Partial defects in transcriptional activity of two novel DAX-1 mutations in childhood-onset adrenal hypoplasia congenita.
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.
Laissue
Paul
P
U567 INSERM and Université Denis Diderot/Paris VII, Paris, France.
Copelli
Silvia
S
Bergada
Ignacio
I
Bergada
Cesar
C
Barrio
Gabriel
G
Karaboga
Sinan
S
Wurtz
Jean Marie
JM
Fellous
Marc
M
Lalli
Enzo
E
Veitia
Reiner A
RA
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
D057137:DAX-1 Orphan Nuclear Receptor; D004268:DNA-Binding Proteins; C539965:NR0B1 protein, human; D018168:Receptors, Retinoic Acid; D012097:Repressor Proteins