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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2008-1219
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The Journal of Clinical Endocrinology & Metabolism Vol. 94, No. 1 314-319
Copyright © 2009 by The Endocrine Society

BRIEF REPORT

OTX2 Loss of Function Mutation Causes Anophthalmia and Combined Pituitary Hormone Deficiency with a Small Anterior and Ectopic Posterior Pituitary

Toshihiro Tajima1, Akira Ohtake1, Masaya Hoshino, Shin Amemiya, Nozomu Sasaki, Katsura Ishizu and Kenji Fujieda

Department of Pediatrics (T.T., K.I.), Hokkaido University School of Medicine, Sapporo, Japan 060-0835; Department of Pediatrics (A.O., M.H., S.A., N.S.), School of Medicine, Saitama Medical University, Saitama, Japan 350-0495; Department of Pediatrics (K.F.), Asahikawa Medical College School of Medicine, Asahikawa, Japan 078-8510

Address all correspondence and requests for reprints to: Toshihiro Tajima, M.D., Ph.D., Department of Pediatrics, Hokkaido University School of Medicine, N15, W7, Sapporo, Japan 060-0835. E-mail: tajeari{at}hokudai.med.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Patient and Methods
 Results
 Discussion
 References
 
Context: Orthodenticle homeobox 2 (OTX2) is a transcription factor necessary for ocular and forebrain development. In humans, heterozygous mutations of OTX2 cause severe ocular malformations. However, whether mutations of OTX2 cause pituitary structural abnormalities or combined pituitary hormone deficiency (CPHD) has not been clarified.

Objectives: We surveyed the functional consequences of a novel OTX2 mutation that was detected in a patient with anophthalmia and CPHD.

Patient: We examined a Japanese patient with growth disturbance, anophthalamia, and severe developmental delay. He showed deficiencies in GH, TSH, LH, FSH, and ACTH. Brain magnetic resonance imaging revealed a small anterior pituitary gland, invisible stalk, ectopic posterior lobe, and Chiari malformation.

Results: Sequence analysis of OTX2 demonstrated a heterozygous two bases insertion [S136fsX178 (c.576-577insCT)] in exon 3. The mutant Otx2 protein localized to the nucleus, but did not activate the promoter of the HESX1 and POU1F1 gene, indicating a loss of function mutation. No dominant negative effect in the presence of wild-type Otx2 was observed.

Conclusion: This case indicates that the OTX2 mutation is a cause of CPHD. Further study of more patients with OTX2 defects is necessary to clarify the clinical phenotypes and endocrine defects caused by OTX2 mutations.


   Introduction
Top
 Abstract
 Introduction
Patient and Methods
 Results
 Discussion
 References
 
The proper development of the anterior lobe of the pituitary gland depends on several transcription factors. Defects in these transcription factors are associated with combined pituitary hormone deficiency (CPHD) (1, 2). In addition to CPHD, individuals with mutations in these transcription factor genes may present with other symptoms related to extrapituitary expression of the altered gene, such as in the nervous system and adrenal gland (1, 2).

Orthodenticle homeobox 2 (OTX2; MIM 600037), a bicoid-type homeodomain gene, is a vertebrate ortholog of Drosophila gene orthodenticle (Otd), which is required for anterior brain, eye, and antenna formation (3, 4, 5, 6, 7). Mouse Otx1 and Otx2 are expressed in developing neural and sensory structures, including the brain, ear, nose, and eye. Homozygous Otx2 knockout mice die at midgestation with severe brain anomalies (3, 4, 5, 6). Heterozygous knockout mice reveal variable phenotypes ranging from anencephaly, micrognathia, anophthalmia, and microphthalmia to normal, depending on the genetic background (3, 4, 5, 6).

Consistent with these findings, heterozygous mutations of OTX2 have been reported recently in patients with severe ocular malformations and/or brain anomalies, seizures, and developmental delay (8). Functional analysis of these OTX2 mutations revealed that they are loss-of-function mutations (9). In humans, a deletion in the 14q22-23 region, including OTX2, causes anophthalmia and hypothalamic-pituitary anomalies (10, 11). Thus, a mutation in OTX2 gene may be the cause of the ophthalmological anomaly observed in patients with CPHD.

