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A Familial Form of Congenital Hypopituitarism Due to a PROP1 Mutation in a Large Kindred: Phenotypic and in Vitro Functional Studies

Laboratoire des Interactions Cellulaires Neuro-Endocriniennes (R.R., S.V.-K., A.B., I.P.-B., 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; Department of Endocrinology, Diabetology, Farhat Hached Hospital (M.C.-C., L.C.), University of Sousse, Sousse, Tunisia 4000; and Departments of Pediatrics (R.R., J.S.) and Endocrinology (S.V.-K., T.B.), Centre Hospitalo-Universitaire Timone, and Laboratoire de Biochimie et Biologie Moleculaire (A.B., A.E.), Hôpital de la Conception, 13385 Marseille, 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

 
We report the natural history of a hypopituitarism in a large Tunisian kindred including 29 subjects from the same consanguineous family. The index case was a 9-yr-old girl with severe growth retardation due to complete GH deficiency and partial corticotroph, lactotroph, and thyrotroph deficiencies. Magnetic resonance imaging showed a hyperplastic anterior pituitary. Thirteen of the 28 relatives examined (10 female subjects) had hypopituitarism. In the 14 patients, previously untreated (aged 6–53 yr), height was –5.7 ± 1.7 SD score, and puberty was spontaneously initiated in only two females. Complete GH deficiency was found in all 12 patients investigated, of whom 11 had thyrotroph and eight of 10 had corticotroph deficiency. A homozygous R73C mutation of PROP1 was present in all 10 patients studied, and a heterozygous mutation was found in six unaffected parents or siblings. In vitro the mutant had 11.5% of the transactivation capacity of the wild type and was unable to bind to a high-affinity DNA sequence. This report showed the deleterious effect of the recessive R73C mutation that affects a hot spot of the PROP1 gene and was associated with severe dwarfism, a lack of spontaneous puberty, and a high incidence of early onset of corticotroph deficiency.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
DURING THE PAST 10 yr, our understanding of anterior pituitary ontogenesis has been considerably broadened by the identification of several transcription factors including Pit-1 (POU1F1), PROP1, HESX1, LHX3, LHX4, PITX1, PITX2, and TPIT (1, 2, 3, 4, 5, 6, 7, 8). These nuclear proteins act in concert with other factors to control the differentiation and the development of one or more anterior pituitary cell lineages (9). Both in humans and in animal models, alterations affecting the genes encoding these factors may lead to sporadic or familial forms of anterior pituitary hormone deficiency (3, 9, 10, 11, 12). These may either affect a single hormone cell type (8) or several cell lineages as in combined pituitary hormone deficiencies (CPHD). PROP1 gene alterations currently represent the most frequently recognized form of genetically determined CPHD in man. PROP1 is a paired like homeobox gene mapped to chromosome 5q in humans. This gene consists of three exons and encodes a 226-amino-acid protein. PROP1 is expressed early, from embryonic d 10.5 in mouse, and only transiently during pituitary ontogenesis. Its expression is necessary to activate gene programs required for ventral proliferation and determination of at least three cell lineages (somatotrophs, lactotrophs, and thyrotrophs). Gonadotroph differentiation and function in Prop1-deficient Ames dwarf mice appear different from what are observed in human PROP1 mutations. Due to interspecies differences or to a hypomorphic mutation in Ames mice, these animals only present with secondary hypofertility, whereas human patients are unable to spontaneously start or complete puberty. The gene activation of Prop1 is dependent on the temporal down-regulation of Hesx1 expression, a paired-like transcriptional repressor (13) that also down-regulates Pit-1 expression. At least 15 PROP1 gene allelic variants have currently been found in association with a human CPHD phenotype transmitted as a recessive autosomal trait. Two sites of hypermutability have been identified at codons 73 and 99 (14, 15). Functional studies of some of the corresponding mutant proteins have been performed including mutation of codon 99 (11, 16, 17), but none has currently been reported on the hot spot codon 73 mutants. Scarce data on the natural history of CPHD due to PROP1 mutations are available, and no genotype-phenotype relationships could be identified in this disease. The present paper reports a phenotypic and genotypic analysis carried out in a large Tunisian kindred with CPHD due to a R73C PROP1 gene mutation. The functional characteristics of the mutant protein were studied in vitro by transient transfection and electromobility shift assay experiments.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Patients

