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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-0119
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 8 4880-4887
Copyright © 2005 by The Endocrine Society

An Uncommon Phenotype with Familial Central Hypogonadism Caused by a Novel PROP1 Gene Mutant Truncated in the Transactivation Domain

Rachel Reynaud, Anne Barlier, Sophie Vallette-Kasic, Alexandru Saveanu, Marie-Pierre Guillet, Gilbert Simonin, Alain Enjalbert, Paul Valensi and Thierry Brue

Laboratoire des Interactions Cellulaires Neuro-Endocriniennes (R.R., A.B., S.V.-K., A.S., M.-P.G., A.E., T.B.), Unité Mixte de Recherche 6544 Centre National de la Recherche Scientifique, Université de la Méditerranée, Institut Fédératif de Recherche Jean-Roche, Faculté de Médecine Nord, 13926 Marseille, France; Departments of Pediatrics (R.R., G.S.), and Endocrinology (A.B., A.S., T.B.), Centre Hospitalo-Universitaire Timone, Laboratoire de Biochimie et Biologie Moléculaire (A.B., A.S., A.E.), Hôpital de la Conception, 13385 Marseille Cedex, France; and Department of Endocrinology, Hôpital Jean Verdier (P.V.), Assistance Publique-Hôpitaux de Paris, 93143 Bondy Cedex, France

Address all correspondence and requests for reprints to: Thierry Brue, M.D., Ph.D., Hôpital de la Timone, 264 rue St. Pierre, 13385 Marseille Cedex 5, France. E-mail: thierry.brue{at}mail.ap-hm.fr.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: PROP1 gene mutations are usually associated with childhood onset GH and TSH deficiencies, whereas gonadotroph deficiency is diagnosed at pubertal age.

Objectives: We report a novel PROP1 mutation revealed by familial normosmic hypogonadotropic hypogonadism. We performed in vitro transactivation and DNA binding experiments to study functional consequences of this mutation.

Setting: Three brothers were followed in the Department of Endocrinology of a French university hospital.

Patients: These patients from a consanguineous kindred were referred for cryptorchidism and/or delayed puberty.

Results: Initial investigations revealed hypogonadotropic hypogonadism. One of the patients had psychomotor retardation, intracranial hypertension, and minor renal malformations. The brothers reached normal adult height and developed GH and TSH deficiencies after age 30. A novel homozygous nonsense mutation (W194X) was found in the PROP1 gene, indicating that the protein is truncated in its transactivation domain. Transfection studies confirmed the deleterious effect of this mutation, whose transactivation capacity was only 34.4% of that of the wild-type. Unexpectedly altered DNA-binding properties suggested that the C-terminal end of the factor plays a role in protein-DNA interaction.

Conclusions: PROP1 mutations should be considered among the growing number of genetic causes of initially isolated hypogonadotropic hypogonadism. This report extends the phenotype variability associated with PROP1 mutations.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
COMBINED PITUITARY HORMONE deficiency (CPHD) in humans may be caused by many acquired disease processes, such as tumors and damage due to trauma, surgery, or radiation. In congenital forms of CPHD, genetic alterations of several pituitary transcription factors (POU1F1, PROP1, LHX3, LHX4, HESX1, RIEG) have been recognized with increased frequency over the past 12 yr, but most genetic causes of CPHD remain unidentified (1). Functional alteration of some of these factors is associated with specific clinical characteristics. The phenotype of POU1F1/Pit-1 mutations was the first to be described. It includes early and severe somatotroph and lactotroph deficiencies, thyrotroph deficiency sometimes of later onset, and hypoplastic or normal size pituitary (2, 3). Mutations of Prophet of Pit-1 (PROP1) now represent the most frequently recognized genetic cause of CPHD (4, 5, 6, 7, 8, 9, 10, 11, 12, 13). These mutations are usually associated with progressive endocrine deficiencies first affecting somatotroph, lactotroph, and thyrotroph axes, with later onset of gonadotroph, and sometimes corticotroph, deficiencies. Delayed puberty thus represents an important, albeit late, feature for discriminating phenotypes of PROP1 vs. POU1F1 mutations., Phenotype, however, varies largely among patients with PROP1 mutations in terms of clinical, hormonal, and neuroradiological presentation, and no clear phenotype-genotype correlations have been identified.

PROP1 is a 226-amino acid transcription factor expressed early during pituitary embryogenesis from embryonic day (e)9.5 to e14. Its protein structure includes two putative functional domains, a paired-like DNA binding domain, and a C-terminal transactivation domain. The first Prop-1 mutation was described in Ames dwarf mice, affected by somatotroph, lactotroph, and thyrotroph deficiencies and hypofertility (14). Inactivating mutations have subsequently been identified in the human homolog PROP1 gene (7, 11). To date, at least 15 distinct human PROP1 mutations have been reported. All of them affect the binding domain, including two hot spots at codons 73 and 99 in exon 2 (12, 15).

