Familial glucocorticoid receptor haploinsufficiency by non-sense mediated mRNA decay, adrenal hyperplasia and apparent mineralocorticoid excess.

Abstract

Primary glucocorticoid resistance (OMIM 138040) is a rare hereditary disease that causes a generalized partial insensitivity to glucocorticoid action, due to genetic alterations of the glucocorticoid receptor (GR). Investigation of adrenal incidentalomas led to the discovery of a family (eight affected individuals spanning three generations), prone to cortisol resistance, bilateral adrenal hyperplasia, arterial hypertension and hypokalemia. This phenotype exacerbated over time, cosegregates with the first heterozygous nonsense mutation p.R469[R,X] reported to date for the GR, replacing an arginine (CGA) by a stop (TGA) at amino-acid 469 in the second zinc finger of the DNA-binding domain of the receptor. In vitro, this mutation leads to a truncated 50-kDa GR lacking hormone and DNA binding capacity, devoid of hormone-dependent nuclear translocation and transactivation properties. In the proband's fibroblasts, we provided evidence for the lack of expression of the defective allele in vivo. The absence of detectable mutated GR mRNA was accompanied by a 50% reduction in wild type GR transcript and protein. This reduced GR expression leads to a significantly below-normal induction of glucocorticoid-induced target genes, FKBP5 in fibroblasts. We demonstrated that the molecular mechanisms of glucocorticoid signaling dysfunction involved GR haploinsufficiency due to the selective degradation of the mutated GR transcript through a nonsense-mediated mRNA Decay that was experimentally validated on emetine-treated propositus' fibroblasts. GR haploinsufficiency leads to hypertension due to illicit occupation of renal mineralocorticoid receptor by elevated cortisol rather than to increased mineralocorticoid production reported in primary glucocorticoid resistance. Indeed, apparent mineralocorticoid excess was demonstrated by a decrease in urinary tetrahydrocortisone-tetrahydrocortisol ratio in affected patients, revealing reduced glucocorticoid degradation by renal activity of the 11β-hydroxysteroid dehydrogenase type 2, a GR regulated gene. We propose thus that GR haploinsufficiency compromises glucocorticoid sensitivity and may represent a novel genetic cause of subclinical hypercortisolism, incidentally revealed bilateral adrenal hyperplasia and mineralocorticoid-independent hypertension.

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Familial Glucocorticoid Receptor Haploinsufficiency by
Non-Sense Mediated mRNA Decay, Adrenal Hyperplasia
and Apparent Mineralocorticoid Excess
Je ´ro ˆme Bouligand1,2,3., Brigitte Delemer4., Annie-Claude Hecart4, Geri Meduri1,2, Say Viengchareun2,3,
Larbi Amazit2,3, Se ´verine Trabado1,2,3, Bruno Fe `ve1,2,5, Anne Guiochon-Mantel1,2,3, Jacques Young1,2,5,
Marc Lombe `s1,2,5*
1Assistance Publique-Ho ˆpitaux de Paris, Ho ˆpital de Bice ˆtre, Service de Ge ´ne ´tique Mole ´culaire, Pharmacoge ´ne ´tique et Hormonologie, Le Kremlin Bice ˆtre, France,
2INSERM, U693, Le Kremlin Bice ˆtre, France, 3Universite ´ Paris-Sud, Faculte ´ de Me ´decine Paris-Sud, UMR-S693, Le Kremlin Bice ˆtre, France, 4Service d’Endocrinologie,
Centre Hospitalier Robert De ´bre ´, Reims, France, 5Assistance Publique-Ho ˆpitaux de Paris, Ho ˆpital de Bice ˆtre, Service d’Endocrinologie et Maladies de la Reproduction, Le
Kremlin Bice ˆtre, France
Abstract
Primary glucocorticoid resistance (OMIM 138040) is a rare hereditary disease that causes a generalized partial insensitivity to
glucocorticoid action, due to genetic alterations of the glucocorticoid receptor (GR). Investigation of adrenal incidentalomas
led to the discovery of a family (eight affected individuals spanning three generations), prone to cortisol resistance, bilateral
adrenal hyperplasia, arterial hypertension and hypokalemia. This phenotype exacerbated over time, cosegregates with the
first heterozygous nonsense mutation p.R469[R,X] reported to date for the GR, replacing an arginine (CGA) by a stop (TGA)
at amino-acid 469 in the second zinc finger of the DNA-binding domain of the receptor. In vitro, this mutation leads to a
truncated 50-kDa GR lacking hormone and DNA binding capacity, devoid of hormone-dependent nuclear translocation and
transactivation properties. In the proband’s fibroblasts, we provided evidence for the lack of expression of the defective
allele in vivo. The absence of detectable mutated GR mRNA was accompanied by a 50% reduction in wild type GR transcript
and protein. This reduced GR expression leads to a significantly below-normal induction of glucocorticoid-induced target
genes, FKBP5 in fibroblasts. We demonstrated that the molecular mechanisms of glucocorticoid signaling dysfunction
involved GR haploinsufficiency due to the selective degradation of the mutated GR transcript through a nonsense-mediated
mRNA Decay that was experimentally validated on emetine-treated propositus’ fibroblasts. GR haploinsufficiency leads to
hypertension due to illicit occupation of renal mineralocorticoid receptor by elevated cortisol rather than to increased
mineralocorticoid production reported in primary glucocorticoid resistance. Indeed, apparent mineralocorticoid excess was
demonstrated by a decrease in urinary tetrahydrocortisone-tetrahydrocortisol ratio in affected patients, revealing reduced
glucocorticoid degradation by renal activity of the 11b-hydroxysteroid dehydrogenase type 2, a GR regulated gene. We
propose thus that GR haploinsufficiency compromises glucocorticoid sensitivity and may represent a novel genetic cause of
subclinical hypercortisolism, incidentally revealed bilateral adrenal hyperplasia and mineralocorticoid-independent
hypertension.
