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A Novel Nonsense Mutation in the Pit-1 Gene: Evidence for a Gene Dosage Effect

Division of Endocrinology, Children’s Hospital and Harvard Medical School (Y.H., L.E.C.), Boston, Massachusetts 02115; and Department of Pediatrics, Instituto di Ricovero e Cura a Carattere Scientifico Policlinico S. Matteo, University of Pavia (M.C.), 27100 Pavia, Italy

Address all correspondence and requests for reprints to: Laurie E. Cohen, M.D., Children’s Hospital, Division of Endocrinology, 300 Longwood Avenue, Boston, Massachusetts 02115. E-mail: laurie.cohen{at}tch harvard.edu.

	Abstract

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The POU transcription factor Pit-1 functions in the development of somatotrophs, lactotrophs, and thyrotrophs of the anterior pituitary gland. It also plays a role in cell-specific gene expression and regulation of the gene products from these cell types, GH, prolactin, and TSH, respectively. In the present report we studied a patient with severe growth failure. Provocative studies revealed undetectable GH, prolactin, and TSH levels, and her pituitary gland was hypoplastic on magnetic resonance imaging. She had a novel homozygous nonsense mutation in the 3' end of the first -helix of the POU-specific domain of the Pit-1 gene. This mutation results in a truncated protein with loss of most of the Pit-1 DNA-binding domains. Interestingly, her parents, who each have one mutant allele, have evidence of mild endocrine dysfunction. Thus, two normal copies of the Pit-1 gene appear necessary for full Pit-1 gene function.

	Introduction

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PIT-1 (OFFICIAL nomenclature now POU1F1) is a member of a family of transcription factors, POU, responsible for mammalian development. Pit-1 expression is restricted to the anterior pituitary lobe (1) and was identified by its specific binding to AT-rich, cell-specific elements in the rat prolactin (PRL) and GH genes (2). Pit-1 usually binds to multiple sites on its target genes (3, 4, 5). High affinity DNA binding of Pit-1 requires two domains, termed POU-specific (POU-S) and POU-homeo (POU-H) (6, 7), and x-ray crystallographic evidence suggests that Pit-1 forms dimers on DNA by interactions between the POU-S domain of one molecule and the C-terminus of the POU-H of the other (8). There are four -helixes present in the POU-S and three -helixes present in the POU-H, and the third -helixes of the POU-S and POU-H make the majority of contacts with the major grooves of DNA (8).

In addition to its role in cell-specific gene expression and regulation, Pit-1 has been shown to be essential for the development of certain anterior pituitary cells. Pit-1 is first detected on embryonic day (e) 13.5 in the mouse in cells occupying a central position in the primordial pars distalis, and then 1 d later in the entire pars distalis (9). Pit-1 transcripts initially appear in cells of the caudomedial region of the anterior pituitary gland on e14.5 and are exclusively in these cell types by e15 (10). Pit-1 protein is detected in the somatotrophs and lactotrophs, preceding GH and PRL gene expression on e16 and e17, respectively, suggesting that Pit-1 is the major cell-specific activator of hormone expression from these cell types (10).

Pit-1 protein is also expressed in the thyrotrophs (10). Thyrotrophs appear to arise from two independent cell populations in mice. The first population is Pit-1 independent and transient; it appears on e12 in the rostral tip of the developing anterior pituitary gland before the first detectable expression of Pit-1 on e14.5, but it phenotypically disappears by the day of birth. The second population is Pit-1 dependent and arises subsequently in the caudomedial portion of the developing pituitary gland on e15.5, after the initial expression of Pit-1 in this area. Pit-1 appears necessary for the appearance of these precursors of the mature thyrotroph cell type, as caudomedial thyrotroph cells are not present in the Pit-1-defective Snell dwarf mouse, and Pit-1 can bind to and transactivate the TSHß promoter (11).

Naturally occurring mutations in the Pit-1 gene have confirmed that Pit-1 is essential for the development of somatotrophs, lactotrophs, and thyrotrophs. The Jackson dwarf mouse has a gross structural alteration of the Pit-1 gene with either an inversion or insertion of a greater than 4-kb segment of DNA. These animals have hypoplastic anterior pituitaries; combined pituitary hormone deficiency (CPHD) of GH, PRL, and TSH; and no Pit-1 gene expression (12). Snell dwarf mice also have hypoplastic anterior pituitaries and CPHD, but they have a low level of Pit-1 expression. These mice have a missense mutation with a tryptophan altered to a cysteine in codon 261 (W261C) in the POU-H. This mutant Pit-1 does not bind a high affinity Pit-1 site in the PRL promoter (12).