All of the patients belonged to the same family characterized by a high degree of consanguinity (Fig. 1A). The index case was initially referred to the Endocrinology Department of the Sousse Hospital for investigation of severe growth retardation. A complete clinical, biological, and neuroradiological workup of this 9-yr-old girl (Figs. 1B and 2) was carried out. Because she was found to have a family history of severe dwarfism affecting several of her relatives, all of the available subjects of the family who had given an informed consent were then investigated. Informed, written consent was obtained from all adult patients and parents of minor patients, including authorization for publication of patients’ photographs. This study was approved by the local ethics committee. Stature was measured in centimeters using a meter attached to the wall with the average of three measurements recorded. Weight was measured with a balance-beam scale. The body mass index (BMI) was calculated weight in kilograms divided by the square of height in meters. The SD score (SDS) for height and height age was estimated from French reference values (18, 19). Roentgenograms of the hand and wrist were compared with the standards of Greulich and Pyle to determine bone age.

View larger version (80K): [in this window] [in a new window]   FIG. 1. A, Pedigree of the index family. Males and females are indicated by squares and circles, respectively. Affected patients with genetic study are indicated by black forms, and affected patients without genetic study are indicated by gray forms. Heterozygous unaffected relatives are indicated by half-black squares or circles when molecular study has been performed. Half-gray squares and circles designate obligatory heterozygous relatives without molecular exploration. *, Index case. B, Sisters and mother of index case. Bottom row, Index case, V39 (age 9 yr). Middle row, Three affected sisters, homozygous for R73C PROP1 mutation (V30, age 31 yr; V35, age 20 yr; and V33, age 24 yr). Top row, One unaffected sister (V32, age 25 yr) and mother (IV24), both heterozygous for the same R73C mutation. C, Views of relatives of index case with nine cases homozygous for the R73C PROP1 mutation. Left panel, Patient V13 (age 20 yr) with her normal sister (age 21 yr) and their mother IV16. Top row, The oldest affected relatives, patients IV8 (age 53 yr) and IV12 (age 43 yr). Second row, Affected siblings V21 (age 30 yr), V22 (age 28 yr), V25 (age 18 yr), and V29 (age 6 yr). Third row, Patient V15 (age 18 yr) and V5 (age 28 yr).

View larger version (100K): [in this window] [in a new window]   FIG. 2. Index case #1 (V39). Front (A) and lateral (C) views of the 9-yr-old index case with immature facies, depressed nasal bridge, and marked frontal bossing. B, Sagittal image obtained by MRI showing a hyperintense, enlarged pituitary gland.

IGF-I concentrations were measured by RIA (Diagnostic Systems Laboratories, Inc., Webster, TX) with a sensitivity of 2 ng/ml; normal range depending on age and Tanner pubertal stage was as follows: 0–4 yr, 50–170 ng/ml; older than 4 yr, 75–500 ng/ml; Tanner II, 245–400 ng/ml; Tanner III, 250–600 ng/ml; Tanner IV and V, 270–550 ng/ml; adult, 100–310 ng/ml). GH was measured by a commercial RIA kit. The GH response was studied with two provocative test [arginine and insulin tolerance test (ITT), 0.05 U/kg]. Basal plasma ACTH and cortisol were measured at 0800 h. The plasma ACTH and cortisol response was also determined during ITT in the index case. (Normal range for cortisol: 210–560 nmol/liter at 0800 h.) Investigation of the gonadotroph axis was performed by measurement of LH and FSH levels at baseline and 30 and 60 min after a GnRH provocative test (100 µg iv Gonadorelin; Ferring, Gentilly, France), together with determination of basal plasma testosterone or estradiol levels. A TRH test was carried out in the index patient with measurements of prolactin (PRL) and TSH at 0, 30, and 60 min after an iv bolus.

