Common PHOX2B poly-alanine contractions impair RET gene transcription, predisposing to Hirschsprung disease

Abstract

HSCR is a congenital disorder of the enteric nervous system, characterized by the absence of neurons along a variable length of the gut resulting from loss-of-function RET mutations. Congenital Central Hypoventilation Syndrome (CCHS) is a rare neurocristopathy characterized by impaired response to hypercapnia and hypoxemia caused by heterozygous mutations of the PHOX2B gene, mostly polyalanine (polyA) expansions but also missense, nonsense, and frameshift mutations, while polyA contractions are common in the population and believed neutral. HSCR associated CCHS can present in patients carrying PHOX2B mutations. Indeed, RET expression is orchestrated by different transcriptional factors among which PHOX2B, thus suggesting its possible role in HSCR pathogenesis. Following the observation of HSCR patients carrying in frame trinucleotide deletions within the polyalanine stretch in exon 3 (polyA contractions), we have verified the hypothesis that these PHOX2B variants do reduce its transcriptional activity, likely resulting in a down-regulation of RET expression and, consequently, favouring the development of the HSCR phenotype. Using proper reporter constructs, we show here that the in vitro transactivation of the RET promoter by different HSCR-associated PHOX2B polyA variants has resulted significantly lower compared to the effect of PHOX2B wild type protein. In particular, polyA contractions do induce a reduced transactivation of the RET promoter, milder compared to the severe polyA expansions associated with CCHS+HSCR, and correlated with the length of the deleted trait, with a more pronounced effect when contractions are larger.

Causative and common PHOX2B variants define a broad phenotypic spectrum

“…This is in accordance with what already known around RET haploinsufficiency in HSCR . Indeed, reduced RET levels would be caused by the additive effect of large PARMs or frame 2 NPARMs of the PHOX2B gene, impairing RET promoter transactivation, coupled with downregulation of RET expression due to the HSCR predisposing haplotype including the RET+3 enhancer variant …”

“…Nevertheless, PARMs and NPARMs share some common features, thus suggesting that the molecular pathogenesis of CCHS is complex and does not depend on PHOX2B subcellular localization only. Indeed, both PHOX2B PARMs and NPARMs lack, at different extent, the ability to transactivate the promoter of target genes such as dopamine beta hydroxylase ( DBH ), PHOX2A , TLX‐2 , PHOX2B itself, and RET . Finally, both PARMs and NPARMs induce aberrant interactions between PHOX2B and CREBBP, thus preventing their synergic effects on transcription …”

“…Similarly, a role for common polyalanine contractions (ie, in frame deletions within the polyAla stretch present in the general population with a frequency of ≈0.015) in PHOX2B exon 3, acting in concert with the RET predisposing haplotype, has been postulated. Although their effect in reducing RET transcription is milder than expansions, it seems to be sufficient to increase HSCR risk when the missing polyalanine stretch is large (ie, −13Ala), while cooperation with the HSCR‐associated RET haplotype is required when the polyalanine contraction is short (ie, −7Ala to −5Ala) …”

“…Overall, when PHOX2B mutation is severe and penetrance is complete (PARMs, NPARMs) HSCR presents as syndromic while, in the presence of a common PHOX2B variant (ie, contractions) the HSCR phenotype is isolated …”

What is new about the genetic background of Hirschsprung disease?

“…The interaction among this transcription factor and RET HSCR‐associated SNPs increases the susceptibility to this pathology . Different mutations in this gene have been associated with HSCR and/or CCHS …”