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Division of Endocrinology, Department of Medicine, Universidade Federal de Sao Paulo (T.C.V., M.R.D.d.S., J.M.C., E.B., M.B., L.T.A., J.A.), Sao Paulo, Brazil; and Division of Endocrinology, Metabolism and Molecular Medicine, Feinberg School of Medicine, Northwestern University (P.K.), Chicago, Illinois 60611
Address all correspondence and requests for reprints to: Julio Abucham, M.D., Division of Endocrinology, Department of Medicine, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Pedro de Toledo 910, Sao Paulo SP 04039-002, Brazil. E-mail: julioabucham{at}nw.com.br.
Abstract
Combined pituitary hormone deficiency (CPHD) is characterized by impaired production of GH and one or more of the other anterior pituitary hormones. Prophet of Pit-1 (PROP-1), one of the pituitary specific homeodomain transcription factors, is involved in the differentiation of the anterior pituitary cells (somatotrophs, lactotrophs, thyrotrophs, and gonadotrophs), and PROP-1 gene mutations may interfere with the development of these cells, resulting in CPHD.
We performed molecular analyses of the PROP-1 gene in two siblings, born to consanguineous parents, who presented with short stature. The index patient, a boy, was initially diagnosed with constitutional growth delay based on familial short stature, low parental target height, normal GH secretion, and imaging of the pituitary gland. On follow-up, auxological data and pubertal delay prompted a thorough reevaluation, which documented GH, TSH, and gonadotropin deficiencies. Direct sequencing of the PROP-1 gene revealed a novel homozygous transition 296G
A in exon 2 in the two affected siblings. The mutation substitutes a highly conserved arginine by a glutamine at codon 99 (R99Q) in the second helix of the DNA-binding domain of the PROP-1 protein. Compared with wild-type PROP-1, R99Q displays a significant decrease in DNA binding on a paired box response element (PRDQ9) and trans-activation of a luciferase reporter gene.
The findings emphasize the importance of repeated evaluations and illustrate that patients with CPHD associated with PROP-1 mutations present with a phenotypic spectrum, suggesting that the consequences of distinct PROP-1 mutations may be diverse and/or that additional factors, such as modifier genes, may have an impact on their expressivity.
COMBINED PITUITARY HORMONE deficiency (CPHD) is a disorder characterized by impaired production of GH and one or more of the other anterior pituitary hormones. Aside from short stature, clinical features may include hypothyroidism, impaired sexual maturation, and hypocortisolism, either individually or simultaneously. CPHD may result from acquired lesions in the hypothalamic-pituitary area (tumor, trauma, surgery, or irradiation) or from genetically defined conditions, or they may be idiopathic. Congenital CPHD is usually sporadic, but nearly 10% of cases are familial (1, 2).
Genetic defects in the development and function of the pituitary gland can result in various forms of CPHD (3, 4, 5). Mutations in the homeobox genes Pit-1, PROP-1, Rpx, Lhx-3, and Lhx-4, which encode transcription factors expressed in the anterior pituitary gland, have been shown, first in mice and then in humans, to result in CPHD (6, 7, 8, 9, 10, 11, 12, 13). In humans, PROP-1 mutations appear to be one of the most common causes of genetically determined CPHD and may account for up to 50% of all cases (2, 14, 15, 16). PROP-1 is necessary for activation of the POU1F-1 gene, the human homolog of mouse Pit-1 (8, 17). Defects in the PROP-1 gene may cause deficiencies in pituitary hormones produced by POU1F-1-dependent cell lineages (somatotrophs, thyrotrophs, and lactotrophs), as well as gonadotropin and corticotropin deficiencies (3, 18, 19).
The most frequent mutation, which accounts for more than 50% of all PROP-1 mutations, is a 2-bp AG deletion (301–302delAG) in a region containing three AG repeats in exon 2 (nucleotides 297–302) (2, 9, 16, 19, 20, 21, 22, 23, 24, 25, 26). This and other less frequent mutations give rise to abnormal proteins with reduced DNA binding and trans-activation properties (9, 20, 27, 28).