Citation: Bouligand J, Delemer B, Hecart A-C, Meduri G, Viengchareun S, et al. (2010) Familial Glucocorticoid Receptor Haploinsufficiency by Non-Sense Mediated
mRNA Decay, Adrenal Hyperplasia and Apparent Mineralocorticoid Excess. PLoS ONE 5(10): e13563. doi:10.1371/journal.pone.0013563
Editor: Pieter H. Reitsma, Leiden University Medical Center, Netherlands
Received June 9, 2010; Accepted September 29, 2010; Published October 22, 2010
Copyright: ? 2010 Bouligand et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work has been supported by fundings from Institut National de la Sante ´ et de la Recherche Me ´dicale (INSERM), the Universite ´ Paris-Sud 11. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: marc.lombes@u-psud.fr
. These authors contributed equally to this work.
Introduction
Glucocorticoids are one of the most important class of steroid
hormones that regulate essential biological processes including
development, metabolism, growth, inflammatory processes, be-
havior and apoptosis. Glucocorticoid actions are mediated by the
glucocorticoid receptor (GR), a nuclear receptor encoded by the
NR3C1 gene [1]. GR is ubiquitously expressed as two distinct
isoforms GRa and GRb. These isoforms are generated by
alternative splicing of the last exon 9a or exon 9b of NR3C1
and differ by their last carboxy terminal amino acids and their
transcriptional activities [2]. GR regulates a large variety of gene
expression in responses to glucocorticoid hormones [3,4]. A closely
related nuclear receptor, the mineralocorticoid receptor also binds
glucocorticoid hormones. However, in classical aldosterone target
tissues, such as the distal nephron and the colon, the enzyme 11b-
hydroxysteroid dehydrogenase type 2 (11bHSD2) metabolizes
cortisol into inactive derivative cortisone and prevents permanent
MR activation leading to inappropriate sodium retention and
subsequent arterial hypertension [5].
To date, twelve germinal mutations of the human GR have
been identified; most of them are heterozygous missense mutations
causing partial loss of function of the GR [6,7,8]. These NR3C1
mutations compromise various steps of the glucocorticoid
signalling pathway and cause primary glucocorticoid resistance
(OMIM 138040), a rare hereditary disease with generalized partial
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insensitivity to glucocorticoid action [6]. However, the clinical
presentation of primary glucocorticoid resistance is very diverse
from severe signs of mineralocorticoid excess due to elevated
aldosterone levels, and hyperandrogenia, to mild or asymptomatic
forms. This variable phenotype is associated with a wide genetic
heterogeneity due to various missense mutations affecting distinct
functional domains of the nuclear receptor and responsible for
impaired glucocorticoid signalling [9].
Up to date, non-sense mutation of NR3C1 has never been
described. We report herein, the discovery of the first heterozygous
nonsense mutation in the human GR. We describe a clear-cut
clinical, cellular and molecular characterization of a stop mutation
and demonstrate that an impaired translation of mutant mRNA in
vivo
causes GRhaploinsufficiency.
p.R469[R,X] identified in a French family with eight affected
siblings spanning three generations gives a unique opportunity to
study the natural history of GR haploinsufficiency as previously
reported in mouse models [10]. We thus infer that GR haploinsuffi-
ciency inhumans is responsibleof a discrete phenotype of subclinical
hypercortisolism, bilateral adrenal hyperplasia and arterial hyper-
tension. This unusual clinical presentation, most notably incidentally
discovered adrenal hyperplasia, progresses gradually throughout the
life cycle, and clearly differs from that previously described during
primary glucocorticoid resistance associated with other NR3C1
missense mutations. We finally provide evidence that arterial
hypertension in this family is related to an illicit MR activation in
the kidney, due to altered renal 11bHSD2 activity and hypercor-
tisolism rather than to elevated mineralocorticoids as previously
proposed for primary glucocorticoid resistance. Thus, GR haploin-
sufficiency might be an underestimated cause of bilateral adrenal
hyperplasia and arterial hypertension [11].