In the present paper, we report on the first PROP1 mutation localized in the transactivation domain. Unexpectedly for PROP1 mutations, this novel mutation was found in three brothers who came to medical attention because of central hypogonadism. Their presentation, uncommon in this condition, was characterized by a normal final height and at least partially maintained somatotroph, lactotroph, and thyrotroph functions in adult age. One of the patients had psychomotor retardation, intracranial hypertension, and minor renal malformation. To analyze the functional consequences of this W194X PROP1 mutation, we performed transactivation and DNA binding studies on this mutant.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patients

All of the patients belonged to the same consanguineous family originating from Tunisia (Fig. 1Go). Informed written consent was obtained from all adults and from parents of minors. This study was approved by the local Ethics Committee. All three affected brothers (patients 1–3) were referred to the Department of Endocrinology of Jean Verdier Hospital for investigation or follow-up of hypogonadotropic hypogonadism. A complete clinical, biological, and (when possible) neuroradiological workup was carried out. Hormonal investigations were also performed in two clinically unaffected sisters (individuals 4 and 5). PROP1 gene sequencing was carried out in subjects 1–3, 5, and 8.



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  		FIG. 1. Pedigree of a family including three brothers who first presented with hypogonadotropic hypogonadism. Males and females are indicated by squares and circles, respectively. Affected patients, indicated by black squares (patients 1, 2, and 3), were found to have a homozygous W194X PROP1 mutation. Heterozygous unaffected relatives are indicated by half-black circles (patients 5 and 8). In patient 4 (clinically unaffected sister), endocrine investigations were performed, but the patient refused genetic analysis. All other subjects were unavailable for molecular testing. Patients 6 and 7 died during childhood.

 
Height was measured in centimeters using a meter attached to the wall; the average of three measurements was recorded. Weight was measured with a balance-beam scale. The body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters. The SD scores (SDS) for height and height age were determined from French reference values (16, 17). To determine bone age, roentgenograms of the hand and wrist were compared with the standards of Greulich and Pyle. Pubertal stage was estimated according to Tanner (18). Final height was determined when growth velocity dropped to less than 2 cm/yr and/or bone age exceeded 17 yr.

Hormonal investigations

GH response was studied by at least one provocative test: insulin tolerance test (ITT) (insulin, 0.05 U/kg; normal peak value, >5 ng/ml) in adult cases 1 and 3; GHRH infusion test (80 µg Somatoreline Choay, Gentilly France; normal peak value, >10 ng/ml) in cases 1, 2, 4, and 5; arginine stimulation (0.5 g/kg iv arginine, Veyron et Froment, Marseille, France) in case 2; or arginine-insulin stimulation test in case 3 during childhood (normal peak value, >10 ng/ml). Basal plasma cortisol (cases 1–5) and ACTH (case 1) were measured at 0800 h (normal range for cortisol and ACTH, respectively: 210–560 nmol/liter and 10–50 pg/ml at 0800 h). The plasma cortisol response (normal peak value ≥ 496 nmol/liter) was also determined during ITT (case 1), ACTH stimulation (250 µg iv Synacthene; Novartis, Inc., Rueil Malmaison, France; cases 1 and 2), or metyrapone (2 g at midnight and measurement of serum level of 11-deoxycortisol at 0800 h the next morning). The gonadotroph axis was investigated by measuring LH and FSH levels at baseline and 30 and 60 min after a GnRH provocative test (100 µg iv Gonadorelin; Ferring, Gentilly, France) and by determining basal levels of plasma testosterone or estradiol. Plasma thyroid hormone levels (normal range of free T3 and T4, 3.0–9.0 pmol/liter and 10–30 pmol/liter, respectively) and TSH concentrations (normal range, 0.2–6 mUI/liter) were measured repeatedly during follow-up. A TRH test (200 µg Protirelin; Roussel, Paris, France) was carried out with measurements of prolactin (PRL) and TSH at 0, 30, 60, and 120 min after an iv bolus. PRL was measured during the domperidone test (10 mg iv) in case 3.

Pituitary magnetic resonance imaging (MRI)

Pituitary MRI was performed in the oldest patient (case 1), using precontrast sagittal and axial spin echo T1-weighted images, followed by T2-weighted imaging.

Genomic analysis of the PROP1 gene (NM 006261)

All three exons and exon-intron boundaries of the PROP1 gene were PCR amplified from genomic DNA by primers flanking the exons for direct sequencing. DNA was extracted from peripheral lymphocytes by the QIAamp Blood kit (QIAGEN, Hilden, Germany). Exons 2, and then 1 and 3 of the PROP1 gene of each affected patient were amplified by PCR, using three sets of flanking intronic primers: F1, 5'-ACC TAC ACA CAC ATT CAG AGA CAG-3'; R1, 5'-TGG AGC CTA TGC TTT CAG C-3'; F2, 5'-AAA GAC TGG AGC AGC ACA GGA CGC A-3'; R2, 5'-CTC AAT GCA GTT GCT CCG ATG-3'; F3, 5'-GCC TTG TGG AAG AGC TTT ACT CC-3'; R3, 5'-ATT TCT AAT CGG TGA GCT GAC CC-3'. Amplification was carried out in a 50-µl reaction, using 200 ng genomic DNA, 0.25 nmol/liter of each deoxy-NTP, 25 pmol of each primer, and 1.5 U Pfu DNA polymerase (Promega Corp. Madison,WI). The reaction consisted of 3 min at 95 C, followed by 30 cycles of 30 sec at 95 C, 30 sec at 55 C for exons 1 and 3 and 60 C for exon 2, 2 min at 72 C, and 5 min at 72 C. PCR products were purified using the Qiaquick PCR purification kit (QIAGEN). Direct sequencing of the double-stranded PCR fragments was carried out according to the thermal cycle sequencing big dye terminator protocol (ABI Prism 310 Genetic Analyzer; PerkinElmer Applied Biosystems, Foster City, CA) using the same PCR primers. Mutations were confirmed by repeat PCR and subsequent sequencing of PCR products.