In this study we report clinical and molecular analyses of two siblings who presented with short stature. Initially, they were thought to have familial short stature and constitutional growth delay based on short stature with low parental target height, normal GH secretion on dynamic testing, and a normal computed tomography (CT) scan of the sella in the index patient. On clinical follow-up, however, auxological data and pubertal delay prompted a thorough reevaluation that revealed GH, TSH, and gonadotropin deficiencies. Molecular analyses led to the detection of a novel mutation in the PROP-1 gene as the underlying cause of their CPHD.
Materials and Methods
After obtaining approval for the study protocol by the institutional ethics committee, written informed consent was obtained from the parents.
Clinical and hormonal evaluation
The index patient is a Caucasian boy, the son of first degree cousins, born at term by normal delivery, with a birth weight of 3.8 kg and a length of 47 cm. He had bilateral cryptorchidism and was submitted to orchidopexy at the age of 4 yr. Decreased linear growth was noticed at the age of 6 yr, and he was first evaluated at the age of 8.5 yr. At this time, his height was 115 cm (-2.6 SD), his bone age was 5 yr (-4.0 SD) (29), and his parental target height was 161.5 cm (-2.1 SD). Initial hormonal evaluation showed normal free T4 and TSH levels and blunted peak GH response during insulin tolerance testing, but a normal peak GH response to clonidine (Table 1
). Basal and stimulated cortisol levels were normal (Table 1). A CT scan of the sella turcica was normal, and the initial assessment was constitutional growth delay and familial short stature.
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At the age of 17 yr, his height was 145.5 cm (-4.0 SD), his bone age was 12 yr (-4.6 SD), and his Tanner stage was G2P3. Hormonal reevaluation showed even lower serum testosterone levels of 3.5 nmol/liter and blunted LH and FSH peak levels in response to GnRH (Table 1
). Furthermore, he had a low free T4 level with an inappropriately normal basal TSH and a prolonged TSH response to TRH (TSH at 60 min, 9.8 mU/liter vs. 9.5 at 15 min and 10.4 at 30 min; Table 1) (31). His IGF-I levels were low, and his GH levels did not rise adequately in response to stimulation with clonidine and insulin tolerance testing. In contrast, his basal cortisol level was normal, and it increased normally after inducing hypoglycemia. His PRL level was normal and responded normally to stimulation with TRH (Table 1). Magnetic resonance imaging (MRI) of the pituitary gland showed a decreased volume of the anterior pituitary lobe (height, 2.0 mm; normal, 4–8 mm), with no abnormalities in the pituitary stalk or the optic chiasm, and a normal posterior pituitary bright spot on T1-weighted images (Fig. 1). Recombinant human GH (hGH; 0.3 mg/kg·wk) and T4 (75 µg/d) were started, and growth velocity increased to 8.7 cm/yr. Testosterone replacement was reinitiated 1 yr later. At 19.5 yr of age, he attained Tanner pubertal development G4P4 with testicular volumes of approximately 7.5 ml, and a height of 164 cm (-1.8 SD), i.e. 2.5 cm above target height. His bone age was 14.5 yr (-4.0 SD), and he continued to grow at 3.5 cm/yr.
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). Hormone replacement therapy with hGH (0.3 mg/kg·wk) and T4 (75 µg/d) were started, but she only received 8 months of hGH treatment. After 1 yr, estrogen replacement was started, and complete breast development occurred after 2 yr. Under estrogen therapy, PRL levels rose to 20 and 26 µg/liter, slightly above the upper limit of the normal reference range (2–15 µg/liter). An MRI scan of the hypophysis also revealed a slightly decreased volume of the anterior pituitary gland, but no other abnormalities (Fig. 1). Her final height was 150 cm (-2.1 SD) for a target height of 148.5 cm (-2.2 SD).
The parents were first degree cousins, and both had short stature. The father’s height was 160 cm (-2.2 SD), and the mother’s height was 150 cm (-2.1 SD). Neither of the parents had any clinical or biochemical evidence of anterior pituitary hormone deficiencies (Table 1).