To date, 14 other point mutations in the Pit-1 gene resulting in CPHD have been described in humans (Fig. 1) (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25). The inheritance pattern and phenotypic presentation are quite different among these patients, reflecting the location of the mutation in Pit-1. Most of the mutations are found in a homozygous or compound heterozygous state and alter residues important for DNA binding (R172X, E174G, W193R, E250X, 747delA) and/or alter the predicted -helical nature of the Pit-1 protein (F135C, R143Q) (26, 27).

View larger version (21K): [in this window] [in a new window]   Figure 1. Naturally occurring point mutations of Pit-1. The -helical regions of the POU-specific and POU-homeodomains are depicted as rectangles. The arrows indicate mutated codons.

In the present report we studied a patient with severe growth failure. Provocative studies revealed undetectable GH, TSH, and PRL levels, and her pituitary gland was hypoplastic on magnetic resonance imaging (MRI). She had a homozygous nonsense mutation in the 3' end of the first -helix of the POU-specific domain of the Pit-1 gene. This mutation results in a truncated protein with loss of most of the POU-S and all of the POU-H. Interestingly, her parents, who each have one mutant allele, have evidence of mild endocrine dysfunction. Thus, two normal copies of the Pit-1 gene appear necessary for full Pit-1 gene function.

	Materials and Methods

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Clinical studies and hormonal assays

TSH and PRL were sampled in response to TRH stimulation (200 µg/m², iv; Reflact, Hoechst, Frankfurt, Germany). GH was sampled in response to glucagon (0.1 mg/kg, im; Glucagone Novo, Novo Industri A/S, Copenhagen, Denmark), arginine (0.5 g/kg, iv; S.A.L.F., Bergamo, Italy), and GHRH-(1–44) (1 µg/kg, iv; Sanofi Pharmaceuticals, Inc., Paris, France). LH and FSH were measured in response to GnRH (100 µg/m², iv; Lutrelef, Ferring Pharmaceuticals Ltd., Milano, Italy).

Hormonal assays were performed using commercially available RIA kits: GH, PRL, cortisol, ACTH, and estradiol (Sorin Biomedica, Saluggia, Italy), TSH (Cis, Gif-sur-Yvette, France), free T₄ (Clinical Assays, Cambridge, MA), and IGF-I (Nichols Institute Diagnostics, San Juan Capistrano, CA). LH and FSH levels were determined using a fluorometric enzyme immunoassay (Baxter Diagnostics, Deerfield, IL).

Genomic analysis of the Pit-1 gene

This study was approved by the Children’s Hospital committee on clinical investigation. The PCR was used to amplify genomic DNA fragments from the human Pit-1 gene. Oligonucleotide primer pairs (16) amplified regions that included each of the six exons and the exon-intron boundaries. PCR conditions were as previously described (17). PCR products were run on a 1.5% agarose gel, and bands of the appropriate size were excised. The PCR product was ligated into the pGEM-T vector (Promega Corp., Madison, WI) and transformed into DH5 cells. The Sanger method of dideoxy sequencing with Sequenase (U.S. Biochemical Corp., Cleveland, OH) was used to determine the sequence of each exon using the SP6 and T7 primers (28). Repeat PCR and subsequent cloning and sequencing of the DNA fragments confirmed the mutation.

Plasmids

The Pit-1 mutant K145X was created using site-directed mutagenesis of wild-type rat Pit-1 (CLONTECH Laboratories, Inc., Palo Alto, CA). Wild-type and mutant Pit-1 cDNA, confirmed by DNA sequencing, were cloned into the EcoRI/BamHI sites of the simian virus 40 viral expression vector, pSG5. Empty vector (EV) pSG5 was used as a control. Human (h) GH promoter (195 bp) or hPRL promoter (164 bp) was inserted upstream of the luciferase reporter gene in pA₃luc.

Transfections

Experiments were carried out in triplicate in 24-well plates. A calcium phosphate precipitation technique (Specialty Media, Inc., Lavallette, NJ) was used in CV-1 cells (Pit-1 deficient). A total of 0.6 µg or 0.3 µg pSG5-Pit-1 expression construct or pSG5 EV in PRL or GH experiments, respectively, and 3 µg luciferase reporter construct were transfected per 3 wells. Luciferase activity was measured after 48 h. Statistical analysis was performed by ANOVA with a post hoc analysis using the Fisher test.

DNA binding studies

Recombinant wild-type and mutant K145X Pit-1 were synthesized in a coupled in vitro transcription/translation reaction (Promega Corp., Madison, WI). As a negative control, EV was used in these experiments. Products were run on SDS-PAGE to confirm protein production and for size determination.