Radiological imaging

Lateral skull films were examined for sellar area. Pituitary magnetic resonance imaging (MRI) was performed in the index patient, using precontrast coronal spin echo T1-weighted images, followed by postgadolinium T1-weighted imaging.

Genomic analysis of the PROP1 gene

All three exons of the PROP1 gene were PCR amplified from genomic DNA using primers flanking the exons for direct sequencing. DNA was extracted from the peripheral lymphocytes using the QIAamp Blood kit (QIAGEN, Hilden, Germany). Exons 2, 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 of Pfu DNA polymerase (Promega, 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. The 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, Boston, MA) using the same PCR primers. Mutations were confirmed by repeat PCR and subsequent sequencing of PCR products. Except in the index case, when a mutation was found in exon 2, exons 1 and 3 were not sequenced.

Plasmid constructs and mutagenesis

The target vector was a ptk-luc (promoter thymidine kinase-luciferase) construct (gift of Dr. Joseph Martial, Liège, 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 reporter gene 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 CMV promoter. The empty pcDNA3T7+ vector was included as a negative control. The R73C mutant PROP1 vector was obtained by site-directed mutagenesis using the Quickchange kit (Stratagene Cloning System, La Jolla, CA); the mutagenesis nucleotide sequence was G CGC CGC CAC TGC ACC ACC TTC AGC).

Cell culture and transfection

The transactivation capacities of wild-type and mutant PROP1 were studied by transient cotransfection experiments in HeLa cells using the liposome technique (Polyfect Transfection Reagent, QIAGEN) according to the manufacturer’s instructions. Briefly, 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 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 of reporter lysis buffer (Promega). 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 (luciferase assay system, Promega) and ß-galactosidase assays (the colored reaction was obtained using 50 mM ß-mercaptoethanol and 25 mM orthonitrophenyl galactopyranoside, and the OD was read at 420 nm). For each control, the total luciferase activity normalized against ß-galactosidase activity was taken as one, 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 R73C PROP1 mutant. Wild-type and mutant PROP1 were transcribed and translated using the TNT-coupled reticulocyte lysate system with T7 polymerase (Promega). 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 nonradioactive amino acid mixture minus methionine and [³⁵S]methionine. The 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