Here, we report a patient with anophthalamia and CPHD, who had a novel mutation of OTX2.


   Patient and Methods
Top
 Abstract
 Introduction
 Patient and Methods
Results
 Discussion
 References
 
Pituitary hormone assessment

GH provocative tests were performed using insulin (0.1 IU/kg), arginine (0.5 g/kg), and GHRH (1 µg/kg). Serum levels of LH and FSH were determined in response to GnRH (100 µg/m2). Serum levels of TSH and PRL were determined in response to TRH (10 µg/kg).

DNA amplification and sequence analysis of OTX2

Informed consent to participate in the study was obtained from the parents. The ethical committee of our hospital approved this study. Genomic DNA was extracted from peripheral leukocytes, and each exon of the OTX2 gene was amplified by PCR as described previously (8). After amplification, the PCR products were purified and sequenced directly using an ABI PRISM Dye Terminator Cycle Sequencing Kit and an ABI 373A automated fluorescent sequencer (Applied Biosystems, Foster City, CA).

Wild-type and mutant Otx2 cDNA construction and plasmid construction

Only eight amino acids (codon 33–40) in the N terminus of mouse Otx2 are different from the amino acid sequence of human OTX2 (96% identity). Thus, to examine the effects of this mutation on OTX2 activity, we created a corresponding mutation in the mouse Otx2 cDNA. Mouse Otx2 cDNA was inserted into pcDNA 3.1 (Wt-Otx2). The mutant cDNA was created by site-directed mutagenesis using an overlapping PCR strategy and was designated MT-Otx2. The mutation was verified by direct DNA sequencing.

Reporter plasmids

A DNA fragment containing –819 to +119 bp of the human HESX1 upstream sequence was generated by PCR amplification using human genomic DNA as a template (12). The PCR product was cloned into the luciferase vector pGL3 by a standard technique. This HESX1 upstream regulatory sequence construct was designated pGL3-HESX1. The 5' upstream region of the human POU1F1 sequence contains the putative DNA binding site to OTX2 (GGATTA, at position –134 to –129) (13, 14). The promoter of POU1F1 was also generated by PCR amplification using human genomic DNA as a template according to a previous report (14). The PCR product was cloned into the luciferase vector pGL3 and was designated pGL3-POU1F1.

Cell culture

COS cells were obtained from American Type Cell Culture (Manassas, VA) and grown in DMEM supplemented with 10% fetal bovine serum.

Transient gene expression

To assay HESX1 and POU1F1 gene promoter activity, COS cells were plated in six-well plates, grown to 70% confluency, and transiently transfected by lipofectamine with: 1) empty expression vector (pCDNA3, 0.5 µg); 2) WT-Otx2 (0.5 µg); 3) MT-Otx2 (0.5 µg); or 4) WT-Otx2 (0.25 µg) plus MT-Otx2 (0.25 µg) together with each reporter vector (1.0 µg) (pGL3-HESX1 or pGL3-POU1F1).

Cell extracts were prepared 48 h after transfection, and luciferase assays were performed. Luciferase measurements were divided by the respective β-galactosidase activity to control for transfection efficiency. The mean of each triplicate reaction was expressed as a percentage of the empty vector control to allow comparison of data from different experiments.

Fluorescence analysis and microscopy

COS cells transfected with either WT-Otx2-green fluorescent protein (GFP) or MT-Otx2-GFP were placed onto glass coverslips and fixed in 4% (vol/vol) formaldehyde/PBS 24 h after transfection. Cells were permeabilized in 0.1% Triton/PBS and then examined on a Fuji fluorescence microscope (Tokyo, Japan).


   Results
Top
 Abstract
 Introduction
 Patient and Methods
 Results
Discussion
 References
 
Case report

The patient is a Japanese boy, currently 6 yr old. He was born after 40 wk gestation by normal vaginal delivery and was the first child of nonconsanguineous parents. The patient had no siblings, and his parents were healthy. His birth weight was 3490 g and length was 49.5 cm. At birth he was noted to have bilateral anophthalmia. At 1 d after birth, he showed failure to thrive and hypoglycemia, but these symptoms improved by iv glucose supplementation. He also showed prolonged jaundice and was treated with UV light for 1 d. Further investigation was not performed until at 4 yr of age he was referred to our hospital because of short stature. On physical examination, his height was 81.8 cm (–5.3 SD for a normal Japanese boy), and his weight was 10.7 kg (–2.5 SD for a normal Japanese boy). His head circumference was 47.2 cm (–2.7 SD for a normal Japanese boy). He had a small penis and bilateral undescended testes. His psychomotor development was markedly delayed. Hormonal data showed central hypothyroidism, GH deficiency, gonadotropin deficiency, and cortisol deficiency (Table 1Go).


