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OTX2 Mutation in a Patient with Anophthalmia, Short Stature, and Partial Growth Hormone Deficiency: Functional Studies Using the IRBP, HESX1, and POU1F1 Promoters

Department of Endocrinology and Metabolism (S.D., M.F., N.S., T.O.), National Research Institute for Child Health and Development, Tokyo 157-8535, Japan; Department of Pediatrics (S.D.), Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8501; and Division of Endocrinology and Metabolism (K.M., M.A.), Kanagawa Children’s Medical Center, Yokohama 232-8555, Japan

Address all correspondence and requests for reprints to: Dr. T. Ogata, Department of Endocrinology and Metabolism, National Research Institute for Child Health and Development, 2-10-1 Ohkura, Setagaya, Tokyo 157-8535, Japan. E-mail: tomogata{at}nch.go.jp.

	Abstract

Top Abstract Introduction Patient and Methods Results Discussion References

 
Context: OTX2 is a transcription factor gene essential for eye development. Although recent studies suggest the involvement of OTX2 in pituitary function, there is no report demonstrating a positive role of OTX2 in the pituitary function.

Objective: The objective of the study was to report the results of functional studies indicating the relevance of OTX2 to pituitary function.

Patient: A Japanese female patient with bilateral anophthalmia was found to have short stature (height, –3.3 SD) and isolated partial GH deficiency (peak serum GH 3.1 and 9.7 µg/liter after insulin and arginine stimulations, respectively; serum IGF-I 37 ng/ml) at 3 yr 9 months of age. Magnetic resonance imaging delineated apparently normal pituitary gland.

Results: Mutation analysis showed a de novo heterozygous frameshift mutation (c.402insC) that is predicted to retain the homeodomain but lose the transactivation domain. Functional studies revealed that the wild-type and mutant OTX2 proteins localized to the nucleus and bound to the target sequences within the IRBP (interstitial retinoid-binding protein), HESX1 (HESX homeobox 1), and POU1F1 promoters. Furthermore, the wild-type OTX2 protein markedly transactivated the promoters of IRBP (27-fold), HESX1 (4.5-fold), and POU1F1 (19-fold), whereas the mutant OTX2 protein barely retained the transactivation activities and had no dominant-negative effects.

Conclusions: The results provide direct evidence for OTX2 being involved in the pituitary function. It is likely that the heterozygous severe OTX2 loss-of-function mutation caused GH deficiency and short stature, primarily because of decreased transactivation function for HESX1 and POU1F1.

	Introduction

Top Abstract Introduction Patient and Methods Results Discussion References

 
Orthodenticle homeobox 2 (OTX2) is a transcription factor gene primary involved in eye development (1). It encodes a paired type homeodomain (HD) on the N-terminal side and a transactivation domain (TD) on the C-terminal side, together with a conserved SIWSPA motif of unknown function and two tandem tail motifs that may be involved in transactivation (1, 2). To date, eight types of heterozygous mutations or rare variants have been identified in patients with ocular abnormalities such as anophthalmia and microphthalmia (3, 4). In this context, previous studies have shown that the mutations have loss-of-function effects for the promoter of interstitial retinoid-binding protein (IRBP) and are associated with a wide phenotypic spectrum ranging from anophthalmia to apparently normal eyes even in the affected family members (2, 3). In addition, developmental retardation is also often exhibited by mutation-positive patients (3).

OTX2 may also be involved in pituitary function. Pituitary dysfunction has been described in two patients with heterozygous 14q22–23 microdeletions involving OTX2 (5, 6). Furthermore, a putative OTX2 binding site has been identified in the promoter of HESX homeobox 1 (HESX1) that plays a critical role in the pituitary development and function (7). However, there has been no report describing functional studies of OTX2 using the HESX1 promoter, and it remains to be clarified whether OTX2 is relevant to the pituitary function.

Thus, we performed functional studies of a mutant OTX2 identified in a patient with anophthalmia, short stature, and partial GH deficiency, using the promoters of IRBP and HESX1. Furthermore, we also carried out similar studies using the promoter of POU class 1 homeobox 1 (POU1F1, alias PIT1) that is also involved in the pituitary development and function. The results argue for a positive role of OTX2 in the pituitary function.

