A novel mutation within the conserved eh-1 domain of HESX1 causes evolving CPHD due to the loss of interaction with the co-repressor TLE1
KS Woods1, L Carvalho2, AL Zamparini3, S Stifani4, N Marcal4, JPG Turton1, BB Mendonca2, JM Brickman3, IJ Arnhold2 & MT Dattani1
The paired-like homeodomain transcriptional repressor HESX1 is implicated in forebrain and pituitary embryogenesis. A homozygous mutation (R160C) was identified in two siblings with septo-optic dysplasia (SOD), with consequent loss of DNA-binding. We have now identified a second homozygous mutation (I26T) within the highly conserved engrailed homology domain (eh-1) of HESX1 that is crucial for the repressor function of HESX1. We aimed to investigate the functional consequences of this mutation.
The mutation was identified in a girl born to consanguineous parents who developed evolving endocrinopathy: GHD at 6 years, gonadotrophin deficiency at 14 years and ACTH and TSH deficiencies at 21 years. MRI revealed a hypoplastic anterior pituitary, thin pituitary stalk, EPP, and normal forebrain and optic nerves. I26 lies within the HESX1 eh-1 domain, which is implicated in transcriptional repression via interaction with TLE1, the mammalian homologue of the Drosophila co-repressor Groucho. Recombinant HESX1(I26T) bound a consensus P3 site in a similar manner to wild type (WTHESX1). Transient transfection assays using a luciferase reporter showed that WTHESX1 fused to the heterologous DNA-binding domain of GAL4 repressed transcription from a reporter containing GAL4-binding sites upstream of the SV40 promoter, whereas HESX1(I26T) failed to do so. WTHESX1, but not HESX1(I26T), suppressed activity of the paired class homeodomain-containing activator BIX from a reporter containing P3 sites upstream from the E4 promoter. WTHESX1 interacted with TLE1 fused to the activation domain of the Herpes Simplex Transactivator VP16 whereas HESX1(I26T) did not.
We have demonstrated the first homozygous human mutation in the critical highly conserved eh-1 motif of HESX1 in a patient with evolving CPHD, and shown that this mutation results in the loss of interaction with the co-repressor TLE1, with ensuing failure of repression. The lack of a forebrain defect in this patient suggests compensatory mechanisms in forebrain, but not pituitary, development.