Mutational analysis of the PROP-1 gene
DNA was extracted from peripheral lymphocytes of the patient, his sister, and his parents, using a Qiagen Midi Kit (Qiagen, Chatsworth, CA), following the manufacturer’s protocol.
Exons 1, 2, and 3 of the PROP-1 gene were amplified by PCR. One hundred nanograms of human genomic DNA were used as template in a 100-µl PCR mixture containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM deoxy-NTPs, 2.5 U Taq polymerase (PCR Reagent System, Life Technologies, Inc., Grand Island, NY), and 0.1 µM upstream and downstream specific primers. The sequence of the PCR primers and the PCR thermal cycling program have been described elsewhere (25). PCR products were analyzed in 1.8% agarose gels and purified using a PCR Product Purification Kit (Life Technologies, Inc.). Direct sequencing of the PCR products was carried out in both directions, using the ABI PRISM Big Dye terminator cycle sequencing ready reaction version 2.0 (PE Applied Biosystems, Foster City, CA) in an ABI PRISM 3100 DNA Sequencer (Perkin-Elmer Corp., Norwalk, CT).
Functional studies
Plasmid construction. The human PROP-1 cDNA was created using overlapping primers for the three exons and an overlap extension methodology (32). All PCR reactions were performed with the proofreading Pfu polymerase (Stratagene, La Jolla, CA). The wild-type cDNA was cloned into the expression vector pCMX and verified by direct sequencing. The mutation R99Q was subsequently introduced using overlap extension mutagenesis (32). The Ames mouse mutation, mS83P, and the mF85S mutation have been described previously and were included as controls (27).
The reporter gene was generated by inserting the 5'-flanking region of the POU1F1 gene (-1113 to +10) into the KpnI and XhoI sites of the vector pGL3-Basic (Promega Corp., Madison, WI).
The human wild-type and mutant cDNAs were cloned in-frame and without a stop codon into the vector pEGFP-N1 (CLONTECH Laboratories, Inc., Palo Alto, CA) to create fusion proteins of PROP-1 with a carboxyl-terminal green fluorescent protein (GFP). All final constructs were verified by direct DNA sequencing using FS AmpliTaq DNA polymerase with an ABI PRISM dye primer cycle sequencing kit following the protocol of the supplier (PE Applied Biosystems). Sequencing products were analyzed on a 377A Sequencer (PE Applied Biosystems).
DNA binding studies.
Gel mobility shift assays were performed to assess the DNA binding properties of the PROP-1 mutation R99Q. Wild-type and mutant PROP-1 were transcribed and translated using the TNT-coupled reticulolysate system (Promega Corp., Madison, WI). The reticulolysate reaction (2.5 µl) was preincubated at room temperature in a 20-µl reaction with a binding buffer consisting of 20 mM HEPES (pH 7.8), 50 mM KCl, 1 mM EDTA, 10% glycerol, 1 mM dithiothreitol, 50 µg/ml poly(dI-dC), and 50 µg/ml herring sperm DNA for 15 min. Annealed synthetic PRDQ9 oligonucleotides were labeled with [32P]deoxy-CTP (
20 fmol; specific activity, 105–106 cpm) and added to this reaction for 20 min. To exclude nonspecific binding, untranslated lysate was incubated as described above in a separate reaction. To determine the specificity of the protein-DNA interaction, reactions with a 100-fold excess of unlabeled PRDQ9 oligonucleotides were included as controls. The protein-DNA complexes were analyzed by electrophoresis through a 5% polyacrylamide gel containing 2.5% glycerol in a 0.5% Tris borate buffer (45 mM Tris borate and 1 mM EDTA) at 4 C. Gels were dried and exposed to film.
Transient transfection and luciferase assays. TSA-201 cells, a clone of human embryonic kidney 293 cells (33), were maintained in DMEM containing 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml). Cells were split into 12-well plates the day before transfection and grown to 80% confluence. pCMX plasmids containing the wild-type or mutant PROP-1 cDNAs were transfected (100 ng/well) together with the pGL3-POU1F1-luciferase construct (500 ng/well) using the calcium phosphate method. The empty pCMX vector was included as a negative control. Cells were harvested 48 h after transfection for luciferase assays. All experiments were performed in triplicate, and the groups were compared by ANOVA.