Plasmid constructs and mutagenesis

The target vector was a ptk-luc (promoter thymidine kinase-luciferase) construct (gift of Dr. Joseph Martial, Liege, Belgium), in which the paired transcription factor consensus sequence PRDQ9 was inserted as three contiguous copies (PRDQ3) (11), fused to a luciferase reporter gene. The luciferase values were normalized using the ß-galactosidase data to control for transfection efficiency. The coding sequence for ß-galactosidase was inserted into the pCEP4 eukaryotic expression vector (Invitrogen, Cergy Pontoise, France), where it was expressed under control of the cytomegalovirus promoter. The empty pcDNA3T7+ vector was included as a negative control. The W194X mutant PROP1 vector was obtained by site-directed mutagenesis using the Quickchange kit (Stratagene Cloning System; Stratagene, La Jolla, CA); the mutagenesis nucleotide sequence was C CAG TCT GAG GAC TGA TAC CCT ACC (underline indicates mutated nucleotide).

Cell culture and transfection

The transactivation capacities of wild-type and mutant PROP1 were studied by transient cotransfection experiments in Hela cells through the liposome technique (Polyfect Transfection Reagent; QIAGEN) according to the manufacturer’s instructions. In brief, cells were plated at 300,000 cells/well in six-well plates, 24 h before transfection, and grown to 80% confluence in DMEM containing 10% fetal bovine serum, ampicillin, and amphotericin B. Total DNA was constant in each well (1.5 µg), and the amount of DNA transfected was 0.15 µg for the empty pcDNA3T7+, the wild-type (wt), or mutant PROP1 vectors; 1.25 µg for the PRDQ3 target vector; and 0.1 µg for the pCEP4 ß-galactosidase construct. Cells were harvested 48 h after transfection for luciferase and ß-galactosidase assays. All experiments were performed in triplicate and repeated at least three times.

Luciferase and ß-galactosidase assays

Hela cells were lysed in 250-µl reporter lysis buffer (Promega Corp.). After three sequential freeze-thaw cycles, cell debris was pelleted by centrifugation at 10,000 x g for 3 min at 4 C, and 20-µl aliquots of the supernatant were used for subsequent luciferase assays (Luciferase assay system; Promega Corp.) and ß-galactosidase assays (colored reaction was obtained using 50 mM ß-mercaptoethanol and 25 mM Ortho nitro phenyl galactopyranoside, and the OD was read at 420 nm). For each control, the total luciferase activity normalized against ß-galactosidase activity was taken as 1, and results were expressed as fold activation over control.

Gel mobility shift assay

Gel mobility shift assays were performed to assess the DNA binding properties of the W194X PROP1 mutant. Wild-type and mutant PROP1 were transcribed and translated using the TNT-coupled reticulocyte lysate system with T7 polymerase (Promega Corp.). Annealed synthetic PRDQ9 oligonucleotides were labeled with [32P]deoxy-CTP. 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 0.5x Tris borate buffer at 4 C.

Protein translation study

Wild-type and mutant PROP1 were transcribed and translated using the same reticulocyte lysate system but with [35S]methionine and a nonradioactive amino acid mixture devoid of methionine. Protein expression was analyzed by electrophoresis on a 10% sodium dodecyl sulfate polyacrylamide gel and then detected by autoradiography.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Three brothers (patients 1–3) were sequentially followed in the same adult endocrinology department for hypogonadism. They belonged to a kindred of nine born to healthy consanguineous parents who emigrated from Tunisia to France (Fig. 1Go). Among them, one brother (patient 6) died in infancy of unknown cause, one sister (patient 7) died of meningitis during childhood, and none of the four other siblings showed signs of endocrine dysfunction. Paternal height was short (161 cm; –2.25 SDS) and maternal height was normal (161 cm; –0.4 SDS).