	Patient and Methods

Top Abstract Introduction Patient and Methods Results Discussion References

 
Patient

This Japanese female patient was born at 40 wk of gestation after an uncomplicated pregnancy and delivery. At birth, her length was 50.0 cm (+0.6 SD), her weight 3.16 kg (+0.2 SD), and her head circumference 33.7 cm (+0.6 SD). The nonconsanguineous parents and the two elder brothers were clinically normal. The 43-yr-old father was 171 cm (±0 SD) tall, and the 36-yr-old mother was 155 cm (–0.6 SD) tall. She had congenital bilateral anophthalmia and received cosmetic repair with artificial eyes at 15 d of age. She also had cleft palate that was surgically repaired at 1 yr 7 months of age.

At 3 yr 9 months of age, she was referred to us because of proportionate short stature. Her height was 85.0 cm (–3.3 SD), her weight 10.1 kg (–2.6 SD), and her head circumference 46.0 cm (–1.9 SD). Endocrine studies indicated partial GH deficiency (Table 1), and her bone age was assessed as 2 yr 9 months. Her karyotype was 46,XX in all 50 lymphocytes examined. Magnetic resonance imaging (MRI) showed bilateral anophthalmia and optic nerve hypoplasia, but pituitary gland was apparently normal with intact stalk and hyperintense signal in the posterior lobe, as was brain structure. She also had developmental retardation. Because the patient showed strong reluctance to the GH injection, she was followed up without GH therapy. On the last examination, she was 8 yr 6 months old, measured 111.1 cm (–3.0 SD), and weighed 17.0 kg (–2.1 SD). Serum IGF-I was 98 ng/ml (12.8 nmol/liter) (age and sex matched normal range 97–477 ng/ml).

The primers and probes used in this study are summarized in supplemental Table 1, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://jcem.endojournals.org.

Plasmid vectors

The expression vector and the fluorescent vector containing the wild-type OTX2 cDNA were constructed by fusing the OTX2 cDNA to the Myc tag in pCMV-Myc and to the green fluorescent protein (GFP) gene in pAcGFO1-C1 (CLONTECH, Palo Alto, CA), respectively (designated as pOTX2-WT and pGFP-WT, respectively). The wild-type OTX2 cDNA was obtained from a human pituitary cDNA sample (CLONTECH), using primers that were designed to lose the first codon to enable the fusion to the C-terminal sides of the Myc tag and the GFP gene. The expression vector and fluorescent vector containing the mutant OTX2 cDNA (designated as pOTX2-MT and pGFP-MT, respectively) were created by site-directed mutagenesis, using the Prime STAR mutagenesis basal kit (Takara, Otsu, Japan).

The luciferase reporter vectors were constructed by inserting the promoter sequences of IRBP (–138 to +82 bp), HESX1 (–405 to +267 bp), and POU1F1 (–541 to +6 bp) into pGL3 basic (designated as pIRBP-luc, pHESX1-luc, and pPOU1F1-luc, respectively). The IRBP and HESX1 promoter sequences were as reported previously (7, 8), and the POU1F1 promoter sequence was identified in this study by in silico analysis for the upstream sequences of genes involved in pituitary development and function, using previously reported OTX2 binding sites as the baits (2, 7, 8).

Mutation analysis of OTX2, HESX1, and POU1F1

This study was approved by the Institutional Review Board Committee at National Center for Child Health and Development. After obtaining written informed consent, leukocyte genomic DNA samples of this patient and the parents were amplified by PCR for the coding exons and their flanking splice sites of OTX2 (exons 3–5), HESX1 (exons 1–4), and POU1F1 (exons 1–6). Subsequently the PCR products were subjected to direct sequencing on a CEQ 8000 autosequencer (Beckman Coulter, Fullerton, CA). To confirm a heterozygous mutation, the corresponding PCR products were subcloned with a TOPO TA cloning kit (Invitrogen, Carlsbad, CA), and normal and mutant alleles were sequenced separately.

