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

Eleonora Di Zanni, Annalisa Adamo, Elga Belligni, Margherita Lerone, Giuseppe Martucciello, Girolamo Mattioli, Alessio Pini Prato, Roberto Ravazzolo, Margherita Silengo, Tiziana Bachetti, Isabella Ceccherini

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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.

Original languageEnglish
Pages (from-to)1770-1777
Number of pages8
JournalBiochimica et Biophysica Acta - General Subjects
Volume1863
Issue number7
DOIs
Publication statusPublished - Jul 2017

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Hirschsprung Disease
Transcription
Alanine
Genes
Mutation
Transcriptional Activation
Enteric Nervous System
Congenital, Hereditary, and Neonatal Diseases and Abnormalities
Frameshift Mutation
Hypercapnia
Nonsense Codon
Neurology
Missense Mutation
polyalanine
Neurons
Exons
Down-Regulation
Observation
Phenotype
Population

Keywords

  • Journal Article

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Common PHOX2B poly-alanine contractions impair RET gene transcription, predisposing to Hirschsprung disease. / Di Zanni, Eleonora; Adamo, Annalisa; Belligni, Elga; Lerone, Margherita; Martucciello, Giuseppe; Mattioli, Girolamo; Pini Prato, Alessio; Ravazzolo, Roberto; Silengo, Margherita; Bachetti, Tiziana; Ceccherini, Isabella.

In: Biochimica et Biophysica Acta - General Subjects, Vol. 1863, No. 7, 07.2017, p. 1770-1777.

Research output: Contribution to journalArticle

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AU - Di Zanni, Eleonora

AU - Adamo, Annalisa

AU - Belligni, Elga

AU - Lerone, Margherita

AU - Martucciello, Giuseppe

AU - Mattioli, Girolamo

AU - Pini Prato, Alessio

AU - Ravazzolo, Roberto

AU - Silengo, Margherita

AU - Bachetti, Tiziana

AU - Ceccherini, Isabella

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N2 - 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.

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