title: Molecular consequences of PHOX2B missense, frameshift and alanine expansion mutations leading to autonomic dysfunction.
abstract: Heterozygous mutations of the PHOX2B gene account for a broad variety of disorders of the autonomic nervous system, either isolated or combined, including congenital central hypoventilation syndrome (CCHS), tumours of the sympathetic nervous system and Hirschsprung disease. In CCHS, the prevalent mutation is an expansion of a 20-alanine stretch ranging from +5 to +13 alanines, whereas frameshift and missense mutations are found occasionally. To determine the molecular basis of impaired PHOX2B function, we assayed the transactivation and DNA binding properties of wild-type and mutant PHOX2B proteins. Furthermore, we investigated aggregate formation by proteins with polyalanine tract expansions ranging from +5 to +13 alanines using immunofluorescence of transfected cells and gel filtration of in vitro translated proteins. We found that transactivation of the dopamine beta-hydroxylase promoter by PHOX2B proteins with frameshift and missense mutations was abolished or severely curtailed, as was in vitro DNA binding although the proteins localized to the nucleus. The transactivation potential of proteins with polyalanine tract expansions declined with increasing length of the polyalanine stretch, and DNA binding was affected for an expansion of +9 alanines and above. Cytoplasmic aggregation in transfected cells was only observed for the longest expansions, whereas even the short expansion mutants were prone to form multimers in vitro. Such a tendency to protein misfolding could explain loss of transactivation for alanine expansion mutations. However, additional mechanisms such as toxic gain-of-function may play a role in the pathogenic process.
authors.affiliation: Université Paris-Descartes, Faculté de Médecine; INSERM; AP-HP, Hôpital Necker-Enfants Malades, INSERM U-393, Paris, France.
authors.forename: Delphine
authors.initials: D
authors.lastname: Trochet
authors.forename: Seok Jong
authors.initials: SJ
authors.lastname: Hong
authors.forename: Jin Kyu
authors.initials: JK
authors.lastname: Lim
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Brunet
authors.forename: Arnold
authors.initials: A
authors.lastname: Munnich
authors.forename: Kwang-Soo
authors.initials: KS
authors.lastname: Kim
authors.forename: Stanislas
authors.initials: S
authors.lastname: Lyonnet
authors.forename: Christo
authors.initials: C
authors.lastname: Goridis
authors.forename: Jeanne
authors.initials: J
authors.lastname: Amiel
mesh_terms: D000818:Animals; D001342:Autonomic Nervous System Diseases / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D004299:Dopamine beta-Hydroxylase / Q000235:genetics; D016368:Frameshift Mutation; D018398:Homeodomain Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D020125:Mutation, Missense; D010455:Peptides / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D014157:Transcription Factors / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics
chemical_list: D018398:Homeodomain Proteins; C109220:NBPhox protein; D010455:Peptides; D014157:Transcription Factors; C019529:polyalanine; D004247:DNA; D004299:Dopamine beta-Hydroxylase