| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
Unidade de Endocrinologia do Desenvolvimento e Laboratório de Hormônios e Genética Molecular, LIM/42, Disciplina de Endocrinologia, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo (M.G.F.O., S.M., A.C.L., B.B.M., I.J.P.A.), Sao Paulo, Brazil; and Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University (P.K.), Chicago, Illinois 60611
Address all correspondence and requests for reprints: Ivo J. P. Arnhold, M.D., Disciplina de Endocrinologia, Hospital das Clínicas, Caixa Postal 3671, CEP 01065–970, São Paulo, Brazil. E-mail: iarnhold{at}usp.br
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Direct sequencing of the PROP-1 gene revealed homozygosity for a novel 263T>C transition that results in the replacement of a highly conserved phenylalanine by serine at codon 88 (F88S). F88 constitutes the hydrophobic core of the first helix of the homeodomain of PROP-1, and the substitution by the polar residue serine is expected to alter the secondary structure and impair binding of the mutated PROP-1 to DNA target sequences. The F88S mutation (which corresponds to murine F85S) was introduced into the murine Prop-1 complementary DNA and its consequences on DNA binding and trans-activation were assessed in vitro. In contrast to wild-type Prop-1, the F88S mutant showed no significant DNA binding to a PRDQ9 Prop-1 response element in gel shift assays. Transcriptional activation of a luciferase reporter gene containing a PRDQ9 site upstream of a simian virus 40 promoter was reduced to approximately 34% compared with that of wild-type Prop-1 in transiently transfected TSA-201 human embryonic kidney cells. The F88S mutation further expands the repertoire of mutations in PROP-1.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In this study we report clinical and molecular studies of a Brazilian girl who came to medical attention because of short stature and who was found to have deficiencies of GH, TSH, PRL, LH, and FSH. Molecular analysis of her PROP-1 gene revealed a novel mutation in the homeodomain, F88S. Consistent with the clinical phenotype, this mutation results in loss of DNA binding and reduced trans-activation properties in vitro. Furthermore, of all the reported human PROP-1 mutations, F88S, which corresponds to F85S in the murine protein, is most similar to that of the Ames dwarf mouse (S83P).
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Informed parental consent, patient assent, and approval of the hospital ethics committee were obtained before initiating the studies.
The patient was a Caucasian girl born at term by cesarean section. At
birth, her weight was 3.65 kg (50–90th percentile), and her length was
51 cm (50th percentile). There were no signs of hypoglycemia or
respiratory distress during the neonatal period, and her psychomotor
development was normal. Growth failure was first noticed at the age of
1.5 yr. At the age of 4.9 yr she had a height of 92.5 cm (-3.1
SD), and her bone age was severely retarded at 2.5 yr. She
was diagnosed with GH and TSH deficiencies (Table 1
), and treatment with
levo-T4 (6 µg/kg·day) and recombinant GH (0.1
U/kg day) sc resulted in adequate catch-up growth. At the age of 14.1
yr (bone age, 12 yr), her height was 150.0 cm (-1.5 SD),
her breast development was Tanner stage I, and her pubic hair was
Tanner stage II. A GnRH test revealed gonadotropin deficiency (Table 1), and administration of conjugated estrogens (0.3 mg/day) was added
to the hormone replacement regimen with levo-T4
and GH.
|
Heights were measured with a stadiometer, and height SD was calculated using British reference standards (17). Bone age was determined by the standards of Greulich and Pyle, and pubertal development was rated using Tanner stages.
Hormonal assays
T3, T4, insulin-like
growth factor I (IGF-I), estradiol, and testosterone levels were
measured at baseline. To assess anterior pituitary function, a combined
pituitary stimulation test was performed; glucose, GH, TSH, PRL, LH,
and FSH were measured before and at 15, 30, 45, 60, and 90 min after iv
administration of 0.1 U/kg insulin, 200 µg TRH, and 100 µg GnRH. GH
was also measured at 0, 60, 90, and 120 min after clonidine stimulation
(0.1 mg/m2, orally). Initially, GH was measured
by immunoradiometric assay, and TSH, PRL, and cortisol were determined
by RIA. Later these hormones were measured by immunofluorometric assays
(Table 1
). LH and FSH were measured by immunofluorometric assay. All
reagents were obtained from Wallac, Inc. (Turku, Finland),
with the exception of the TSH RIA (Abbott, Chicago, IL) and the
cortisol assay (INCSTAR Corp., Stillwater, MN). IGF-I was
measured by RIA after extraction with a commercial kit (Nichols Institute Diagnostics, San Juan Capistrano, CA).