Western blot analysis

The Myc-tagged pOTX2-WT and pOTX2-MT (20 µg) were transfected into HEK293 cells in a plate with a diameter of 10 cm. Cell lysates were obtained at 48 h after transfection, and probed with antibodies for Myc and β-actin used as an internal control.

Subcellular localization analysis

The pGFP-WT and pGFP-MT (4 µg) were transfected into HEK293 cells in a dish with a diameter of 3.5 cm. The fluorescent signals were observed at 48–72 h after the transfection using a laser-scanning microscope LSM510 (version 3.2; Carl Zeiss, Oberkochen, Germany) shortly after nuclear staining with 4, 6 diamidino-2-phenylindole.

DNA binding analysis

EMSA was performed, using a 22-bp probe (designated as IP1-WT) with two OTX2 binding sites of high (TAATCC) and low (TAAGCC) affinities within the IRBP promoter sequence (8); a 24-bp probe (designated as HX1-WT) with a high (TAATCC) affinity within the HESX1 promoter sequence (7); and a 20-bp probe (designated as PF1-WT) with a putative OTX2 binding site (GGATTA) within the POU1F1 promoter sequence. For comparison, EMSA was also performed with mutant probes in which the OTX2 binding sites were destroyed (designated as IP1-MT, HX1-MT, and PF1-MT). Each biotin-labeled probe (20 fmol) was incubated with a small amount of nuclear extract with wild-type or mutant OTX2 protein, and the incubation mixture was subjected to gel electrophoresis. The biotin-labeled probe was detected by chemiluminescence on a nylon membrane with Lightshift chemiluminescent EMSA kit (Pierce, Rockford, IL).

Transactivation analysis

Transactivation analysis was performed with dual-luciferase reporter assay system (Promega, Madison, WI). COS1 cells seeded in 12-well dishes (1.5 x 10⁵ cells/well) were transiently transfected using lipofectamine 2000 (Invitrogen) with: 1) the empty expression vector (0.6 µg), 2) pOTX2-WT (0.6 µg), 3) pOTX2-MT (0.6 µg), or 4) pOTX2-WT (0.3 µg) plus pOTX2-MT (0.3 µg), together with each reporter vector (0.6 µg) (pIRBP-luc, pHESX1-luc, or pPOU1F1-luc) and pRL-CMV vector (20 ng) used as an internal control for the transfection. Luciferase assays were performed at 48 h after the transfection with Lumat LB9507 (Berthold, Bad Wildbad, Germany). Transfections were performed in triplicate within a single experiment, and the experiment was repeated three times.

PCR-based expression analysis of OTX2

PCR amplification was performed for human cDNA samples (0.5 ng; Invitrogen), using the primers hybridizing to exon 3 and 5 of OTX2 and those hybridizing to exons 2/3 and 4/5 (boundaries) of GAPDH used as an internal control.

	Results

Top Abstract Introduction Patient and Methods Results Discussion References

 
Mutation analysis of OTX2, HESX1, and POU1F1

This patient had a heterozygous single base pair insertion at exon 5 (c.402insC) of OTX2 that is predicted to cause a frameshift at the 135th codon for the leucine and resultant termination at the 136th codon (p.L135fsX136) (Fig. 1A). The mutant protein is predicted to retain the HD but lose the TD as well as the SIWSPA motif and the two-tail motif. This mutation was absent from the parents. By contrast, no mutation was identified in HESX1 and POU1F1.