The oldest brother was born in 1949, after a normal pregnancy. His medical history remained uneventful until age 19 yr, when he was evaluated for delayed puberty. Hypogonadotropic hypogonadism was diagnosed, and testosterone treatment was started. Two years later, the patient discontinued replacement therapy and did not return to the endocrinologist until familial screening of hypogonadism was carried out. At the age of 35 yr, a new hormonal evaluation was also performed with pituitary tomodensitometry. An empty sella turcica was found. Hormone replacement was reinitiated but again rapidly stopped by the patient. In 1987, at age 38 yr, he presented to an orthopedic surgeon with lombosciatalgia and vertebral fracture. Bone investigations, including bone densitometry (lumbar Z score = –0.96) and biopsy (no osteoporosis) suggested metabolic osteopathy. He was referred to the endocrine department for exploration. Physical examination showed the patient to be short (height, 160 cm; –2.4 SDS) and obese (weight, 85 kg; BMI, 33.2 kg/m2), without facial or axillary hair. Pubic hair was Tanner 3, with infantile genitalia, and he reported lack of erection and libido. He was not anosmic. Serum gonadotropin, testosterone, and dihydrotestosterone levels were low (Table 1Go). Basal serum TSH, FT4, ACTH, cortisol, and PRL levels were normal (Table 1Go). Dynamic tests showed decreased LH and FSH response to GnRH stimulation and a normal GH secretion to GHRH; PRL and TSH responses to TRH stimulation were normal (Table 1Go). MRI of the pituitary area confirmed an empty sella turcica with eutopic posterior lobe and pituitary stalk. No olfactory lobe malformation was seen. Testosterone replacement therapy was administered during 2 yr. A new hormonal exploration revealed a partial thyrotroph deficiency; L-T4 therapy was initiated. Nine years later, undetectable GH concentrations after ITT tests performed before and during testosterone treatment revealed complete GHD. The other pituitary hormone functions were stable. The onset of a type 2 diabetes mellitus was then diagnosed (fasting blood glucose, 8.1 mmol/liter; fasting insulin, 48 pmol/liter).


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  		TABLE 1. Evaluation of somatotroph, lactotroph, thyrotroph, and corticotroph functions in three affected brothers (patients 1–3) with hypogonadotropic hypogonadism at different time points during follow-up (age in years at each investigation is indicated), and two of their unaffected sisters (4 and 5)

 
The second affected brother was born in 1957. His medical history was uneventful until the age of 18 yr, when he was operated on for undescended testes. Endocrine explorations revealed hypogonadotropic hypogonadism (Table 1Go). Administration of human chorionic gonadotropin caused serum testosterone levels to increase to normal adult male levels (testosterone, 21.1 nmol/liter). He was subsequently lost to follow-up; and in 1998 (age 41 yr), he was referred to the adult endocrine clinic because of diabetes mellitus (fasting blood glucose, 9.3 mmol/liter; fasting insulin, 118 pmol/liter). His height was normal (175 cm; 0.1 SDS) with a large excess of weight (134 kg; BMI, 43.7 kg/m2). His obesity had begun during the second decade of life. Androgen replacement was given every 2 wk. Both TSH and serum T4 levels were close to the lower limit of normal range, in keeping with a diagnosis of secondary hypothyroidism that was confirmed 1 yr later. Arginine and GHRH stimulation revealed a complete GHD (peak, 0.05 mg/liter and 0.09 mg/liter, respectively). Anterior pituitary function was otherwise normal.

The third affected brother, the seventh sibling, was born in 1960. Unlike the others, he was brought to medical attention during childhood (Fig. 2Go). At age 8, he was referred to a pediatric endocrinologist because of undescended testes. Physical examination showed genital abnormalities, including left cryptorchidism and micropenis, obesity (BMI, +3.5 SDS), short stature (height, –2.4 SDS), with delayed bone age (6 yr) and psychomotor retardation. A constitutional delay of growth was diagnosed, and no endocrine explorations were carried out. Surgical orchidopexy was performed on the left side and, 1 yr later, on the right side. Right testicular biopsy was normal. Endocrine status was evaluated when he was 12 yr old. He remained obese (BMI, +3 SDS), short (height, –2.9 SDS), and at prepubertal stage. Bone age was delayed by 3 yr. GnRH provocative test failed to increase LH and FSH (Table 2Go). Serum testosterone was low before and after human chorionic gonadotropin stimulation (testosterone, 3 nmol/liter and 9.3 nmol/liter, respectively), and poor GH response to stimulation performed without sex steroid priming was attributed to obesity. Serum IGF-I was normal for age. Corticotroph and thyrotroph stimulation tests were normal. It was again concluded that this patient had constitutional delay of growth and puberty. No hormone replacement was started. Minor renal malformations were found, including the left kidney in lumbar position and right malrotated kidney. The left testis was removed after necrosis at age 16. When he was 18 yr old, psychomotor retardation was considered worsened by a neuropsychologist, and food intake was increased. He was 155 cm high (–3.25 SDS), weighed 77 kg (BMI, 32 kg/m2), without pubertal development (no pubic and axillary hair, infantile penis, and atrophic right testis). Cranial standard roentgenogram showed an enlarged sella turcica. A Sylvius duct stenosis with intracranial hypertension was found at tomodensitometry. A ventriculo-atrial derivation transiently decreased behavior and food intake disorders; but later, neuroleptic treatment was added. He still gained height until he reached his final adult height at age 24 (161.4 cm; –2.2 SDS). Obesity was even more severe (BMI, 38.5 kg/m2) at Tanner’s pubertal stage A2P3G1. Thyroid dysfunction required initiation of L-T4 treatment at age 30. No MRI could be performed during follow-up because of the ventriculo-atrial derivation.



