title: Partial defects in transcriptional activity of two novel DAX-1 mutations in childhood-onset adrenal hypoplasia congenita.
abstract: OBJECTIVE
  Mutations in DAX-1, an X-linked gene encoding an orphan nuclear receptor, have been associated with adrenal hypoplasia congenita and hypogonadotropic hypogonadism. Here we describe two novel DAX-1 mutations, Y214X and I361T, associated with childhood-onset primary adrenal failure. We aimed at analysing their effects on protein localization, transcriptional activity and propose a structural-function relationship.
  
  
  METHODS
  We have directly sequenced the DAX-1 gene from PCR-amplified DNA. The effect of the mutations on protein localization was assessed by immunocytochemistry in transfected cells. The impact of mutations on transcriptional activity was determined by transfection using a StAR promoter-luciferase reporter system.
  
  
  RESULTS
  The mutation Y214X produces a protein lacking the C-terminal half of DAX-1. The other one, I361T, affects a highly conserved amino acid within the putative ligand-binding domain. The mutant Y214X displayed a wild-type subcellular localization while I361T was partially retained in the cytoplasm. Both mutants retained subtantial transcriptional repressive activity, compared to mutants producing early onset adrenal failure. Interestingly, I361T displayed similar in vitro transcriptional repressive activity to the mutant I439S previously described in a case of late-onset AHC. This shows that molecular alterations of DAX-1 leading to similar in vitro activities may result in very different ages of onset of adrenal failure, which suggests that additional genetic and epigenetic factors are important in determining the clinical course of AHC.
  
  
  CONCLUSIONS
  Our results help the understanding of structure-function relationships in the DAX-1 molecule, suggesting that the N-terminal domain is relatively autonomous and add credence to presumed molecular interactions within ligand binding domain of the protein.
authors.affiliation: U567 INSERM and Université Denis Diderot/Paris VII, Paris, France.
authors.forename: Paul
authors.initials: P
authors.lastname: Laissue
authors.forename: Silvia
authors.initials: S
authors.lastname: Copelli
authors.forename: Ignacio
authors.initials: I
authors.lastname: Bergada
authors.forename: Cesar
authors.initials: C
authors.lastname: Bergada
authors.forename: Gabriel
authors.initials: G
authors.lastname: Barrio
authors.forename: Sinan
authors.initials: S
authors.lastname: Karaboga
authors.forename: Jean Marie
authors.initials: JM
authors.lastname: Wurtz
authors.forename: Marc
authors.initials: M
authors.lastname: Fellous
authors.forename: Enzo
authors.initials: E
authors.lastname: Lalli
authors.forename: Reiner A
authors.initials: RA
authors.lastname: Veitia
mesh_terms: D000311:Adrenal Glands / Q000737:chemistry / Q000378:metabolism; D000309:Adrenal Insufficiency / Q000151:congenital* / Q000378:metabolism; D002648:Child; D002675:Child, Preschool; D057137:DAX-1 Orphan Nuclear Receptor; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007223:Infant; D008297:Male; D009154:Mutation; D017434:Protein Structure, Tertiary; D018168:Receptors, Retinoic Acid / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D012097:Repressor Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods
chemical_list: D057137:DAX-1 Orphan Nuclear Receptor; D004268:DNA-Binding Proteins; C539965:NR0B1 protein, human; D018168:Receptors, Retinoic Acid; D012097:Repressor Proteins