 
Patients’ characteristics

We have studied a large consanguineous family from a geographically isolated rural area in central Tunisia (region of Sidi Bouzid) (Fig. 1A). The index case was a 9-yr, 3-month-old girl referred for severe growth retardation to the Endocrinology Department of the Farhat Hached Hospital, University of Sousse. Born to consanguineous parents, her height was 91 cm (–8.0 SDS), with a markedly delayed bone age (1.8 yr) (Table 1). She had the so-called doll-like aspect and marked frontal bossing (Fig. 2, A and C) suggestive of somatotroph deficiency. This was confirmed by endocrine investigations (Table 2) showing complete GH deficiency, with undetectable GH concentrations (peak <0.2 µg/liter) during both arginine and ITT tests (blood glucose nadir, 1.1 mmol/liter at ITT). At this time, partial corticotroph deficiency (cortisol peak at ITT, 476 nmol/liter; ACTH, 6 pg/ml) and partial lactotroph deficiency (peak PRL under TRH, 4.5 µg/liter) were present. TSH was normal, and free T₄ remained in the lower limit of normal range (1.5 mIU/liter and 10.1 pmol/liter, respectively); a delayed (90 min) and blunted (peak, 3.8 mIU/liter) TSH response to TRH suggested partial central hypothyroidism. Pituitary MRI revealed a homogeneous pituitary enlargement (Fig. 2B). Glucocorticoid replacement was initiated at age 9 yr 3 months. Under recombinant GH therapy, given at age 10, growth velocity rose to 7 cm/yr (+5.7 SDS). Thyroid function monitoring revealed thyroid function deficiency at age 10 yr 5 months (TSH, 0.26 mIU/liter; free T₄, 5.54 pmol/liter) requiring T₄ therapy. The familial screening examined 28 relatives of the index case, of whom 13 had a clinical presentation of hypopituitarism (dwarfism and lack of pubertal development): three male and 10 female patients (Fig. 1, A and C). A total of 14 affected subjects were thus identified in this large consanguineous family. Their auxological characteristics presented in Table 1 can be compared with those of their clinically normal relatives. In the 14 affected individuals aged 6–53 yr, the mean height SDS was –5.7 ± 1.7 and height age was 8.7 ± 2.5 yr. None of the patients had increased BMI; BMI for height age was normal in 11 and under –2 SDS in three. In the 12 subjects aged at least 18 yr, height was markedly decreased compared with their adult relatives: –5.5 ± 1.5 SDS (n = 9) vs. 1.2 ± 0.9 SDS (n = 8) in women; –6.7 ± 0.5 SDS (n = 3) vs. 0.6 ± 0.5 SDS (n = 5) in men. Among the 12 patients beyond pubertal age, signs of puberty were absent in 10, and two female patients are Tanner classification B2P1 (Table 3). In addition to hormonal investigations performed in the index case, 11 adult patients were available for an endocrine workup (Tables 2 and 3). All had complete GH deficiency evidenced by undetectable GH and IGF-I levels, and low IGF binding protein 3 values ranging from 188 to 650 ng/ml (normal range, 1000–3300 ng/ml) (Table 2). Corticotroph deficiency was unexpectedly present in seven of nine investigated patients with such a high incidence. Thyrotroph deficiency was present in 10 of 11.

Complete sequencing of the PROP1 gene in the index case allowed identification of a missense mutation of this gene, by transition of a C to a T at position 217. This single nucleotide change predicted substitution of an Arg to a Cys at codon 73 (R73C) (Fig. 3A). PROP1 gene analysis performed in 14 other family members showed the same homozygous R73C mutation in eight relatives who all presented with a CPHD phenotype. In the six remaining subjects a heterozygous R73C mutation was present, and none of the four examined had clinical features of CPHD, in keeping with a recessive mode of inheritance (Fig. 1A).

View larger version (21K): [in this window] [in a new window]   FIG. 3. A, Direct sequencing of the coding exons of the PROP1 gene revealed a single homozygous C to T change at position 217. B, Cotransfection experiment using expression vectors of wild-type or mutant R73C PROP1 with ptk-PRDQ3-luciferase reporter gene. Wild-type PROP1 strongly stimulated the ptk-PRDQ3-luciferase reporter gene compared with pcDNA3 empty vector. Relative luciferase activity was expressed as fold increase ± SD from the empty vector (pcDNA3) on three distinct experiments. Relative luciferase activity of R73C was reduced to 11.5% of wild-type activity.

Cotransfection of wild-type PROP1 resulted in a strong stimulation of the ptk-PRDQ9-luciferase reporter gene compared with that of the empty vector. Activation by PROP1 R73C mutant was dramatically reduced to 11.5% of the wild type (Fig. 3B). To ascertain that the stimulation of reporter gene activity was due to the effect of wild-type or mutant PROP1 on the PRDQ3 paired factor target sequence, and not to a nonspecific effect on the ptk-luc empty vector, we checked that this empty vector had low basal activity and was not regulated by PROP1 (data not shown).

