system development. In humans, heterozygous mutations of the PHOX2B gene lead to Congenital Central Hypoventilation Syndrome (CCHS), a rare disorder characterized by a broad variety of symptoms of autonomic nervous system dysfunction including inadequate control of breathing. The vast majority of patients with CCHS are heterozygous for a poly-alanine repeat expansion mutation of a twenty residues poly-alanine tract in the C-terminus of PHOX2B. Although several lines of evidence support a dominant-negative mechanism for PHOX2B mutations in CCHS, the molecular effects of PHOX2B mutant proteins on the transcriptional activity of the wild-type protein have not yet been elucidated. One of the targets of PHOX2B is the PHOX2B gene itself, and we have recently demonstrated that mutated PHOX2B variants can actually negatively interfere with the expression of the normal allele. Since in vitro the poly-alanine expanded proteins alter the regulation of other three PHOX2B target genes (PHOX2A, DBH, TLX2) in a promoter-specific manner and that, in CCHS patients, different PHOX2B mutations may affect different target genes, this highlights the importance of identifying PHOX2B target genes in order to get new insights into the molecular pathogenesis of the disease. To this purpose, we carried out Chromatin Immunoprecipitation experiments from IMR32 neuroblastoma cell line, followed by massively parallel sequencing of the co-immunoprecipitated genomic DNA fragments (ChIP-Seq). We are currently validating the most promising candidate target genes by biochemical and functional approaches.