ThismutationNR3C1
Results
Case Reports
The 46-year-old French Caucasian male propositus (subject
II.3, Fig. 1A) was referred for evaluation of bilateral adrenal
hyperplasia discovered incidentally during computerized tomog-
raphy (CT) performed for lumbago. His personal history was
marked by recent onset of arterial hypertension (190 mm Hg
systolic values). Physical examination showed no signs of Cushing’s
syndrome [12] such as striae bruises, amyotrophy or faciotroncular
obesity (Fig. 1B). Biochemical evaluation showed normal glycemia
(4.1 mmol/L), moderate hypokalemia (3.5 mmol/L) with inap-
propriate kaliuresis (55 mmol/d) and normal renal function.
Twenty-four-hour urinary free cortisol (UFC) excretion, measured
by mass spectrometry-HPLC, was 2- to 4-higher than normal
while serum ACTH levels were inappropriate and increased after
the CRH test (Table 1). Further endocrine evaluation showed a
preserved circardian cortisol cycle but markedly elevated midnight
cortisolemia (248 nmol/L). An overnight 1-mg dexamethasone
(DEX) suppression test failed to completely suppress plasma
cortisol (nadir 119 nmol/L, N ,50). Supine aldosterone and
active renin levels were undetectable (Table 1) and deoxycortico-
sterone values were low (40 pg/ml, N 40-200). In contrast, the
urinary tetrahydrocortisone/tetrahydrocortisol ratio (THE/THF)
was low (0.92, N.1.5), suggesting impaired renal 11b-hydroxy-
steroid dehydrogenase type 2 (11bHSD2) activity [13]. Abdominal
CT revealed bilateral macronodular adrenal hyperplasia (Fig. 1C,
middle panel). Normal bone density and osteocalcin levels ruled
out detrimental consequences of the cortisol excess on bone
structure and metabolism. Magnetic resonance imaging showed
normal pituitary and no muscle atrophy and no abdominal or
subcutaneous adipose depots.
The two affected brothers of the propositus (subjects II.5 and
II.7, Fig. 1A) had no clinical features of Cushing’s syndrome
despite hypercortisolism and insufficiently suppressed plasma or
salivary cortisol with low or undetectable renin and aldosterone
values (Table 1). The THE/THF ratio was abnormally low in
both cases (Table 1). CT also revealed bilateral nodular adrenal
hyperplasia in subject II.5 (not shown).
The 72-year-old mother (I.2, Fig. 1A) had a history of severe
uncontrolled arterial hypertension and hypokalemia (Table 1),
with no clinical features of hypercortisolism or hyperandrogenism
(testosterone 0.31 ng/mL, N 0.1-0.4, D4-androstenedione 1.3 ng/
mL, N 0.6-1.6, DHEAS 400 ng/ml, N 200-2300). Her bone
mineral density was at the upper limit of normal for her age
despite probable longstanding glucocorticoid excess. UFC and
midnight plasma cortisol levels were elevated and remained high
after an overnight DEX suppression test (Table 1). Aldosterone
was undetectable. Her THE/THF ratio was also abnormally low
(Table 1). CT revealed bilateral nodular adrenal hyperplasia
(Fig. 1C, upper panel).
The proband’s 9-year-old affected daughter (III.2, Fig. 1A) has
normal height, growth and prepubertal development (S2, P1),
Figure 1. A family with glucocorticoid resistance. A) Structure of
the pedigree. Three generations (I, II and III) of the kindred are
represented. Individuals carrying the heterozygous R469X mutation are
shown in black. Black arrow indicates the proband. B) Phenotype of the
propositus. C) Bilateral adrenal hyperplasia was readily visible by
computerized tomography (CT) in three affected individuals belonging
to three generations (I.2, the mother of the propositus; II.3, the
propositus; and III.2, his daughter, white arrows indicate adrenal
glands).
doi:10.1371/journal.pone.0013563.g001
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without clinical hypercortisolism or hyperandrogenism. Her UFC
was increased, with inappropriately high ACTH levels (Table 1).
Androgen levels were very low. CT also revealed slightly enlarged
adrenal glands for her age (Fig. 1C, lower panel). Subject III.5, a
14-year-old daughter of subject II.7, had normal development and
regular menses that started at age 12 years. She shows no signs of
hypercorticism despite a 4-fold increase in UFC and a negative
suppression test (Table 1).
All these clinical and endocrine abnormalities raised the
diagnosis of familial generalized glucocorticoid resistance and
prompted us to search for genetic defects. We also studied some
unaffected family members, including three with normal hormone
levels (Table 1) and one with a normal abdominal CT (not shown),
in order to demonstrate phenotype and genotype co-segregation.