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  		FIG. 2. Auxological data from the third affected brother with all retrospectively retrieved measurements until 22 yr. Height (with SD values) and weight (percentiles) curves from French reference values [Sempé (17 )]. Inset, BMI curve expressed in percentiles from French reference values [Rolland-Cachera et al. (16 )]. Bone age (diamonds) determinations are connected to each corresponding height by dotted lines. TH, Target height.

 

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  		TABLE 2. Evaluation of gonadotroph function in three brothers with hypogonadotropic hypogonadism (patients 1–3) at different stages and in two unaffected sisters (4 and 5)

 
Genetic studies

Complete sequencing of the PROP1 gene served to identify the same nonsense mutation in all three brothers; it consisted of a transition from a G to an A at position 582 (Fig. 3AGo). This single nucleotide change predicted substitution of a tryptophan residue for a stop codon at codon 194 (W194X). The rest of the coding sequence was normal. Genetic studies in one sister and her daughter did not reveal any PROP1 mutation except an already described (7, 12, 15) heterozygous polymorphism at codon 142 (A142T). No other family member was available for genetic studies (Fig. 1Go).



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  		FIG. 3. In vitro studies. A, Direct sequencing of the coding exons of the PROP1 gene revealed a single homozygous C-to-T change at position 582 (reverse sequence shown). B, Cotransfection experiment using expression vectors of wild-type or mutant W194X PROP1 with ptk-PRDQ3-luciferase reporter gene. Wild-type PROP1 strongly stimulated the ptk-PRDQ3-luciferase reporter gene as compared with pcDNA3 empty vector. Relative luciferase activity is expressed as fold increase ± SD from the empty vector (pcDNA3) on three distinct experiments. Relative luciferase activity of W194X was reduced to 34.4% of wild-type activity. C, EMSA. Lane 1, Labeled DNA-binding site PRDQ9 (free probe). Lane 2, Empty pcDNA3 vector (without PROP1 cDNA) after incubation with labeled PRDQ9. Lane 3, Without competition (–), wild-type PROP1 binds strongly to the PRDQ9 response element. Lane 4, Specific PROP1 binding was displaced by the addition of a 100-fold excess of cold oligonucleotide (+). Lanes 5 and 6, For the W194X PROP1 mutant, before (–) or after (+) cold probe competition, no specific protein-DNA interactions were detectable. D, Autoradiogram of labeled wild-type (lane 2) and W194X PROP1 (lane 3) proteins translated in vitro in the presence of 35S methionine. Lane 1, Negative control using the empty pcDNA3 vector. Molecular weight markers are indicated on the right.

 
Transfection studies

Cotransfection of wild-type PROP1 and the paired-like transcriptional factor response element PRDQ9 resulted in a strong stimulation of luciferase reporter gene relative to that of the empty vector. Activation by PROP1 W194X mutant was reduced to 34.4% of the wild-type (Fig. 3BGo).

Gel retardation assay

DNA binding of the wild-type PROP1 and of the W194X mutant was tested on the PRDQ9 response element. Wild-type PROP1 had a detectable DNA binding that was specifically displaced by addition of an excess of unlabeled PRDQ9 oligonucleotide. No detectable DNA binding was observed with W194X in our experimental conditions (Fig. 3CGo).


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
In the present report, we describe three brothers with the W194X PROP1 mutation, representing the first case of alteration of this gene as revealed by familial idiopathic hypogonadotropic hypogonadism (IHH). This unusual phenotype was associated with the first mutation of PROP1 found in the transactivation domain. CPHD, induced by PROP1 mutations, currently represents the most frequently identifiable genetic cause of congenital hypopituitarism (19). Prop1 mutations were first associated in Ames mice with somatotroph, lactotroph, and thyrotroph deficiencies (20). This phenotype of triple anterior pituitary hormone deficit was highly similar to that of Snell and Jackson mice that harbor Pit-1 gene alterations. For Ames mice, immunohistochemical analyses highlighted low expression of somatotroph cells, whereas Pit-1-deficient mice had no detectable somatotroph cells. Although morphologically normal corticotroph and gonadotroph cells are observed in Prop1-deficient Ames mice (20), these mice display a mild gonadotroph phenotype with decreased plasma gonadotrophin levels and low fertility (21). In humans, gonadotrophin deficiency was described as an associated feature in the early clinical descriptions of PROP1 mutation (7, 11). This insufficiency, however, occurred later than somatotroph, thyrotroph, or lactotroph deficiencies (12, 15). In contrast, our patients were first found to have IHH, and other deficiencies were diagnosed later. Despite cryptorchidism the younger brother was first diagnosed as having constitutional delay in growth and puberty. Indeed, his height and predicted height were appropriate for his parental and target heights, respectively, bone age was delayed, and plasma IGF-I levels were normal. Blunted GH response to provocative tests was attributed to the fact that testing had been performed without testosterone priming and to obesity. GH deficiency was thus ruled out. As in the case report by Vieira et al. (22), the lack of spontaneous onset of puberty a posteriori ruled out the initial diagnosis of constitutional delay of puberty, and the patient was subsequently considered to have IHH. Because androgen replacement therapy was initiated after 18 yr, delayed bone maturation allowed ongoing growth beyond this age. He indeed gained 6 cm between 18 and 23 yr of age. Note that he had psychomotor retardation associated with minor cerebral and renal malformations not previously reported in patients with PROP1 mutations. Given the consanguinity of the pedigree, such an association may however represent an unrelated phenomenon. The variable age of onset of pituitary hormone deficits in patients harboring the same PROP1 gene mutations, such as R120C (23) or R73C (24), illustrates the variability in phenotype expression usually attributed to the influence of other genetic and/or environmental factors.