Gel mobility shift analysis was performed with these protein products and a ³²P-labeled DNA-binding fragment of the hGH gene containing a Pit-1-binding site (underlined), GH2: 5'-CTTCTAAATTATCCATTAG-3'. The Pit-1 complexes were supershifted with a Pit-1 monoclonal antibody (Transduction Laboratories, Inc., Lexington, KY).

	Results

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Clinical evaluation (Table 1)

The patient is a 15-yr-old girl, the first of three children of healthy unrelated Italian parents. She was born after an uneventful pregnancy (birth weight, 2550 g) by a cesarean delivery due to lack of head engagement. Her postnatal course was complicated by jaundice and failure to thrive. On admission to the Pediatrics Department in Pavia at the age of 2 months she had severe growth failure (weight, 2750 g; length, 46 cm), hypotonia, somnolence, and a poor cry. Typical signs of hypothyroidism were also present, such as a large tongue, myxedema, umbilical hernia, jaundice, and widely open anterior and posterior fontanels. Laboratory investigation revealed severe hypoglycemia with blood sugar ranging 10–40 mg/dl (0.6–2.3 mmol/liter), hyperbilirubinemia with a total bilirubin of 12.7 mg/dl (217 µmol/liter), and hypothyroidism with a free T₄ level of 0.17 ng/dl (2.2 pmol/liter). A TRH stimulation test revealed undetectable basal and stimulated TSH and PRL levels. Cortisol and ACTH levels were normal at 10 µg/dl (0.28 nmol/liter) and 44 pg/ml (9.6 pmol/liter), respectively. After L-T₄ replacement and establishment of euthyroidism at 4 months of age, the IGF-I level was low at 0.12 U/ml, and peak GH responses to arginine, glucagon, and GHRH were extremely low at less than 0.5 (<0.02 pmol/liter), 0.25 (0.01 pmol/liter), and 0.93 (0.04 pmol/liter) ng/ml, respectively. Cranial computed tomography scan revealed hypoplasia of the anterior pituitary gland with maximum height of 3 mm and a normal pituitary stalk. This finding was later confirmed by MRI examination at the age of 7 yr (Fig. 2).

View larger version (170K): [in this window] [in a new window]   Figure 2. Cranial MRI of a patient with CPHD of GH, PRL, and TSH. The arrow points to the hypoplastic anterior pituitary gland.

The patient’s mother is 157 cm tall (10th-25th percentile), while her father is 165.5 cm (3rd-10th percentile). The mother had hypogalactorrhea and was unable to breast feed her three children for more than 1 month each. Her PRL and TSH levels were on the lower side of normal: 4.2 ng/ml (normal, 3.3–13.4; 186 pmol/liter) and 0.6 µU/ml (normal, 0.4–3.1), respectively. The father’s PRL and TSH levels were also in the low normal range: 2.9 ng/ml (normal, 2.6–7.2; 129 pmol/liter) and 0.5 µU/ml (normal, 0.4–3.1), respectively. The father had a low free T₄ level of 4.1 ng/dl (normal, 7–20; 5.3 pmol/liter) on one occasion. Other family members were not available for study.

Genomic analysis of the Pit-1 gene

PCR was used to amplify genomic DNA fragments from all 6 exons of the Pit-1 gene. An A to T mutation in codon 145 was found in all 10 independent clones sequenced from the patient (Fig. 3). Approximately half of the clones from each parent contained this mutation (5 of 9 in the mother and 8 of 11 in the father), with the other clones containing wild-type sequence (Fig. 3).

View larger version (33K): [in this window] [in a new window]   Figure 3. The human Pit-1 gene and a point mutation in the POU-specific domain in a patient with CPHD. Representative radiograph of a wild-type allele from the mother and the A to T mutation in the patient. The arrow denotes the nucleotide mutated. The amino acid sequence is illustrated, noting the mutation of a lysine to a stop codon at codon 145 in this patient.

Mutant Pit-1 is able to be transcribed and translated into protein

³⁵S-Labeled Pit-1 protein was generated in an in vitro transcription/translation system and run on an SDS-PAGE gel. Figure 4 demonstrates that wild-type Pit-1 proteins of the expected 31- and 33-kDa isoforms were detected. The mutant Pit-1 protein was approximately 14 kDa in size, consistent with a truncated protein.

View larger version (66K): [in this window] [in a new window]   Figure 4. Wild-type and mutant K145X Pit-1 protein. Protein was synthesized by a coupled transcription/translation reaction and run on SDS-PAGE. The small arrow points to the expected 31- and 33-kDa wild-type isoforms. The large arrow points to the mutant product, which is approximately 14 kDa in size, consistent with a truncated protein.

