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Case Reports
. 2008 Nov;93(11):4351-9.
doi: 10.1210/jc.2008-1189. Epub 2008 Aug 26.

A novel dominant negative mutation of OTX2 associated with combined pituitary hormone deficiency

Daniel Diaczok  1 , Christopher Romero, Janice Zunich, Ian Marshall, Sally Radovick
Affiliations
Case Reports

A novel dominant negative mutation of OTX2 associated with combined pituitary hormone deficiency

Daniel Diaczok et al. J Clin Endocrinol Metab. 2008 Nov.

Abstract

Context: Combined pituitary hormone deficiency (CPHD) is characterized by deficiencies in more than one anterior pituitary hormone. Mutations in developmental factors responsible for pituitary cell specification and gene expression have been found in CPHD patients. OTX2, a bicoid class homeodomain protein, is necessary for both forebrain development and transactivation of the HESX1 promoter, but as of yet, has not been associated with CPHD.

Objective: The goal of this study was to identify and characterize novel mutations in pituitary specific transcription factors from CPHD patients.

Design: Genomic DNA was isolated from patients with hypopituitarism to amplify and sequence eight pituitary specific transcription factors (HESX1, LHX3, LHX4, OTX2, PITX2, POU1F1, PROP1, and SIX6). Characterization of novel mutations is based on structural and functional studies.

Results: We describe two unrelated children with CPHD who presented with neonatal hypoglycemia, and deficiencies of GH, TSH, LH, FSH, and ACTH. Magnetic resonance imaging revealed anterior pituitary hypoplasia with an ectopic posterior pituitary. A novel heterozygous OTX2 mutation (N233S) was identified. Wild-type and mutant OTX2 proteins bind equivalently to bicoid binding sites, whereas mutant OTX2 revealed decreased transactivation.

Conclusions: A novel mutation in OTX2 binds normally to target genes and acts as a dominant negative inhibitor of HESX1 gene expression. This suggests that the expression of HESX1, required for spaciotemporal development of anterior pituitary cell types, when disrupted, results in an absent or underdeveloped anterior pituitary with diminished hormonal expression. These results demonstrate a novel mechanism for CPHD and extend our knowledge of the spectrum of gene mutations causing CPHD.

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Figures

Figure 1
Figure 1
Sagittal MRI before and after contrast of patient 1 performed at 6.25 yr. A, Bright spot representing an ectopic neurohypophysis (arrow). B, Hypoplastic adenohypophysis along with absent or severely hypoplastic stalk (arrow).
Figure 2
Figure 2
Schematic illustration of OTX2 (297aa) showing the N-terminal domain (1–47aa), homeodomain (HD) (47–102aa), poly-glutamine stretch (PolyQ) (102–109aa), SIWSPA region (158–163aa), and repeated OTX-tail motif (Otx1 TF) (263–275aa and 281–293aa). A heterozygous mutation, A698G, was visualized by electropherogram and results in a change from an asparagine to a serine residue (codon 233). This mutation is located in the previously described OTX1 transcription factor region and was found in two unrelated patients of nonconsanguineous mating. ex, Exon.
Figure 3
Figure 3
A, An EMSA was performed using the consensus OTX2 binding sequence incubated with in vitro transcribed and translated empty vector, WT or N233S (MUT) OTX2 protein. Lanes 2–4 and 5–7 demonstrate increased intensity of binding with the addition of increasing quantities of in vitro translated OTX2 (2, 4, or 8 μl). The OTX2 complex is denoted by an arrowhead. B, Probes containing consensus, BDI, BDII, and BDIII sites were incubated with in vitro translated WT or MUT OTX2. Binding of both WT and MUT OTX2 was observed, as shown in lanes 1 and 2 (CON), 3 and 4 (BDI), and 7 and 8 (BDIII), whereas no binding was seen in lanes 5 and 6 (BDII) of the EMSA (denoted by arrowhead). Lane 9 is empty vector incubated with consensus sequence. C, Consensus, BDI, and BDIII probes were incubated with WT or MUT OTX2 protein, followed by addition of a polyclonal OTX2 antibody resulting in “supershifted” (SS) protein-DNA complexes by EMSA (denoted by the double arrowhead).
Figure 4
Figure 4
A and B, Transient transfection studies were performed in a heterologous cell line, 293T (A), or a GH expressing cell line, GH3 (B). The HESX1 proximal promoter and 5′ untranslated region (−819 to +119 bp) were fused to the luciferase reporter construct and transfected along with expression vectors containing WT OTX2, MUT OTX2, or an empty pSG5 (EV). The promoter activity seen with WT OTX2 overexpression or equal amounts of either EV/WT or WT/MUT OTX2 were compared with that seen with EV. In addition, in 293T cells WT concentration was held constant as the amount of MUT expression vector was increased. Percent expression of HESX1 reporter plasmid is calculated with respect to WT. Each independent experiment was performed in triplicate. The graphs show the mean ± sem of the fold change from at least 10 representative experiments. For each experiment the coefficient of variation values were less than 10%. C and D, Transient transfection studies were performed in a heterologous cell line, 293T (C), or a GH expressing cell line, GH3 (D). A multiple bicoid binding site luciferase reporter construct was transfected along with expression vectors containing WT OTX2, MUT OTX2, or empty pSG5 (EV). The promoter activity seen with OTX2 expression or equal amounts of either EV/WT or WT/MUT OTX2 were compared with that seen with EV. In addition, in 293T cells WT OTX2 concentration is held constant as the concentration of MUT OTX2 expression vector was increased. Percent expression of the multiple bicoid binding site reporter plasmid is calculated with respect to WT. Each independent experiment was performed in triplicate. The graphs show the mean ± sem of the fold change from at least 10 representative experiments. For each experiment the coefficient of variation values were less than 10%. *, P < 0.01; **, P < 0.001.
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