Only 10–20% of patients with IHH currently have identified mutations in genes associated with variable inherited transmission: X-linked (KAL1 and NROB1), autosomal recessive (GnRHR, LEP, LEPR, GPR54), or autosomal dominant (FGFR1) (12, 15, 25, 26). Among 99 normosmic IHH males screened by Park et al. (27), no PROP1 mutations, deletions, or polymorphisms were identified. We report three male patients with IHH revealed by cryptorchidism and/or delayed puberty who later proved to harbor other anterior pituitary deficiencies. Because such a deficiency remained isolated until the fourth decade of life, PROP1 mutations should be considered among genetic causes of normosmic IHH, even though they are rare in this setting. To our knowledge, only one previously reported patient harboring PROP1 mutation (R120C) also had hypogonadism as a presenting feature. This 28-yr-old Mexican-American woman with normal height and lack of puberty (28) had been referred to an endocrinology department for primary amenorrhea. Like our two older brothers, she had normal psychomotor development and uneventful childhood. Her prepubertal growth velocity without pubertal spurt was associated with being overweight. Thyrotroph and corticotroph functions remained normal, whereas gonadotroph and, in contrast to our cases, somatotroph functions were deficient at the time of presentation with GH unresponsive to two stimulation tests and low serum IGF-I levels. Our three brothers were also constantly overweight, their puberty failed to start, but their final height was normal (case 2) or at the lower limit of the normal range, with androgen replacement as the only treatment. None of them had thyrotroph deficiency or GHD during puberty. In contrast with most previously described patients with PROP1 mutants, these patients are characterized by a late onset GHD. Indeed GHD manifested after hypogonadotropic hypogonadism during the 4th decade of life, accounting for a normal height at adult age. Obesity, which was present during puberty in all three patients, presumably through associated hyperinsulism, may have contributed to their growth. Although detailed longitudinal biological and auxological data were not available in our two older patients, the notion that onset of GHD is delayed in this kindred is supported by their reaching normal final height without any GH treatment. In the older patient (no. 1) the GH response to GHRH test at age 38 yr was above the 75th percentile of the GH responses to a GHRH-arginine test in normal males (29). Such a good response was remarkable because the amplitude of GH response to stimulation tests is usually blunted in obese subjects (29, 30).

Previously described mutations of PROP1 gene that most frequently occur in exon 2 (12, 15, 31) were found to alter either the DNA-binding domain or the adjacent nuclear localization sequences (32) of this transcription factor. Our three patients shared a homozygous G-to-A transition at codon 582 in the PROP1 gene, representing the first mutation in the sequence encoding the transactivation domain. A frameshift was generated that yielded a truncated protein with preserved DNA-binding domain and nuclear localization sequences but lacking the transactivation domain. In our in vitro functional studies, we found this mutation markedly alters the transactivation capacity of the factor. The unexpected loss of DNA binding on a consensus response element suggests the so-called transactivation domain is directly or indirectly involved in DNA interaction. Because the exact physiological response elements of PROP1 and the three-dimensional structure of the protein are currently unknown, the mechanisms whereby such a mutation might alter the functional properties of the mutant remain speculative.

In conclusion, our in vitro studies confirmed the critical functional role of the C-terminal end of the transcription factor. Our observations underline interspecies phenotype differences and illustrate the phenotypic variability of PROP1 mutations in humans. These familial cases demonstrate that PROP1 mutations should be considered among possible genetic causes of initially isolated hypogonadotropic hypogonadism. Because patients with a PROP1 mutation may develop additional pituitary hormone deficiencies, they should undergo regular hormone evaluations to allow timely treatment of these deficits.


   Acknowledgments
 
We thank Jean-Louis Franc, Jean-Paul Herman, and Isabelle Pellegrini-Bouiller for fruitful team discussions. We are thankful to Prof. Michel Polak (Hôpital Necker Enfants Malades, Paris) for giving us access to the pediatric files of the younger affected brother previously followed in Prof. Raphaël Rappaport’s Pediatric Endocrinology Department.


   Footnotes
 
The GENHYPOPIT network for the study of genetic determinants of hypopituitarism was funded by the Groupement d’Intérêt Scientifique Institut des Maladies Rares (GISMR0201) and the Programe Hospitalier de Recherche Clinique (PHRC 25/2003, French Ministry of Health). The present study was supported by the Association pour le Développement des Recherches Biologiques et Médicales au Centre Hospitalier Régional de Marseille, Pharmacia International Fund, and Pfizer France, and grants (to R.R.) from Serono France and from Evian through the Société Française de Pédiatrie.