Gel retardation assay

DNA binding of the PROP1 wild type and the R73C mutant was tested on the PRDQ9 response element. The wild type had a strong detectable DNA binding that is specifically displaced with an excess of unlabeled PRDQ9 oligonucleotide. No detectable DNA binding was observed with R73C under the chosen experimental conditions (Fig. 4A), whereas in vitro translated PROP1 and R73C proteins were similar for their expression and electrophoretic properties (Fig. 4B).

View larger version (47K): [in this window] [in a new window]   FIG. 4. A, 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 (band indicated by arrow). Lane 4, Specific PROP1 binding (–) was displaced by the addition of a 100-fold excess of cold nucleotide (+). Lanes 5 and 6, For the R73C PROP1 mutant, before (–) or after (+) cold-probe competition, no specific protein-DNA interactions were detectable. B, Autoradiogram of labeled wild-type and R73C PROP1 proteins translated in vitro in the presence of [35S]methionine. Lane 1 denotes negative control using the empty pcDNA3 vector.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

In addition to codon 99, codon 73 was shown to be another hot spot of the PROP1 gene in our previous study that did not include the family currently screened (15). Remarkably, all four homozygous R73C mutations found in families not known to be related were diagnosed in patients of Tunisian origin (15, 29). It is not clear whether this finding may be due to a founder effect or to a site of hypermutability due to the presence of CpG doublet. This missense mutation affects a highly conserved region among paired like homeodomain proteins involved in the binding of the transcription factor to its cognate DNA target sites. However, no functional study had been performed to ascertain the deleterious effect of this gene defect. Our in vitro data, although to some degree reflecting studies performed in a heterologous system, showed that this mutation virtually abolished both transactivating and binding capacities of the PROP1 protein. We had previously reported another mutation of this codon, R7³H. This mutation induces a similar alteration of the transcriptional activating properties of the protein compared with the R73C mutant (data not shown). At difference with cysteine, histidine is unable to form a new disulfide bond that could change the three-dimensional conformation of Prop1. In the absence of crystallographic data on the Prop1-DNA complex, however, the hypothesis of a newly formed disulfide bond involving the cysteine that replaces the normal arginine residue remains speculative.

This large familial study provided an opportunity to study the natural history of congenital hypopituitarism due to the R73C PROP1 mutation. All of the 14 affected patients examined had severe dwarfism and a lack of spontaneous puberty. None of them presented with blue sclera or limitation of elbow extensibility described in other families (20, 21). It is noteworthy that corticotroph deficiency was found with a high incidence at a relatively early age in this population. Although largely asymptomatic, this finding requires particular attention in such patients and warrants regular follow-up. Genetic screening of the PROP1 gene in hypopituitary patients is useful to the clinical management of the affected children because it permits an earlier diagnosis of the hormone deficits of gradual onset, especially thyrotroph and corticotroph deficiencies, and also allows anticipating gonadotroph deficiency at pubertal age. Our functional studies revealed that this mutant was defective in its ability to bind to a paired like factor response element, thereby preventing activation of its target genes. Such phenotypic and genotypic studies will broaden our knowledge of the role of transcription factors in pituitary development and function.

	Footnotes

 
This work was supported by the Association pour le Développement des Recherches Biologiques et Médicales au Centre Hospitalier Régional de Marseille (ADEREM), Pharmacia International Fund and Pharmacia/Pfizer France, Groupement d’Intérêt Scientifique (GIS) Institut des Maladies Rares (GISMR0201), the Programe Hospitalier de Recherche Clinique (PHRC 2003, French Ministry of Health), and grants (to R.R.) from Serono France and Evian through the Société Française de Pédiatrie.

Abbreviations: BMI, Body mass index; CPHD, combined pituitary hormone deficiencies; ITT, insulin tolerance test; MRI, magnetic resonance imaging; PRL, prolactin; SDS, SD score.

Received December 11, 2003.

Accepted August 4, 2004.

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Top Abstract Introduction Patients and Methods Results Discussion References

 

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