Skull radiography and pituitary magnetic resonance imaging (MRI)
Radiological studies of the sella turcica were first performed with conventional posterior-anterior and lateral radiographs of the skull. Sellar volume was calculated according to the method of Underwood et al. (18). MRI scans were performed in a 1.5-Tesla unit (GE, Milwaukee, WI) using T1-weighted sagittal and coronal scans with repetition times of 366–433 ms and echo times of 20–23 ms. The maximal height of the pituitary gland was measured perpendicular to the sella turcica floor and compared to that in normal controls (19).
DNA analysis
Genomic DNA was isolated from peripheral blood from the proposita, her parents, the two normal sisters, and 20 normal subjects. Exons 1, 2, and 3 of the PROP-1 gene were amplified by PCR using the following three sets of primers: F1 (5'-GGAAGCAGAGAAATCTCAAGTC-3') and R1 (5'-GACTGGAGCACCCCTTGG-3'), F2 (5'-TGGTCCAGCACCGAGGAG-3') and R2 (5'-GCTATCATAGAATGTTGGGC-3'), and F3 (5'-GTGTCACCACCTATGTCAAGTGTG-3') and R3 (5'-GTCAGCTCACCGATTAGAA-3'). Amplification of each exon was carried out in 50-µL reactions using 200 ng genomic DNA, 10 mmol/L Tris-HCl (pH 8.3), 1.5 mmol/L MgCl2, 25 mmol/L KCl, 200 mmol/L of each deoxy-NTP, 15 pmol of each primer set, and 1.25 U Taq polymerase (Pharmacia, Uppsala, Sweden). PCR products were purified by enzymatic pretreatment with 10 U exonuclease I and 2 U shrimp alkaline phosphatase (Amersham Pharmacia Biotech and U.S. Biochemical Corp., Cleveland, OH). All PCR products were sequenced using the ABI Prism BigDye terminator kit (Perkin-Elmer Corp., Foster City, CA) in an ABI Prism Genetic Analyzer 310 automatic DNA sequencer (Perkin Elmer Corp.).
Plasmids
The murine wild-type Prop-1 complementary DNA (cDNA) in the expression vector pCMX was a gift from Prof. M. G. Rosenfeld (University of California-San Diego, La Jolla, CA) (4). The human F88 corresponds to F85 in the murine Prop-1. The murine mutant F85S was created using the overlap extension methodology with Pfu polymerase (Stratagene, La Jolla, CA) (20). The Ames mouse mutation, mS83P, which is located two codons amino-terminal from the mF85S mutation (3), was created by the same approach and included as control.
The reporter gene was generated by inserting a single copy of the Prop-1 response element PRDQ9 (ACTAATTGAATTAGC) into the BglII site of the vector pGL3 promoter (Promega Corp.) upstream of a simian virus 40 promoter and a luciferase gene (3).
The wild-type and the mutant cDNAs were cloned in-frame and without 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. Sequencing products were analyzed on a 377A Sequencer (PE Applied Biosystems, Foster City, CA).
DNA binding studies
Gel mobility shift assays were performed to assess the DNA
binding properties of the Prop-1 mutation mF85S. Wild-type and mutant
Prop-1 were transcribed and translated using the TNT-coupled
reticulolysate system (Promega Corp., Madison, WI). Part
(2.5 µL) of the reticulolysate reaction was preincubated at room
temperature in a 20-µL reaction with a binding buffer consisting of
20 mmol/L HEPES (pH 7.8), 50 mmol/L KCl, 1 mmol/L ethylenediamine
tetraacetate, 10% glycerol, 1 mmol/L 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; SA,
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 mmol/L Tris borate and 1
mmol/L ethylenediamine tetraacetate) 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 (21), 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 (2 µg/well) together with the pGL3-PRDQ9-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 in more than six independent experiments, and the groups were compared by ANOVA.
Green fluorescent fusion protein
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, expression and localization of the fusion protein were analyzed under an inverted fluorescent microscope.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The relevant hormone values are summarized in Table 1. At 4.9 yr
of age, the patient’s plasma IGF-I level was low (49 ng/mL). Serum GH
levels were also very low and failed to reach normal levels after
stimulation by hypoglycemia and clonidine (peak GH, 1.9 ng/mL; normal,
>7 ng/mL). Basal TSH and PRL levels were normal, but they displayed a
blunted response to stimulation with TRH and hypoglycemia. Of note,
basal cortisol levels were normal, and they rose adequately in response
to hypoglycemia (Table 1). At 10.7 yr (bone age, 10 yr), the patient
was prepubertal, and gonadotropin levels were below the limit of
detection of the assay both before and after GnRH administration (Table 1). A repeated evaluation at 14.1 yr of age again documented GH, TSH,
PRL, and gonadotropin deficiencies and confirmed normal function of the
ACTH-adrenal axis (Table 1).