The effect of the K145X Pit-1 mutant on the expression of the hGH and hPRL genes was investigated in CV-1 cells (Pit-1 deficient). The hGH and hPRL genes were chosen for these studies because their responses to Pit-1 have been well characterized. Constructs containing 195 bp of the proximal hGH promoter or 164 bp of the proximal hPRL promoter were fused to the luciferase reporter gene. The proximal GH promoter contains both GH1 and GH2 sites (Pit-1-binding sites) and the intervening Z-box sequence (29). The proximal PRL promoter contains two high affinity Pit-1-binding sites (30). The rat Pit-1 cDNA was chosen because rat Pit-1 and its isoforms are much better characterized than human Pit-1, and rat and human Pit-1 are virtually identical at the amino acid level.

Figure 5 illustrates the effect of wild-type Pit-1 and the human mutant of Pit-1, K145X, on PRL and GH gene activation. Although wild-type Pit-1 activated the hPRL promoter 7.4-fold and the hGH promoter 2.4-fold relative to EV, the K145X mutant Pit-1 was completely defective in stimulation (1.1- and 0.8-fold, respectively), similar to EV. The relatively low basal activation of the hGH promoter by Pit-1 is similar to results previously reported (23, 31) and suggests the importance of interactions with other factors, such as cAMP response element binding protein on GH gene regulation (31). When a 1:1 ratio of wild-type and mutant Pit-1 was used, keeping total Pit-1 levels constant, there was a 50% decrease in activation of the hPRL promoter (3.7-fold) relative to the wild type. The response was similar when the mutant was replaced with EV (3.4-fold) as a control. There was a modest, but significant, decrease in stimulation when the same experiment was performed with the hGH promoter.

View larger version (24K): [in this window] [in a new window]   Figure 5. Mutant K145X Pit-1 cannot transactivate target genes. CV-1 cells were transfected with simian virus 40 expression vectors (pSG5) containing wild-type (WT) or mutant (mut) Pit-1 or EV as a control in the presence of 164 bp of the proximal hPRL promoter (A) or 195 bp of the proximal hGH promoter (B). Data are expressed as the mean activation ± SE relative to EV Pit-1. In addition, WT and EV or WT and mutant Pit-1 were transfected in equal proportions, keeping total expression vector quantity the same. *, P < 0.05, by ANOVA with a post hoc analysis using the Fisher test. There was no statistical significance between WT/mut and WT/EV.

We next determined the DNA-binding properties of the K145X mutant Pit-1 molecule, as the truncated protein would be expected to decrease or lack DNA binding, which would explain the functional changes observed. Wild-type and K145X mutant Pit-1 proteins were generated in an in vitro transcription/translation system, and the binding properties were evaluated in a gel mobility shift assay using one of the Pit-1-binding sites of the hGH promoter (GH2).

As shown in Fig. 6, wild-type Pit-1 formed a distinct complex with the hGH probe, which was absent with the use of the mutant K145X protein. The wild-type complex was specifically shifted with a Pit-1 monoclonal antibody, confirming its identity.

View larger version (85K): [in this window] [in a new window]   Figure 6. Mutant K145X Pit-1 does not bind to target genes. Wild-type or mutant K145 X in vitro translated Pit-1 protein was exposed to a radiolabeled hGH promoter fragment known to bind Pit-1, the GH2 site. The large arrow indicates the binding complex with wild-type Pit-1, and the small arrow indicates the supershifted complex with the addition of Pit-1 antibody (Pit-1 AS). No binding complexes were seen using the K145X mutant Pit-1 protein.

	Discussion

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The K145X mutation leads to a truncation of the Pit-1 protein. The mutant Pit-1 is unable to bind to target genes with resultant inability to transactivate target reporter genes, resulting in CPHD. Stimulation tests confirmed GH, PRL, and TSH deficiencies in the patient, and cranial MRI revealed a hypoplastic pituitary gland.

Sporadic mutations on one allele of the Pit-1 gene (R271W, K216E) result in dominant negative inhibition of wild-type Pit-1 function. DNA binding mutations result in CPHD when found on both alleles or in compound heterozygosity (R172X, E174G, W193R, E250X, 747delA). However, the phenotype of family members heterozygous for these mutations has not been well characterized.