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  		TABLE 1. Endocrinological findings in the patient

 
Brain magnetic resonance imaging (MRI) revealed a small anterior pituitary, invisible pituitary stalk, ectopic posterior lobe, bilateral anophthalamia, defect of the bilateral optic nerve, and Chiari malformation (Fig. 1Go). Thus, he was diagnosed as having CPHD, and replacement therapy with L-T4 and hydrocortisone was started at that time. Treatment with GH replacement therapy was started 2 months later. At present, his body weight is 13 kg and height is 93.5 cm (–4.3 SD for a normal Japanese boy). The patient’s development remains severely delayed.


Figure 1
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  		FIG. 1. A sagittal image showing a small anterior pituitary (arrow) and an ectopic posterior gland (arrowhead).

 
Gene sequencing results

Sequence analysis of OTX2 demonstrated a heterozygous two bases insertion in exon 3 [S136fsX178 (c.576-577insCT)] (Fig. 2AGo). This insertion mutation caused amino acid changes and a premature stop codon 184 bases downstream (codon 178). Thus, this mutant lacks the C-terminal region of OTX2 (Fig. 2BGo). Neither the patient’s parents nor 50 normal Japanese subjects showed these base changes.


Figure 2
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  		FIG. 2. A, Sequence analysis demonstrated an insertion of CT nucleotides. Note the double bands present after the mutation site [c.576-577insCT (S136fsX178)]. B, Schematic representation of the OTX2 gene. The hatched box represents the homeodomain. The SIWSPA motif is conserved between OTX families. The two black boxes in the C terminus represent the tandem repeated conserved tail motif. The mutation reported in this study introduces a premature stop codon at 178, denoted by an arrow. Six previously reported mutations are shown by arrowheads. C, Transactivation functions of WT-Otx2 and MT-Otx2. Cotransfection of WT-Otx2 with either HESX1 or POU1F1 promoter stimulated the luciferase reporter gene relative to the empty vector. In the HESX1 promoter, MT-Otx2 abolished the activation function; however, MT-Otx2 was hypomorphic using the POU1F1 promoter. Cotransfection of MT-Otx2 and WT-Otx2 did not impair the transactivation capacity of WT-Otx2, suggesting no dominant negative effect of the mutant protein. D, Subcellular localization of WT-Otx2 and MT-Otx2. COS cells expressing WT-Otx2-GFP or MT-Otx2-GFP were visualized directly by GFP fluorescence. Both WT-Otx2 and Mt-Otx2 are localized to the nucleus.

 
Functional analysis and subcellular localization

Although we have studied this mutation in the context of the mouse Otx2 cDNA, the findings are likely to apply to human OTX2 because the amino acids sequence of the relevant regions of the human OTX2 and mouse Otx2 are identical.

WT-Otx2 activated the HESX1 promoter activity, whereas MT-Otx2 did not (Fig. 2CGo). Using the POU1F1 promoter, MT-Otx2 partially activated promoter activity compared with empty vector; however, activation was less than 50% of that by WT-Otx2 (Fig. 2CGo). Cotransfection of MT-Otx2 and equivalent amounts of WT-Otx2 did not affect WT-Otx2, which excluded dominant negative effects (Fig. 2CGo).

We analyzed the subcellular localization of WT-Otx2 and MT-Otx2. As shown in Fig. 2DGo, both WT-Otx2 and MT-Otx2 proteins localized to the nucleus. These results are in accordance with the results reported by Chatelain et al. (9).


   Discussion
Top
 Abstract
 Introduction
 Patient and Methods
 Results
 Discussion
References
 
We identified a novel OTX2 mutation in a patient with anophthalmia and CPHD. By the nature and the location of the mutation, impaired function is expected, and our in vitro experimental evidence confirms this.