Pituitary imaging studies
At 9 yr, radiography of the skull revealed a wide, open sella with
a volume of 484 mm3, which is above the average
for age (Fig. 1). At the same age, a MRI
showed a normal pituitary stalk, a normally located posterior lobe, and
a hypoplastic and asymmetric anterior pituitary lobe with a maximal
height of 3 mm (normal range in age-matched controls, 4.5 ± 0.6
mm; Fig. 1).
|
Direct sequencing of PCR fragments derived from exon 2 of the
patient revealed a homozygous transition of nucleotide 263T>C (Fig. 2). This mutation leads to substitution
of the highly conserved phenylalanine 88 by serine (F88S) in the first
-helix of the homeodomain of PROP-1. Consistent with an autosomal
recessive mode of inheritance, the parents and sisters of our patient
were heterozygous for this mutation and clinically normal (Fig. 2). In
contrast, sequence analysis of exon 2 of the PROP-1 gene
from 20 normal subjects (40 alleles) did not reveal this alteration,
suggesting that it is not a polymorphic variant.
|
The DNA-binding properties of the murine Prop-1 wild-type, the
mF85S mutant, and the Ames mouse mutant mS83P were tested on the PRDQ9
response element. In contrast to the wild-type, which showed strong
binding to this response element, the mF85S mutant had no detectable
DNA binding under the chosen experimental conditions, and the
protein/DNA complexes of mS83P were very weak (Fig. 3). Adequate transcription/translation of
the various constructs was confirmed by transcription/translation of
the proteins in the presence of 35S-labeled
methionine and subsequent analysis by SDS-PAGE (data not shown).
|
Cotransfection of murine wild-type Prop-1 resulted in strong
stimulation of a pGL3-PRDQ9-luciferase reporter gene compared with that
of empty vector (Fig. 4). Activation by
the Prop-1 mutant mF85S was significantly reduced to about 34% of the
wild-type and to about 43% in the case of the Ames dwarf mutant.
|
The wild-type and the two mutants, mF85S and mS83P, 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
two mutants (Fig. 5).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). Using the tightly
linked polymorphic microsatellite marker D5S408, Cogan et
al. demonstrated that the affected families with the 301–302delAG
mutation are not related and that this mutation is recurring
independently, an observation arguing against a founder effect (6). The
frequent occurrence of this mutation suggests that the AG repeat
in exon 2 is prone to misalignment during replication.
|
Although overt ACTH deficiency appears to be uncommon in patients with PROP-1 mutations, corticotroph function may also be affected in some of these patients. Parks et al. reported that partial ACTH deficiency eventually developed in 2 of 9 patients with PROP-1 mutations (22). Mendonca et al. reported a patient with the 301–302delAG mutation in the PROP-1 gene who had high normal basal cortisol levels at 6.6 yr, but evidence of partial cortisol deficiency when tested again at 15.2 yr (9). Moreover, Deladoëy et al. found low basal cortisol and ACTH levels in 7 of 35 patients, but their responses to insulin-induced hypoglycemia were normal (10). The blunted response in cortisol secretion in 1 individual studied by Nogueira et al. was thought to be the consequence of long-term treatment with prednisone (23). Recently, Pernasetti et al. reported a large consanguineous Brazilian family with 10 members affected by the 301–302delAG mutation in the PROP-1 gene, ranging in age from 8–67 yr (15). An impaired cortisol response to hypoglycemia was present in an 11-yr-old and in 5 of 6 of the older patients (43–67 yr old), suggesting late-onset impairment of the ACTH-adrenal axis. This suggests that partial ACTH deficiency may develop with age in a subset of patients with PROP-1 mutations (9). Repeated evaluation of the pituitary-adrenal axis indicates that the patient reported here has maintained a normal cortisol response to hypoglycemia up to age 14.1 yr.
Underwood et al. measured sella turcica volume on
skull radiographs in 34 patients with idiopathic hypopituitarism, and
all had sellar volumes below the normal mean for age, suggesting that a
smaller pituitary gland size is frequently found in this disorder (18).
In contrast, Parks et al. observed large sellae turcicae and
pituitary enlargement in patients with the 301–302delAG mutation in
the PROP-1 gene (22). One of these patients underwent
surgery to remove a pituitary mass with suprasellar extension that
showed amorphous material with occasional fibroblasts on histopathology
(22). Rosenbloom et al. reported increased sella turcica
area for height age on lateral skull radiographs in 3 of 8 patients
with 301–302del AG in PROP-1 from the Dominican Republic (8). We have
previously reported a patient with CPHD due to the 301–302delAG in
PROP-1 gene mutation who had a large sella turcica on skull
x-rays at the age of 8.8 yr as well as an enlarged pituitary gland with
a diffuse hyperintense signal on T1-weighted MRI images (9). Six years
later, reevaluation by MRI demonstrated a marked reduction of the
anterior pituitary gland (9). Our patient with the F88S mutation has a
wide, open sella turcica with a sellar volume above the normal mean for
age, but a small anterior pituitary lobe on MRI. These findings suggest
that some patients with PROP-1 deficiency might have a period of
pituitary enlargement followed by involution. Although the majority of
patients with CPHD caused by molecular alterations in the
PROP-1 gene were found to have hypoplastic pituitary glands
on MRI (4, 5, 14, 15, 23), conventional sellar radiographs were rarely
reported (Table 2). To determine
whether a round or large sella turcica may be common in patients with
PROP-1 gene mutations will require further studies.