GFP
Plasmids encoding fusion proteins of wild-type or mutant PROP-1 and a carboxyl-terminal GFP were transfected into TSA-201 cells as described above. Forty-eight hours after transfection, the expression and localization of the fusion protein were analyzed under an inverted fluorescent microscope.
Results
Mutational analysis of the PROP-1 gene
As shown in Fig. 2, direct sequencing of the PCR products showed that both affected siblings harbor a homozygous 296GA transition in exon 2 of the PROP-1 gene. The parents are heterozygous for this mutation. The mutation 296GA predicts an amino acid change at codon 99 replacing a highly conserved arginine with a glutamine (R99Q) in the second helix of the paired DNA-binding homeodomain of the PROP-1 protein. Genotyping of 100 unrelated control subjects without pituitary dysfunction only showed the wild-type sequence 296G.
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DNA binding studies.
The DNA binding properties of the human PROP-1 wild-type and the mutants hR99Q and mF85S were tested on the PRDQ9 response element. In contrast to the wild type, which showed strong binding to this response element, the F85S mutant introduced into murine PROP-1 had no detectable DNA binding under the chosen experimental conditions, a finding consistent with our previous results (27). In comparison with the human wild type, the protein/DNA complexes of the R99Q were significantly reduced (Fig. 3).
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). In contrast, the two mutations hR99Q and mF85S were unable to trans-activate this reporter gene (Fig. 4).
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GFPs of the human PROP-1 wild-type and the mutant R99Q were expressed at similar levels in transfected TSA-201 cells. Strong fluorescence was limited to the nucleus and did not differ between the wild type and the mutant (Fig. 5).
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CPHD, impaired production, and secretion of one or several anterior pituitary hormones may be caused by mutations in several transcription factors (4, 34). Among them, mutations in the paired-like homeodomain factor PROP-1, first discovered in the Ames mouse (8), lead to a clinically heterogeneous form of CPHD that typically includes GH, TSH, LH, FSH, and PRL and, more rarely, also ACTH (3, 35, 36).
The two patients reported here are homozygous for a novel transition 296GA in the PROP-1 gene that is associated with a progressive deficiency of GH, TSH, LH, and FSH. It is immediately adjacent to the 297-AGAGAG-302 repeat in exon 2, which is a well known hot spot for AG deletions (25), and it forms the first missense mutation in this region. The 296GA transition found in our patients results in substitution of arginine by glutamine at position 99 (R99Q) of the PROP-1 protein. The arginine residue at that position is highly conserved in several species and among all classes of homeodomain proteins, which indicates its importance to the functional integrity of the PROP-1 protein (9). As this new mutation is located in the DNA-binding region of PROP-1, it was anticipated that it may disrupt or diminish interaction with paired domain response elements. However, in contrast to the 301–302AG deletion, which generates a frameshift with a prematurely truncated and completely nonfunctional PROP-1 protein (9), R99Q is a full-length protein that may still preserve some functional activity, as suggested by gel-shift experiments that revealed very weak residual binding on a PRDQ9 response element.
The progressive development of anterior pituitary hormone deficiencies associated with the novel R99Q mutation is reminiscent of the patients with progressive CPHD homozygous for another missense mutation (358CT, R120C) in exon 3 of PROP-1 gene (35). The resultant mutated protein, R120C, has been shown to retain some in vitro DNA binding and trans-activation capacity. These clinical and molecular observations contrast with the generally severe presentation of patients with the more disruptive 301–302delAG mutation (9, 16, 19, 22, 24, 26). Interestingly, the PROP-1 mutation responsible for the Ames dwarf mouse phenotype is also a missense Prop-1 mutation that generates a protein with decreased, but not abolished, DNA binding and trans-activation (9, 27). The Ames mouse is deficient for GH, PRL, and TSH, but not ACTH, and the gonadotropin deficiency is variable among individual mice. Although the phenotypic differences between Ames mice and human patients with PROP-1 mutations may in part represent species differences in the requirement of PROP-1 for the development and maintenance of the various pituitary cell lineages, they may also reflect the phenotypic spectrum between normal and completely nonfunctional PROP-1 proteins.