Identification of GR mutation and in vitro
characterization
After sequencing the ten exons and the exon-intron boundaries
of the proband’s hGR gene, a single heterozygous cytosine to
thymidine substitution was identified in exon 4 at nucleotide
position 1405 (Fig. 2A), replacing a CGA (arginine) by a TGA stop
codon at amino acid 469 in the second zinc finger of the DNA-
binding domain of GR. This p.R469X mutation results, as
expected, in a truncated GR molecule of 468 amino-acids (Fig. 2B).
Interestingly, this nucleotide substitution abrogates the Bsp119I
restriction site (TTCGAA), thus facilitating rapid identification of
the mutation in the amplified exon 4 sequence (Fig. 2C). This
heterozygous nonsense mutation was detected in eight heterozy-
gous individuals (see Fig. 1A: I.2, II.3, II.5, II.7, III.2, III.3, III.4
and III.5), absent in unaffected family members and in 100
unrelated Caucasians.
The functional properties of the hGRa-R469X mutant were
assayed in human HEK 293 cells by transient transfection
experiments. As expected from the DBD truncation, the hGRa-
R469X mutant migrated as a ,50-kDa band as shown by
Western blot with an antibody recognizing the N-terminal part of
GR (Fig. 3A) and by autoradiography of the radiolabeled receptor
obtained in a translation-coupled-to-transcription assay (Fig. 3B).
The mutant receptor was unable to bind DNA as shown by gel
retardation assay (Fig. 3C) neither to translocate to the nucleus
after dexamethasone exposure (Fig. 3D). Finally, the mutant was
unable to transactivate the GRE2-Luc reporter gene in the
absence or presence of hormone (Fig. 3E).
In vivo characterization of GR mutation and GR
Haploinsufficiency
Fibroblasts cultured from a proband skin biopsy sample were
used for molecular characterization of the endogenous GR
Table 1. Clinical and biological features of the affected and unaffected individuals of the kindred.
# SexAge
BMI
(kg/m2)
Blood
Pressure
(Hg mm) Kalemia
UFCa
(m mg/d)
Fb(nmol/L)
DXM sup
Test Nadir
ACTH/CRHd
(pmol/L)
GR targets
Osteocalcine
(ng/mL) THE/THFf
Aldo/rennin Aldog(pg/
mL) Reninh(pg/mL)
DOCi(pg/mL)
Affected individuals
I.2F 7134160/70 3.9 mmol/l* 65 (x2)Basal 1107
Nadir 1236
ND Osteocalcin 3.5
THE/THF 0.72
A,27
R 20*
II.3 M 4626 190/100 3.5 mmol/l 94 (x2) Basal 654
Nadir 119
0’ 2.4
30’ 16.9
Osteocalcin 11
THE/THF 0.92
A,27
R undetect
DOC 40
II.5M 4130.4176/1024.0 mmol/l 192 (x4)Basal 9800’ 8.4
30’ 16.3
Osteocalcin 9.5
THE/THF 0.94
A,27
R 6.8
II.7M3628 120/804.0 mmol/l54 (.N)Sal Fc48 (x2)
Basal 733
0’ 9.2
15’ 27.1
THE/THF 0.84A ND
R ND
III.2F918.2 96/593.6 mmol/l41 (x2)Basal 845 0’ 11.7THE/THF 2.37A 125
R 6.3
DOC 76
III.5F14 20.2 110/603.7 mmol/l124 (x4)Basal 675
Nadir 528
0’ 24.9 THE/THF 2.98 A 37
R 12.3
DOC 307
Unaffected individuals
II.1F5023 115/67ND18Basal 410 NDTHE/THF 1.32A 71
R 2.8
III.1M 1621110/70NDND Basal 413ND NDA 97
R 12.8
III.6F1017100/70ND 33 NDNDTHE/THF 2.51A ND
R ND
a: Urinary free cortisol, UFC, normal ranges: age.59 y, 7–33 mg/d, 15 to 59 y 13–53 mg/d for males and 11–38 mg/d for females,10–14 y 7–34 mg/d, 5–9 y 4–25 mg/d.
b: Plasma cortisol (F), 8.00 am basal values, normal ranges in adult and children (N,540 nmol/l); overnight 1-mg DEX suppression test, F,50 nmol/L.
c: Salivary cortisol (Sal F), normal ranges 2–19 nmol/L.
d: ACTH, normal values basal 2.2–13.2 pmol/L.
e: Osteocalcin, normal values 5.3–24 ng/mL.
f: THE/THF ratio (Normal values 1.5–2.5).
g: Aldosterone (A), normal resting values 19–117 pg/mL, normal supine values 41–323 pg/ml, 100–800 pg/mL (children).
h: Renin (R), normal resting values, 3–16 pg/ml, normal supine values 3–33 pg/ml, 7.5–25 pg/ml (children)i: deoxycorticosterone (DOC), normal values, 40–200 pg/mL
(adult).
*on therapy, ND not determined; NA not available, undetect: below detectable level.