View larger version (39K): [in this window] [in a new window]   FIG. 1. Mutation and functional analyses. A, Mutation analysis of OTX2 in this patient. Left, Electrochromatograms delineating a c.402insC mutation. The mutation has been indicated by the direct sequencing and confirmed by the subsequently performed sequencing of the subcloned normal and mutant alleles. Right, Schematic presentation of the position of the mutation. The black and white boxes on genomic DNA (gDNA) denote the coding regions on exons 3–5 (E3–E5) and the untranslated regions, respectively. OTX2 cDNA encodes the HD (a blue region), the SIWSPA conserved motif (an orange region), and the two tandem tail motifs (green triangles). The transactivation domain (TD) (a gray triangle) is assigned to the C-terminal side; deletion of each tail motif drastically reduces the transactivation function, and that of a region distal to the SIWSPA motif nearly abolish the transactivation function (2 ). In addition, another TD may also reside in the 5' side of the HD (2 ). B, Western blot analysis. Both wild-type (WT) and mutant (MT) OTX2 proteins are detected with different molecular masses. Shown are the molecular masses of wild-type and mutant OTX2 proteins after excluding Myc tag. C, Subcellular localization analysis. Whereas GFP alone is diffusely distributed throughout the cell, the GFP-fused wild-type and mutant OTX2 proteins localize to the nuclei. D, EMSA using the wild-type and mutated probes derived from the IRBP promoter (IP1-WT and IP1-MT), the HESX1 promoter (HX-WT and HX-MT), and the POU1F1 promoter (PF1-WT and PF1-MT). The + and – symbols indicate the presence and absence of the corresponding probes, respectively. Both wild-type and mutant OTX2 protein bind to the wild-type probes but not to the mutant probes. For the IP1-WT, two shifted bands are found for both OTX2-WT and OTX2-MT, as reported previously (2 ). E, Transactivation analysis of the wild-type and mutant OTX2 proteins, using the promoter sequences of IPBP, HESX1, and POU1F1. The + and – symbols represent the presence and the absence of the corresponding expression vectors. The results are expressed using the mean and SD. For each promoter, the difference between four bars is significant (P < 0.05 by the t test).

Western blot analysis detected wild-type OTX2 protein of 31.6 kDa and mutant OTX2 protein of 15.4 kDa (Fig. 1B). The molecular mass of the mutant protein was as expected from the frameshift mutation. Both the wild-type and the mutant OTX2 proteins localized to the nucleus (Fig. 1C), and bound to the IP1-WT, HX1-WT, and PF1-WT but not to the IP1-MT, HX1-MT, and PF1-MT (Fig. 1D). The band shift in the EMSA was more obvious for the wild-type OTX2 protein than for the mutant OTX2 protein, consistent with the difference in the molecular masses. Furthermore, the wild-type OTX2 protein markedly transactivated not only the IRBP reporter (27-fold) but also the HESX1 (4.5-fold) and the POU1F1 reporter (19-fold), whereas the mutant OTX2 protein barely retained transactivation activities and had no dominant-negative effects (Fig. 1E).

Expression analysis of OTX2

OTX2 was expressed in the pituitary gland as well as the brain and the thalamus but not in other tissues examined (Fig. 2).

View larger version (36K): [in this window] [in a new window]   FIG. 2. PCR-based human cDNA library screening for OTX2 (35 cycles). GAPDH has been used as an internal control. F, Fetus; A, adult.

	Discussion

Top Abstract Introduction Patient and Methods Results Discussion References

This patient had short stature and partial GH deficiency, in addition to bilateral anophthalmia. In this regard, several findings are noteworthy. First, OTX2 was shown to be expressed in the pituitary gland. Second, the mutant OTX2 protein barely transactivated the HESX1 and POU1F1 promoters and had no dominant-negative effect. Third, heterozygous loss-of-function mutations of HESX1 are associated with a wide phenotypic spectrum that ranges from combined pituitary hormone deficiency to apparently normal phenotype and includes isolated GH deficiency, although homozygous mutations usually lead to combined pituitary hormone deficiency (9, 10, 11, 12, 13, 14). Fourth, heterozygous loss-of-function mutations of POU1F1 usually permit apparently normal pituitary function, although homozygous loss-of-function mutations and heterozygous dominant-negative mutations usually cause GH, TSH, and prolactin deficiencies (9, 15, 16, 17, 18). Lastly, heterozygosity for recessive mutations of genes involved in the GH-IGF-I axis, such as GH1, GHR, IGF1, IGF1R, and POU1F1, variably affects statural growth in the absence of discernible endocrine abnormalities (17, 18, 19, 20, 21, 22). Collectively, it is inferred that the heterozygous OTX2 mutation in this patient caused GH deficiency and resultant short stature primarily because of decreased transactivation function for the HESX1 promoter and that decreased transactivation function for the POU1F1 promoter could also be relevant to the short stature phenotype. It should be pointed out, however, that this notion does not exclude the relevance of other factors, such as a possible transactivation function of OTX2 for the GH1 promoter, to the short stature and partial GH deficiency of this patient.