|
-helix forming the homeodomain. Replacement of the
hydrophobic phenylalanine residue by the polar serine amino acid is
predicted to alter the structure of the first -helix and impair
binding of PROP-1 to target DNA sequences (24). Furthermore, this
phenylalanine is highly conserved among at least 529 homeodomain
transcription factors (25, 26). F88 in human PROP-1 corresponds to F85
in murine Prop-1, which is two codons carboxyl-terminal to the S83P
mutation that causes CPHD in the Ames dwarf mouse (3). The mF85S/hF88S
mutation shows severely impaired DNA binding and significantly reduced
trans-activation properties in vitro, supporting
the important role of F88 in formation of helix 1 of the
homeodomain. In conclusion, the F88S mutation reported here further expands the repertoire of PROP-1 mutations that cause CPHD and underscores the critical role of this highly conserved hydrophobic phenylalanine in the structure and function of the homeodomain of PROP-1.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received September 3, 1999.
Revised October 7, 1999.
Revised May 5, 2000.
Accepted May 14, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cys at codon 120 (R120C). J Clin Endocrinol
Metab. 83:3727–3734.This article has been cited by other articles:
 |
|
 |
|
  D. Kelberman and M. T. Dattani Hypothalamic and pituitary development: novel insights into the aetiology Eur. J. Endocrinol., August 1, 2007; 157(suppl_1): S3 - S14. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Heathcote, C. Braybrook, L. Abushaban, M. Guy, M. E. Khetyar, M. A. Patton, N. D. Carter, P. J. Scambler, and P. Syrris Common arterial trunk associated with a homeodomain mutation of NKX2.6 Hum. Mol. Genet., March 1, 2005; 14(5): 585 - 593. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Reynaud, M. Chadli-Chaieb, S. Vallette-Kasic, A. Barlier, J. Sarles, I. Pellegrini-Bouiller, A. Enjalbert, L. Chaieb, and T. Brue 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., November 1, 2004; 89(11): 5779 - 5786. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. K. Lee, Y.-S. Zhu, J. J. Cordero, L.-Q. Cai, I. Labour, C. Herrera, and J. Imperato-McGinley Long-Term Growth Hormone Therapy in Adulthood Results in Significant Linear Growth in Siblings with a PROP-1 Gene Mutation J. Clin. Endocrinol. Metab., October 1, 2004; 89(10): 4850 - 4856. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Voutetakis, M. Argyropoulou, A. Sertedaki, S. Livadas, P. Xekouki, M. Maniati-Christidi, I. Bossis, N. Thalassinos, N. Patronas, and C. Dacou-Voutetakis Pituitary Magnetic Resonance Imaging in 15 Patients with Prop1 Gene Mutations: Pituitary Enlargement May Originate from the Intermediate Lobe J. Clin. Endocrinol. Metab., May 1, 2004; 89(5): 2200 - 2206. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. N. Cohen, L. E. Cohen, D. Botero, C. Yu, A. Sagar, M. Jurkiewicz, and S. Radovick Enhanced Repression by HESX1 as a Cause of Hypopituitarism and Septooptic Dysplasia J. Clin. Endocrinol. Metab., October 1, 2003; 88(10): 4832 - 4839. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. C. Vieira, M. R. Dias da Silva, J. M. Cerutti, E. Brunner, M. Borges, L. T. Arnaldi, P. Kopp, and J. Abucham 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., January 1, 2003; 88(1): 38 - 44. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. G. F. Osorio, S. Marui, A. A. L. Jorge, A. C. Latronico, L. S. S. Lo, C. C. Leite, V. Estefan, B. B. Mendonca, and I. J. P. Arnhold 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., November 1, 2002; 87(11): 5076 - 5084. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. E. Cohen and S. Radovick Molecular Basis of Combined Pituitary Hormone Deficiencies Endocr. Rev., August 1, 2002; 23(4): 431 - 442. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Vallette-Kasic, A. Barlier, C. Teinturier, A. Diaz, M. Manavela, F. Berthezene, P. Bouchard, J. L. Chaussain, R. Brauner, I. Pellegrini-Bouiller, et al. 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., September 1, 2001; 86(9): 4529 - 4535. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