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Figure 2. Identification of the GR R469X mutation in the kindred. A) Identification of the heterozygous 1405C.T transition. Sequencing of
exon 4 in genomic DNA of individual II.1 confirmed the existence of the normal GR coding region, whereas the corresponding sequence of the
proband DNA indicated that patient II.3 was heterozygous for a single C.T nucleotide change at position 1405, converting the amino acid arginine
(R) into a premature stop codon (X) at position 469 of GR in all affected individuals. B) Genomic organization of the human GR and functional
domains of the wildtype GRa. The hGR gene is composed of 10 exons, the two last two of which exon 9a and exon 9b are alternatively spliced. The
C.T substitution at position 1405 in exon 4 results in a premature termination of translation and gives rise to a 468-amino-acid truncated GR mutant
which lacks the C-terminal region of the receptor including part of the DNA-binding domain (DBD) and the ligand-binding domain (LBD). C) The
1405C.T substitution abrogated the Bsp119I restriction site in exon 4 of GR, thus allowing rapid identification of the heterozygous mutation. PCR-
amplified exon 4 fragments from all individuals in the kindred were digested with Bsp119I and loaded on agarose gel: (upper panel individuals I.2 to
II.7, lower panel individuals III.1 to III.6). The presence of a 286-bp fragment resistant to Bsp119I digestion confirmed the C.T substitution.
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mutation in vivo. The presence of the heterozygous GR mutation in
genomic DNA from fibroblasts of individual II.3 was confirmed.
However, direct sequencing of cDNA failed to detect any mutated
transcripts (Fig. 4A, upper panel). This was consistent with
selective degradation of the mutated GR mRNA through a
nonsense-mediated mRNA Decay (NMD), a specific quality-
control mechanism that eliminates aberrant mRNAs harboring a
premature termination codon before the last exon [14]. The
involvement of this active process was unambiguously demon-
strated as treatment of patient fibroblasts with either emetine or
cycloheximide, two potent NMD inhibitors [15], stabilized the
mutant mRNA expressed from the defective allele (Fig. 4A) and
significantly increased the total amount of GR mRNA levels as
measured by quantitative real-time PCR (Fig. 4B). Furthermore,
GR mRNA levels expressed in patient fibroblasts were approx-
imately half those measured in two controls (Fig. 4C). DEX
binding assays accordingly demonstrated a 50% decrease in the
number of fibroblast hormone-binding sites as compared to
controls (Fig. 4D). Given the drastic reduction in GR expression at
both the mRNA and protein levels and the absence of defective
allele expression, we suspected that the mutated GR protein was
not expressed in vivo. Indeed, the 50-kDa mutated GR species was
undetectable by western blot whereas a 50% reduction in the 90-
kDa WT GR molecule was observed (Fig. 4E). Finally, owing to
this decrease in the functional GR concentration in the proband’s
fibroblasts, significantly below-normal induction of FKBP5, a
glucocorticoid-induced target gene [16], was observed after DEX
exposure (Fig. 4F).
Altogether, these findings unambiguously establish that the
heterozygous nonsense mutation p.R469[R,X] results in GR
haploinsufficiency that ultimately compromises glucocorticoid
signaling in vivo.
Discussion
We describe a family carrying the first heterozygous nonsense
mutation (R469X) in the GR gene. This germinal mutation
results in partial glucocorticoid resistance associated with
Figure 3. In vitro characterization of the GR mutant. A) Western blot analysis of GR. Four micrograms of protein obtained from homogenates of
HEK293 cells transiently transfected with WT hGRa (WT) and hGRa-R469X mutant were processed for immunoblotting with an anti-GR antibody. Note
the presence of a specific 90-kDa band for WT GR and a 50-kDa band for hGRa-R469X. B) Protein expression of in vitro-translated [35S]-labeled WT
hGRa and hGRa-R469X separated by 10% SDS-PAGE. GR migrated as a major 90-kDa form whereas the GR mutant, as expected, had a lower
molecular mass of approximately 50 kDa. C) Binding of in vitro-translated WT hGRa and hGRa-R469X to GRE consensus sequence by gel retardation
assay. Specific GR-radiolabeled GRE complexes (arrow) were detected in the absence of unlabeled competitor (-), which were abolished in the presence
of 50 ng unlabeled probe (50). As expected, the GR mutant was unable to bind DNA. P: free probe. D) Intracellular trafficking of WT hGRa and hGRa-
R469X in transfected COS7 cells by Immunocytochemistry. Cells were counterstained with DAPI in blue. WT GR translocates from the cytoplasm to the
nucleus after 5 min incubation with 1 mM DXM whereas GR mutant remains exclusively in the cytoplasmic compartment either in the absence or
presence of DXM. E) Transcriptional activity of the WT hGRaand the truncated hGRa-R469X mutant. HEK 293 cells were transfected using Lipofectamin
2000 with either WT hGRa or hGRa-R469X together with the glucocorticoid-responsive reporter gene pGL3-GRE2-TATA-Luc and pSV.b-Gal plasmids.