Growth data are available in five patients with heterozygous OTX2 mutations, although pituitary function studies remain poor (cases 1–5 in Table 2). Notably, case 1 with hypomorphic mutation has low-normal height, whereas cases 3–5 and the present case with severe loss-of-function mutations have short stature. Whereas case 2 with severe loss-of-function mutation has normal height and apparently intact pituitary function, haploinsufficiency of genes involved in human development is usually associated with a wide range of penetrance and expressivity, probably depending on other genetic and environmental factors (23). Indeed, ocular phenotype is variable in patients with OTX2 mutations (3). Thus, the growth data would primarily be consistent with OTX2 mutations exerting deleterious effects on statural growth.

Two matters should also be pointed out for the clinical phenotypes (Table 2). First, developmental retardation is often exhibited by mutation-positive patients. This may be directly related to the OTX2 mutations because OTX2 is expressed in the brain (1) (Fig. 2). Second, defective optic nerve and/or corpus callosum is frequently identified in mutation-positive patients. This may partly be due to impaired transactivation for HESX1 because heterozygous HESX1 mutations are frequently associated with septooptic dysplasia and optic nerve hypoplasia (10, 11, 12, 13, 14). This notion, however, would not explain the development of anophthalmia and microphthalmia in OTX2 mutations.

At this time, however, the phenotypic spectrum remains to be determined in patients with heterozygous OTX2 mutations. Indeed, heterozygous Otx2 knockout mice have highly variable brain and ocular phenotypes, although pituitary structure and function have not been studied (homozygous knockout mice are embryonically lethal) (26, 27, 28). Thus, heterozygous OTX2 mutations may also be associated with diverse clinical features ranging from severely impaired ocular and pituitary development to nearly normal phenotype, including pituitary dysfunction only phenotype.

In summary, the results provide for the first time direct evidence for OTX2 being involved in the pituitary function and statural growth, primarily via transactivation function for HESX1 and POU1F1. Further studies will permit a better clarification of clinical and molecular findings in patients with OTX2 mutations.

	Footnotes

 
This work was supported by grants for Child Health and Development (20C-2) and Research on Children and Families (H18-005) from the Ministry of Health, Labor, and Welfare and by Grants-in-Aid for Scientific Research (priority areas: 16086215; category B: 19390290) from the Ministry of Education, Culture, Sports, Science, and Technology.

Disclosure Statement: The authors have nothing to declare.

First Published Online July 15, 2008

Abbreviations: GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; HD, homeodomain; HESX1, HESX homeobox 1; HX1-WT/MT, a probe with wild-type/mutant OTX2 binding sites within the HESX1 promoter; IP1-WT/MT, a probe with wild-type/mutant OTX2 binding sites within the IRBP promoter; IRBP, interstitial retinoid-binding protein; MRI, magnetic resonance imaging; OTX2, orthodenticle homeobox 2; PF1-WT/MT, a probe with a wild-type/mutant OTX2 binding site within the POU1F1 promoter; pGFP-WT/MT, a fluorescent vector with wild-type/mutant OTX2 cDNA; pIRBP-/pHESX1-/pPOU1F1-luc, a luciferase reporter vector with an IRBP/HESX1/POU1F1 promoter sequence; pOTX2-WT/MT, an expression vector with wild-type/mutant OTX2 cDNA; POU1F1, POU class 1 homeobox 1; TD, transactivation domain.

Received April 1, 2008.

Accepted July 7, 2008.

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Top Abstract Introduction Patient and Methods Results Discussion References

 

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