Clinically, the index patient was initially diagnosed with constitutional growth delay and familial short stature based on auxological data, a low target height, normal CT scan of the sella, and a normal GH response to clonidine. Interestingly, patients harboring the R120C mutation also exhibited at least one normal GH response to provocative testing that declined on follow-up (35). In addition, patients harboring the R120C mutation were able to initiate puberty spontaneously, although they failed to complete sexual development (35). In our male patient, a short course of testosterone treatment at age 14 yr resulted in increased penis size and pubic hair from Tanner stage G1P1 to G2P3. Six months after cessation of testosterone substitution, testicular size increased, and serum testosterone levels reached the midpubertal range, suggesting that biologically relevant gonadotropin secretion was still present. In the following years, however, no additional sexual development was noticed. Serum testosterone levels progressively fell to the peripubertal range, and LH and FSH responses to LHRH administration were blunted.
At that point, a complete hormonal reevaluation revealed CPHD affecting GH and gonadotropins, whereas the TSH response to TRH was prolonged, but quantitatively normal. This pattern for the TSH response curve is commonly observed in several forms of central hypothyroidism (31). The exact mechanism underlying this phenomenon remains elusive, but it is noteworthy that the TRH receptor contains POU1F1-binding sites in its regulatory region, and it is conceivable that its expression may be altered (37). ACTH and PRL deficiencies were not detected in our patients during a 20-yr follow-up, but they may still develop these deficiencies in the future, as suggested by other patients with CPHD associated with PROP-1 mutations (3). It is well known that ACTH deficiency may not be present at initial diagnosis, but may develop years later in patients with CPHD due to the 301302-delAG or other PROP-1 mutations that severely compromise PROP-1 function (16, 19, 26, 28, 38). On the other hand, ACTH deficiency has not been reported in the few CPHD children with PROP-1 missense mutations and milder progressive clinical presentations (35). In view of the consistently low basal and/or TRH-stimulated PRL levels reported in all CHPD patients with various PROP-1 mutations (9, 16, 22, 24, 26, 27, 28, 35), the preservation of normal PRL secretion in our patients seems to indicate a distinctive feature of that missense mutation compared with the more common AG deletions in PROP-1. Interestingly, Arroyo et al. (36) reported a similar observation in a patient harboring another missense mutation (R120C). In the Ames mouse, PRL was not detectable by immunoprecipitation (39), but PRL-immunoreactive cells could be identified (40).
In conclusion, the patients presented in this report illustrate that CPHD may initially present with growth retardation with normal GH testing and may be misdiagnosed as having constitutional growth delay. This emphasizes that regular clinical and biochemical reevaluations are essential in the care of children with delayed growth. Heightened attention should be present in the case of other clinical findings, such as cryptorchidism and parental consanguinity. The functional data confirm that the R99Q mutation has a significant decrease in DNA-binding and trans-activation properties, whereas the GFP-fusion protein is expressed and targeted to the nucleus. The phenotypic variability among patients with PROP-1 mutations suggests that the consequences of distinct PROP-1 mutations may be diverse and that additional factors, such as modifier genes, could have an impact on their expressivity.
Acknowledgments
Footnotes
This work was supported by Grant 97/14182–9 from Fundação de Amparo à Pesquisa do Estado de São Paulo (to T.C.V. and J.A.) and the Premio Dr. Ettore Balli (to P.K.).
Abbreviations: CPHD, Combined pituitary hormone deficiency; CT, computed tomography; GFP, green fluorescent protein; hGH, human GH; MRI, magnetic resonance imaging; PROP-1, Prophet of Pit-1.
Received November 26, 2001.
Accepted September 24, 2002.
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