Following transfection, cells were exposed to 100 nM DXM for 24 h. Results (Luc/b-gal activity) are expressed as the percentage of relative
transcriptional activity of WT GR arbitrarily set at 100% with 100 nM DXM. Results are means 6 SD of at least 3 independent determinations.
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subclinical hypercortisolism, bilateral adrenal hyperplasia and
hypertension but yet low mineralocorticoid levels, aldosterone
and deoxycorticosterone, defining an apparent mineralocorticoid
excess. In vitro characterization revealed that the GR p.R469X
mutant is unable to bind hormones and DNA. GR p.R469X
mutant does not exhibit any ligand-dependant nuclear translo-
cation nor display transcriptional activity. In vivo functional
characterization of the endogenous heterozygous nonsense
mutation demonstrated that the molecular mechanism underly-
ing glucocorticoid insensitivity in this kindred involves GR
haploinsufficiency and nonsense-mediated
(NMD) [17]. This mechanism of glucocorticoid signaling
dysfunction differs from that induced by other heterozygous
missense GR mutations that impair one or several steps of the
GR activation cascade and/or exert dominant negative effect on
wild-type GR [6].
mRNADecay
Figure 4. Evidence of GR haploinsufficiency in the proband’s fibroblasts due to nonsense-mediated mRNA Decay. A) Demonstration
of GR haploinsufficiency in the fibroblasts of the propositus (II.3). Sequencing of exon 4 genomic DNA prepared from the patient’s fibroblasts
confirmed the presence of the heterozygous C.T substitution as observed in lymphocyte genomic DNA (see Supplemental Fig. S1A SI lower panel).
In contrast, direct sequencing of the cDNA (see specific primers in Supplemental Table S1 SI) prepared from fibroblast RNA revealed only the wildtype
C allele. The absence of mutated GR transcripts (upper panel) in the patient’s fibroblasts is consistent with nonsense-mediated mRNA Decay, a cellular
mechanism that prevents translation of mutated mRNA bearing a premature termination codon. When fibroblasts were treated for 6 h with 100 mg/
ml emetine (lower panel) or for 2 h with 20 mg/ml cycloheximide (not shown), the expression of the defective allele was restored as shown by direct
sequencing of the corresponding cDNA fragment. B) Increase in GR mRNA expression in fibroblasts of patient II.3 after exposure to two inhibitors of
nonsense-mediated mRNA Decay, cycloheximide (20 mg/ml for 2 h) or emetine (100 mg/ml for 6 h). Relative expression of GR, beta-actin ou 18S RNA
was measured by using quantitative real-time RT-PCR. Results are means 6 SEM of 4 determinations and expressed as fold induction relative to
untreated cells. (* P,0.05 Kruskal Wallis followed by Dunn’s post test and Mann Whitney test). C) Reduction of GR mRNA expression in fibroblasts of
patient II.3 compared with two controls C1 and C2. The expression of mRNA was measured by using quantitative real-time RT-PCR. Results are
expressed as attomol/fmol of 18S and are means 6 SEM of 3 independent determinations (*** P,0.001 Mann Whitney test). D) Reduction in specific
[3H]-DXM binding sites. Fibroblasts pre-incubated in steroid-free medium for 24 h, were exposed to 50 nM [3H]-DXM in the absence or presence of a
500-fold excess of unlabeled DEX for 1 h at 37uC. Radioactivity was measured and specific binding was calculated. Data are means 6 SEM of 3
independent determinations performed in triplicate. The estimated GR density in the propositus’ fibroblasts was 36104sites per cell (*** P,0.001 vs
controls). E) Western blot analysis of GR. Thirty micrograms of protein from fibroblast homogenates of controls (C1 and C2) and patient II.3 were
processed for immunoblotting with anti-GR (upper panel) and anti-b actin (lower panel). Note the presence of a specific 90-kDa GR in controls and an
approximately 50% reduction in WT GR expression in patient II.3 whereas the 50-kDa band corresponding to the truncated hGRa-R469X mutant was
not detected. Quantitative analysis of GR signals normalized to b-actin loading was performed using QuantityOne software (Biorad). Results are
means 6 SD of at least 3 independent analyses (* P,0.05 Mann Whitney test). F) Altered glucocorticoid-inducible gene expression in the patient’s
fibroblasts. Fibroblasts from controls (C1 and C2) and from patient II.3 were starved for 24 h in steroid-free medium and then exposed to 100 nM
DXM for 6 h. Relative levels of FKBP5 transcripts were determined by quantitative real-time RT-PCR analysis. Results are expressed as attomol/fmol
18S and are means 6 SEM of 6 independent determinations (** P,0.01, Kruskal Wallis followed by Dunn’s post test).