First Published Online June 7, 2005

Abbreviations: BMI, Body mass index; CPHD, combined pituitary hormone deficiency; IHH, idiopathic hypogonadotropic hypogonadism; ITT, insulin tolerance test; MRI, magnetic resonance imaging; PRL, prolactin; PROP1, Prophet of Pit-1; SDS, SD scores.

Received January 20, 2005.

Accepted May 26, 2005.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

  1. Savage JJ, Yaden BC, Kiratipranon P, Rhodes SJ 2003 Transcriptional control during mammalian anterior pituitary development. Gene 319:1–19[CrossRef][Medline]
  2. Ohta K, Nobukuni Y, Mitsubuchi H, Ohta T, Tohma T, Jinno Y, Endo F, Matsuda I 1992 Characterization of the gene encoding human pituitary-specific transcription factor, Pit-1. Gene 122:387–388[CrossRef][Medline]
  3. Li S, Crenshaw 3rd EB, Rawson EJ, Simmons DM, Swanson LW, Rosenfeld MG 1990 Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene pit-1. Nature 347:528–533[CrossRef][Medline]
  4. Lazar L, Gat-Yablonski G, Kornreich L, Pertzelan A, Phillip M 2003 PROP-1 gene mutation (R120C) causing combined pituitary hormone deficiencies with variable clinical course in eight siblings of one Jewish Moroccan family. Horm Res 60:227–231[Medline]
  5. Paracchini R, Giordano M, Corrias A, Mellone S, Matarazzo P, Bellone J, Momigliano-Richiardi P, Bona G 2003 Two new PROP1 gene mutations responsible for compound pituitary hormone deficiency. Clin Genet 64:142–147[Medline]
  6. Osorio MG, Marui S, Jorge AA, Latronico AC, Lo LS, Leite CC, Estefan V, Mendonca BB, Arnhold IJ 2002 Pituitary magnetic resonance imaging and function in patients with growth hormone deficiency with and without mutations in GHRH-R, GH-1, or PROP-1 genes. J Clin Endocrinol Metab 87:5076–5084[Abstract/Free Full Text]
  7. Duquesnoy P, Roy A, Dastot F, Ghali I, Teinturier C, Netchine I, Cacheux V, Hafez M, Salah N, Chaussain JL, Goossens M, Bougneres P, Amselem S 1998 Human Prop-1: cloning, mapping, genomic structure. Mutations in familial combined pituitary hormone deficiency. FEBS Lett 437:216–220[CrossRef][Medline]
  8. Fofanova O, Takamura N, Kinoshita E, Parks JS, Brown MR, Peterkova VA, Evgrafov OV, Goncharov NP, Bulatov AA, Dedov II, Yamashita S 1998 Compound heterozygous deletion of the PROP-1 gene in children with combined pituitary hormone deficiency. J Clin Endocrinol Metab 83:2601–2604[Abstract/Free Full Text]
  9. Krzisnik C, Kolacio Z, Battelino T, Brown M, Parks JS, Laron Z 1999 The little people of the island of Krk-Revisited. Etiology of hypopituitarism revealed. J Endocr Genet 1:9–19
  10. Nogueira CR, Sabacan L, Jameson JL, Medeiros-Neto G, Kopp P 1999 Combined pituitary hormone deficiency in an inbred Brazilian kindred associated with a mutation in the PROP-1 gene. Mol Genet Metab 67:58–61[CrossRef][Medline]
  11. Wu W, Cogan JD, Pfaffle RW, Dasen JS, Frisch H, O’Connell SM, Flynn SE, Brown MR, Mullis PE, Parks JS, Phillips 3rd JA, Rosenfeld MG 1998 Mutations in PROP1 cause familial combined pituitary hormone deficiency. Nat Genet 18:147–149[CrossRef][Medline]
  12. Vallette-Kasic S, Barlier A, Teinturier C, Diaz A, Manavela M, Berthezene F, Bouchard P, Chaussain JL, Brauner R, Pellegrini-Bouiller I, Jaquet P, Enjalbert A, Brue T 2001 PROP1 gene screening in patients with multiple pituitary hormone deficiency reveals two sites of hypermutability and a high incidence of corticotroph deficiency. J Clin Endocrinol Metab 86:4529–4535[Abstract/Free Full Text]
  13. Mody S, Brown MR, Parks JS 2002 The spectrum of hypopituitarism caused by PROP1 mutations. Best Pract Res Clin Endocrinol Metab 16:421–431[CrossRef][Medline]
  14. Sornson MW, Wu W, Dasen JS, Flynn SE, Norman DJ, O’Connell SM, Gukovsky I, Carriere C, Ryan AK, Miller AP, Zuo L, Gleiberman AS, Andersen B, Beamer WG, Rosenfeld MG 1996 Pituitary lineage determination by the Prophet of Pit-1 homeodomain factor defective in Ames dwarfism. Nature 384:327–333[CrossRef][Medline]
  15. Deladoey J, Fluck C, Buyukgebiz A, Kuhlmann BV, Eble A, Hindmarsh PC, Wu W, Mullis PE 1999 "Hot spot" in the PROP1 gene responsible for combined pituitary hormone deficiency. J Clin Endocrinol Metab 84:1645–1650[Abstract/Free Full Text]