We summarized the clinical phenotypes, MRI findings, mutations, and functional consequences of OTX2 reported thus far (8, 9) (Table 2Go). To date, six heterozygous mutations of OTX2 in seven patients have been reported. The locations of these mutations are shown in Fig. 2BGo. Three mutations cause a frame shift (p.P28fs, p.G40fsX86, and p.L156fsX178), two are nonsense mutations (Q99X and Y179X), and one is a missense mutation (R89G). Chatelain et al. (9) demonstrated that p.P28fs and p.G40fsX86 do not have DNA binding activity, whereas Q99X, p.L156fsX178, and Y179X retain the DNA binding activity but do not activate the target gene promoter. These findings indicate that the homeodomain of Otx2 is sufficient for DNA binding, whereas the C-terminal region is required for the target gene activation. Therefore, although we did not perform a DNA binding study, the functional alteration of our mutant (S136fsX178) is likely due to the lack of C-terminal region and not the lack of DNA binding activity, similar to the p.L156fsX178 and Y179X mutants.


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  		TABLE 2. Clinical, MRI, and molecular findings in seven patients and our patient with OTX2 mutations

 
Clinical phenotypes and MRI findings of patients with mutations of OTX2 are variable, even in the same family, as shown in Table 2Go. For instance, ophthalmological symptoms ranged from bilateral anophthalmia to microphthalmia. In addition, four patients showed developmental delay. In MRI findings, the development of the optic nerve and chiasm varies from normal to absent. Thus, bilateral anophthalmia, developmental delay, and absence of optic nerve in our patient are consistent with a mutation of OTX2.

In regard to endocrine function, our case demonstrated that CPHD was caused by pituitary and hypothalamic disturbance, consistent with the findings of MRI. In a previous report, endocrine investigations of patients with OTX2 mutation were not described (8). Three patients showed short stature; however, two had normal pituitary glands as demonstrated by MRI (Table 2Go). These findings suggest that they did not have severe CPHD.

To explain the hypothalamic-pituitary abnormality and the endocrinological findings in our patient, we investigated potential targets for this transcriptional factor. One possible target is the HESX1 gene, because three OTX2 binding sites are found in the HESX1 promoter region, and these sites are required for gene activation (12). In addition, murine Hesx1 failed to be transcribed in the anterior neural plate in Otx2 knockout mice (15). Furthermore, mutations of HESX1 cause CPHD and pituitary abnormalities in humans (1, 2, 16). In this context, our mutation was unable to activate the HESX1 gene promoter adequately, perhaps resulting in the observed hypothalamic-pituitary abnormality and CPHD observed in our patient.

Another potential target gene is the POU1F1 gene, which also contains an OTX2 binding site. Using the POU1F1 promoter, mutant Otx2 showed poor activity compared with that of WT-Otx2. It is known that mutations of POU1F1 cause GH, TSH, and prolactin (PRL) deficiency in humans (1, 2, 17, 18, 19). Therefore, additive effects of the impairment of HESX1 and POU1F1 gene activation may be involved in the development of CPHD in this patient. Further studies are required to determine whether OTX2 interacts with other genes, leading to the hypothalamic-pituitary abnormality and CPHD.

In conclusion, we report a patient with CPHD, anophthalmia, and developmental delay caused by an OTX2 mutation. To understand clinical phenotypes and endocrinological findings caused by OTX2 mutations, further study of more patients with OTX2 defects is necessary.


   Acknowledgments
 
We thank Dr. D. J. Diaczok and Dr. S. Radovick for the gifts of the pGL3-HESX1 and pCDNA3- WT-Otx2 expression vectors.


   Footnotes
 
Disclosure Statement: The authors have nothing to declare.

First Published Online October 14, 2008

1 T.T. and A.O. contributed equally to this work. Back

Abbreviations: CPHD, Combined pituitary hormone deficiency; GFP, green fluorescent protein; MRI, magnetic resonance imaging; OTX2, orthodenticle homeobox 2; PRL, prolactin.

Received June 5, 2008.

Accepted October 6, 2008.


   References
Top
 Abstract
 Introduction
 Patient and Methods
 Results
 Discussion
 References
 

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