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The rare familial or sporadic cases of primary glucocorticoid
resistance [6] are associated with mild to very severe clinical
presentations. In this family, the clinical presentation was quite
unusual. Indeed, adrenal incidentalomas are emerging as an
increasingly important clinical entity owing to the routine use of
efficient imaging techniques. Its prevalence increases with age,
affecting as much as 10% of the elderly [11]. Among clinically
unapparent adrenal masses, bilateral adrenal hyperplasia often
causes difficulties of diagnostic and management. Investigation of
adrenal incidentalomas leads to the identification of this nonsense
mutation in eight individuals spanning three generations allowing
us to examine the natural history of the disease and to study the
effects of human GR deficiency throughout the life cycle.
Moderate hypercortisolism is associated with an almost normal
phenotype with normal fertility and no virilization in affected
women. A striking observation is the extent of bilateral adrenal
hyperplasia which progresses gradually, probably owing to chronic
exposure to inappropriate ACTH levels; this clinical presentation
resembles that of heterozygous GR+/2mice [10]. Alternatively,
altered intraadrenal glucocorticoid-regulated adrenocortical cell
signaling [18] could play a prominent role in the pathogenesis of
the disease.
Besides GR, glucocorticoids also bind the closely related
mineralocorticoid receptor (MR) [5]. Therefore, despite a drastic
reduction in normal GR abundance, longstanding glucocorticoid
excess may have deleterious effects through MR binding. In
classical aldosterone target tissues such as the distal nephron, the
enzyme 11bHSD2 metabolizes cortisol into inactive cortisone
[13] and prevents permanent MR activation with subsequent
sodium retention and arterial hypertension. Indeed, the THE/
THF ratio was low and fell gradually over the generations in this
kindred, strongly suggesting impaired activity of renal 11bHSD2,
a direct GR target gene [19]. Owing to the absence of elevated
aldosterone and DOC levels, increased cortisol levels induce illicit
occupation and activation of the unprotected MR leading to an
apparent mineralocorticoid excess with hypertension and hypo-
kalemia. Glucocorticoid-activated MR could also trigger proadi-
pogenic effects [20], or affect neuron function [21], although this
needs to be investigated in patients with glucocorticoid receptor
haploinsufficiency.
On the other hand, the absence of hyperglycemia or
hyperlipidemia in the propositus despite chronically high gluco-
corticoid levels, suggests that the reduction in hepatic or adipose
GR levels might somehow protect against glucose intolerance or
metabolic alterations as observed in equivalent animal models
[10,22,23]. It remains to be established how and to which extent
such an unbalanced GR dosage affects metabolic, central nervous
system or cardiovascular functions in these affected individuals.
In conclusion, our results provide first evidence for a new
human genetic defect due to nonsense-mediated mRNA Decay
responsible for GR hapoinsufficiency. Indeed, GR haploinsuffi-
ciency identified in this family compromises glucocorticoid
sensitivity and may represent a novel genetic cause of subclinical
hypercortisolism, incidentally revealed bilateral adrenal hyperpla-
sia and mineralocorticoid-independent hypertension. We propose
that a GR genetic screening should be proposed and particularly
relevant in such patients given the possible therapeutic potential of
MR antagonists [24,25].
Materials and Methods
DNA analysis
All the participants gave their written informed consent for
genetic analyses which were approved by the local ethics
committee (CHU Bice ˆtre). Genomic DNA was extracted from
white blood cells. The entire coding regions of the hGRa and
hGRb genes were amplified and sequenced with primers described
in Supplemental Table S1.
Mutagenesis
Arginine to stop mutation (hGRa-R469X) was obtained with
the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La
Jolla, CA) with pcDNA3-hGRa as a matrix.
Cell culture
Fibroblasts (passages 5–15) were cultured at 37uC with 5% CO2
in DMEM High Glucose (4.5 g/l), 2 mM glutamine, 1 mM
sodium pyruvate, 100 U/ml penicillin, 100 mg/ml streptomycin
and 10% fetal calf serum, pH 7.4.
RT-PCR and quantitative real-time PCR
Total RNA was extracted from cells with Trizol reagent
(InVitrogen, Cergy Pontoise, France) and gene expression was
quantified by real-time RT-PCR, using an ABI 7300 Sequence
Detector (Applied Biosystems, Foster City, CA) as described [26].
Transcription assays
HEK 293 cells starved for 24 h in steroid-free medium were
transfected using Lipofectamin 2000 (InVitrogen) with pcDNA3-
hGRa or pcDNA3 hGRa-R469X together with pSVbgal and
pGRE2-TATA-luc plasmids and then exposed to 100 nM DEX.
b-galactosidase and luciferase activities were assayed as described
elsewhere [27].
In vitro translated GR proteins
In vitro translated WT and mutated GR were prepared by using
the TnT-T7 Quick Coupled Transcription/Translation kit
(Promega, Charbonnie `res-les-Bains, France), and labeled with
[35S]-methionine (Perkin Elmer, Courtaboeuf, France).
Western blot analysis
Total protein extracts were prepared from cells lysed at 4uC, as
previously described [26]. Immunoblots were incubated overnight
with anti-GR antibody (AbC10-G015, AbCys Paris, France)
followed by a peroxidase-conjugated goat anti-mouse antibody
(Vector, Burlingame, CA) for 30 min at room temperature.