  16. Rolland-Cachera MF, Cole TJ, Sempe M, Tichet J, Rossignol C, Charraud A 1991 Body mass index variations: centiles from birth to 87 years. Eur J Clin Nutr 45:13–21[Medline]
  17. Sempe M 1979 Auxologie, méthode et séquences. Theraplix, PARIS
  18. Tanner JM, Goldstein H, Whitehouse RH 1970 Standards for children’s height at ages 2–9 years allowing for heights of parents. Arch Dis Child 45:755–762
  19. Scully KM, Rosenfeld MG 2002 Pituitary development: regulatory codes in mammalian organogenesis. Science 295:2231–2235[Abstract/Free Full Text]
  20. Andersen B, Pearse 2nd RV, Jenne K, Sornson M, Lin SC, Bartke A, Rosenfeld MG 1995 The Ames dwarf gene is required for Pit-1 gene activation. Dev Biol 172:495–503[CrossRef][Medline]
  21. Tang K, Bartke A, Gardiner CS, Wagner TE, Yun JS 1993 Gonadotropin secretion, synthesis, and gene expression in two types of bovine growth hormone transgenic mice. Biol Reprod 49:346–353[Abstract]
  22. Vieira TC, Dias da Silva MR, Cerutti JM, Brunner E, Borges M, Arnaldi LT, Kopp P, Abucham J 2003 Familial combined pituitary hormone deficiency due to a novel mutation R99Q in the hot spot region of Prophet of Pit-1 presenting as constitutional growth delay. J Clin Endocrinol Metab 88:38–44[Abstract/Free Full Text]
  23. Fluck C, Deladoey J, Rutishauser K, Eble A, Marti U, Wu W, Mullis PE 1998 Phenotypic variability in familial combined pituitary hormone deficiency caused by a PROP1 gene mutation resulting in the substitution of Arg–>Cys at codon 120 (R120C). J Clin Endocrinol Metab 83:3727–3734[Abstract/Free Full Text]
  24. Reynaud R, Chadli-Chaieb M, Vallette-Kasic S, Barlier A, Sarles J, Pellegrini-Bouiller I, Enjalbert A, Chaieb L, Brue T 2004 A familial form of congenital hypopituitarism due to a PROP1 mutation in a large kindred: phenotypic and in vitro functional studies. J Clin Endocrinol Metab 89:5779–5786[Abstract/Free Full Text]
  25. Layman LC 2002 Human gene mutations causing infertility. J Med Genet 39:153–161[Abstract/Free Full Text]
  26. de Roux N, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom E 2003 Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc Natl Acad Sci USA 100:10972–10976[Abstract/Free Full Text]
  27. Park JK, Ozata M, Chorich LP, Cheng L, Bick DP, Sherins RJ, Ozdemir IC, Bolu E, Cogan JD, Phillips JA, Layman LC 2004 Analysis of the PROP1 gene in a large cohort of patients with idiopathic hypogonadotropic hypogonadism. Clin Endocrinol (Oxf) 60:147–149[Medline]
  28. Arroyo A, Pernasetti F, Vasilyev VV, Amato P, Yen SS, Mellon PL 2002 A unique case of combined pituitary hormone deficiency caused by a PROP1 gene mutation (R120C) associated with normal height and absent puberty. Clin Endocrinol (Oxf) 57:283–291[CrossRef][Medline]
  29. Qu XD, Gaw Gonzalo IT, Al Sayed MY, Cohan P, Christenson PD, Swerdloff RS, Kelly DF, Wang C 2005 Influence of body mass index and gender on growth hormone (GH) responses to GH-releasing hormone plus arginine and insulin tolerance tests. J Clin Endocrinol Metab 90:1563–1569[Abstract/Free Full Text]
  30. Cordido F, Alvarez-Castro P, Isidro ML, Casanueva FF, Dieguez C 2003 Comparison between insulin tolerance test, growth hormone (GH)-releasing hormone (GHRH), GHRH plus acipimox and GHRH plus GH-releasing peptide-6 for the diagnosis of adult GH deficiency in normal subjects, obese and hypopituitary patients. Eur J Endocrinol 149:117–122[Abstract]
  31. Fofanova OV, Takamura N, Kinoshita E, Parks JS, Brown MR, Peterkova VA, Evgrafov OV, Goncharov NP, Bulatov AA, Dedov, II, Yamashita S 1998 A mutational hot spot in the Prop-1 gene in Russian children with combined pituitary hormone deficiency. Pituitary 1:45–49[CrossRef][Medline]
  32. Guy JC, Hunter CS, Showalter AD, Smith TP, Charoonpatrapong K, Sloop KW, Bidwell JP, Rhodes SJ 2004 Conserved amino acid sequences confer nuclear localization upon the Prophet of Pit-1 pituitary transcription factor protein. Gene 336:263–273[Medline]



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