In the present case the parents, both of whom are below the mean for height, have PRL and TSH levels at the lower range of normal. The mother had hypogalactorrhea, suggesting lactotroph dysfunction, and the father has intermittent low free T₄, suggesting inadequate thyrotroph function. These findings suggest that one normal Pit-1 allele may be insufficient for full function. Transfection experiments on the hPRL promoter support this finding, as there is a 50% decrease in promoter activation when half of the wild-type Pit-1 is replaced with K145X mutant Pit-1. As the response is similar when half of the wild-type Pit-1 is replaced with empty vector, this is probably a haploinsufficient response rather than dominant negative inhibition.

The hypothesis of a gene dosage effect of Pit-1 is supported by the phenotype of patients bearing the alanine to proline transition at codon 158 (A158P). The A158P mutant Pit-1 is a poor transactivator of its target genes. Individuals who were compound heterozygotes of the A158P allele with a null Pit-1 allele had more severe central hypothyroidism than those who were homozygous for the A158P allele. The former individuals had hypoplastic anterior pituitary glands, and the latter had normal size glands (15). In addition, although heterozygous Snell dwarf mice bearing the W261C Pit-1 mutation grow normally, their GH synthesis rate is depressed by 21–25% (32).

The phenotypic characteristics of the other Pit-1 autosomal recessive mutations are less well characterized. No information is provided about the parents of the individuals bearing the R143Q (16), R172X (13), E174G (22), and E250X mutations (33). The mother of the patient with the homozygous F135C mutation was short, at 149 cm (-2.4 SD), but was able to breast feed. The patient’s father was 175 cm (-0.3 SD), but how his stature compared with that of his family members was not revealed (19). Three Saudi Arabian families carry the P239S mutation. The mothers ranged from 149.2–159.2 cm (-2.2 to -0.6 SD score by U.S. standards), whereas the fathers were 162.6–166.4 cm (-2.0 to –1.4 SD score by U.S. standards). These heights are said to be normal stature for the Al-Baha region of Saudi Arabia. However, genotype analysis was not performed unless there was a family member with CPHD, so the prevalence of heterozygous carriers of this mutation and the correlation between heterozygosity and height are not known (20). A patient with compound heterozygosity for W193R and 747delA (yielding a truncated Pit-1 protein similar to E250X) has a mother bearing the heterozygous W193R mutation who was said to have a normal phenotype and normal hormonal studies. However, the father and brother bearing the 747delA mutation were not described.

The phenotype of one inactive Pit-1 allele is likely to be subtle based on findings in studies of other transcription factors. Knockout mice heterozygous for the POU domain transcription factor Brn-2 do not have morphological changes, but they do have a 50% reduction in the levels of vasopressin and oxytocin (34). Similarly, only a small fraction of adult knockout mice heterozygous for another type of homeodomain transcription factor, the bicioid-like homeobox factor Pitx2, exhibit anterior chamber defects of the eye (35). However, many of the mice display thinning of the ventral wall (the complete knockout has failure of closure of the ventral body wall and thorax with visceral herniation) (36). Thus, it appears that various organs have differing sensitivities to Pitx2 deficiency (35).

Our findings support a gene dosage effect of Pit-1. Mild hormonal deficiencies may not be obvious, as signs and symptoms of central hypothyroidism, hypoprolactinemia, and GH deficiency may be subtle in adults. It is also possible that deficiencies may develop over time when age-related decreases get superimposed, such as that seen with TSH (37). Finally, the interactions with other genes may be important, and thus, genetic background may contribute to variable phenotypes in the heterozygous state. Therefore, family members heterozygous for a mutant Pit-1 gene should be screened for pituitary hormone deficits.

	Note Added in Proof

Top Abstract Introduction Materials and Methods Results Discussion Note Added in Proof References

 
Since the submission of this article, Kishimoto et al. (38) found that the P24L Pit-1 mutant can not recruit CBP and loses its responsiveness to cAMP to stimulate the expression of target genes.

	Acknowledgments

 
We thank Dr. Frederick Grant for his aid with the statistical analysis, Melissa Herrmann for her technical assistance, and Dr. Sally Radovick for her advice. The hGH promoter was a gift from N. Eberhardt, and the hPRL promoter was a gift from T. Brue.

	Footnotes

 
This work was supported in part by NIH Grant K11-DK-02329 from the National Institute of Diabetes and Digestive and Kidney Diseases (to L.E.C.) and by the Genentech Foundation for Growth and Development (to L.E.C.).

Present address for Y.H.: Department of Neurology, Gunma University School of Medicine, Maebashi, 371-8711 Gunma, Japan.

Abbreviations: CPHD, Combined pituitary hormone deficiency; e, embryonic day; EV, empty vector; h, human; MRI, magnetic resonance imaging; POU-H, POU-homeo; POU-S, POU-specific; PRL, prolactin.

Received September 26, 2002.

Accepted December 16, 2002.

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