Proteins were visualized with the ECL+detection kit (GE
Heathcare, Buckinghanshire, UK).
Binding assays
Primary cultures of patient fibroblasts were grown for 24 h in
minimal medium (DMEM High Glucose), then incubated for 1 h
at 37uC with 50 nM [3H]-DEX (1517 GBq/mmol; Perkin Elmer)
in the presence or absence of 25 mM unlabeled DEX. Specific
[3H]-DEX binding was determined as the difference between total
and nonspecific binding and was expressed as sites/cell.
Gel retardation assay
Electromobility shift assays were essentially performed as
previously described [26]. Purified complementary oligonucleo-
tides (GRE-forward: 59-AGCTGCTCAGCTAGAACACTCTG-
TTCTCTACT-39 and GRE-reverse 59-AGCTAGTAGAGAA-
CAGAGTGTTCTAGCTAGC-39) were annealed and radiola-
beled with [32P]-dCTP (Perkin-Elmer) using the Klenow fragment
of DNA polymerase (Invitrogen) to a specific activity of approx-
imately 108cpm/mg of DNA. In vitro translated wildtype and
mutated GR were incubated with radiolabeled GRE probe for
GR Haploinsufficiency
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Page 8

15 min at 25uC in the absence or presence of 100-molar excess of
labeled GRE and separated from free dsDNA by non-denaturing
electrophoresis in a 4.5% acrylamide/bisacrylamide (37:1) gel for
1 h at 200V (40 mA) in 0.256TBE (50 mM Tris, 50 mM boric
acid, 1 mM EDTA). Gels were dried and exposed to X-ray film at
280uC.
Immunocytochemistry
WT and mutated GR transfected cells were grown on acid-
etched and poly-d-lysine-coated glass coverslips. After DXM
treatment, cells were rinsed in ice-cold PBS, fixed with a 4%
formaldehyde solution in PEM buffer (0.1 M PIPES, 2 mM
EGTA, 3 mM MgCl2), permeabilized in 0.5% Triton X-100 in
PEM buffer for 30 min, and quenched in sodium borohydride
(0.5 mg/ml in PEM buffer). Coverslips were then incubated for
1 h (RT) in 5% nonfat dry milk in TBST before overnight
incubation at 4uC with anti-GR antibody (AbC10-G015) and
subsequently with goat anti-mouse antibody-Alexa 555 (Molecular
Probes). Cells were postfixed and counterstained with DAPI (49,69-
diamidino-2-phenylindole) (0.5 mg/ml for 1 min), rinsed in water,
and mounted onto slides (ProLong Gold; Molecular Probes).
Statistical analysis
We used nonparametric Mann-Whitney test and Kruskal-Wallis
multi-variance analysis followed by a post-test analysis of Dunn’s
comparison test (Prism 4, GraphPad Software, San Diego, CA).
Supporting Information
Figure S1
R469[R,X] of the glucocorticoid receptor. A) Sequencing of exon
4 in genomic DNA (gDNA) of individual II.1 lymphocytes
confirmed the existence of the normal GR coding region. B and
Genotyping of the heterozygous nonsense mutation
C) Identification of the heterozygous 1405C.T transition.
Sequencing of exon 4 in genomic DNA (gDNA) of lymplocytes
(B) and fibroblasts (C) of patient II.3 DNA indicated that the
proband was heterozygous for a single C.T nucleotide change at
position 1405, converting the amino acid arginine (R) into a
premature stop codon (X) at position 469 of GR in all affected
individuals.
Found at: doi:10.1371/journal.pone.0013563.s001 (4.02 MB TIF)
Table S1
sequencing. The sense and antisense primers were designed, GC%
calculated and Tm estimated with the online version 0.4 of Primer
3. All primers were blasted to check their selectivity on the NCBI
web site using the "Human Genomic Plus Transcript database".
The sequences in bold indicate exonic primers while the others are
intronic. cDNA2-5 refers to the amplified cDNA fragment
encompassing exon 4.
Found at: doi:10.1371/journal.pone.0013563.s002 (0.07 MB
DOC)
Primer sequences of hGR (NR3C1) for PCR, and
Acknowledgments
The authors thank Dr L. N. Dwuayo (CH Saint Dizier) for referring family
members to our institution and Dr A. Boutron for primary cultures of
fibroblasts. We are also indebted to I. Boucly and M. Messina for excellent
technical assistance. L.A was recipient of a fellowship from Fondation pour
la Recherche Me ´dicale (FRM). We also thank l’Association Franc ¸aise des
Malades de la Surre ´nale (AFMS) for its support.
Author Contributions
Conceived and designed the experiments: JB BD AGM JY ML. Performed
the experiments: JB BD ACH GM SVV LA ST AGM ML. Analyzed the
data: JB BD GM SVV LA ST BF AGM JY ML. Wrote the paper: JB JY
ML.
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