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Department of Pediatrics, New York-Presbyterian Hospital, New York Weill Cornell Center (M.I.N., S.N., S.C.-R., R.C.W., R.S.N.), New York, New York 10021; the Department of Medicine, Oregon Health Sciences University (D.D.B., D.L.L.), Portland, Oregon 97201; the Department of Pediatrics, McMaster University (J.V.), Hamilton, Ontario, Canada; and the Department of Cell Biology, Baylor College of Medicine (N.B., B.O.), Houston, Texas 77030
Address all correspondence and requests for reprints to: Maria I. New, M.D., Department of Pediatrics, Chief, Division of Pediatric Endocrinology, New York-Presbyterian Hospital, 525 East 68th Street, Room M-622, New York, New York 10021. E-mail: minew{at}mail.med.cornell.edu
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Abstract |
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Introduction |
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Herein we report a 14-yr-old native American girl from the Iroquois Nation who was referred to this institution as a possible patient with the syndrome of apparent mineralocorticoid excess. Instead, her evaluation revealed resistance to glucocorticoids, mineralocorticoids, and sex steroids, but no resistance to vitamin D or thyroid hormones. She lacked Cushingoid features despite significantly high cortisol levels. Menses were regular, and she did not show signs of masculinization despite high androgen levels in the male range. Partial resistance to exogenous glucocorticoid, mineralocorticoid, and sex steroids was demonstrated. Her 11.5-yr-old sister was equally affected.
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Subjects and Methods |
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Patient 1. A 14-yr-old girl was referred to the Children’s Clinical Research Center at the New York Weill Cornell Center for an evaluation of mild hypertension associated with low PRA, hypokalemia, and undetectable serum aldosterone concentrations.
The proband (patient 1) was the result of a normal, full-term vaginal delivery. Her birth weight was 2.5 kg. There were no perinatal complications. Growth and development were entirely normal, and she had no significant medical problems. Menarche occurred at the age of 11 yr, accompanied by normal pubertal development. Her menstrual periods have been regular.
At the age of 9 yr, the first episode of left facial palsy occurred. She was not seen by a physician until 12 yr of age when she had her third episode. Each episode lasted for about 2 weeks and spontaneously resolved. At the third episode, mild hypertension was found. The patient was followed thereafter intermittently until she was referred to the Children’s Clinical Research Center.
Physical examination revealed the patient to be an attractive girl with
a normal female body habitus (Fig. 1a
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Her height was 161 cm (50th percentile for age), and weight was 55.5 kg
(75th percentile for age). Pertinent physical findings included a
mildly elevated blood pressure of 140–150/83–99 mm Hg (90th
percentile for age is 125/81 mm Hg), a mild residual left-sided facial
weakness, small Tanner stage V breasts and Tanner stage V pubic hair, a
normal vaginal introitus, and no acne, hirsutism, or clitoromegaly.
There was no atrophy of the skin, buffalo hump, truncal obesity,
plethoric moon face, striae, or bruising. Her Ferriman-Gallway score
was 6 (which was within normal limits) (15).
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), was
asymptomatic, but was found to have biochemical findings similar to
those of her older sister (patient 1; see results below). She was the
result of a full-term vaginal delivery, and her past medical history
was unremarkable. Menarche occurred at 11 yr of age, and regular
menstruation ensued. She was 155 cm tall (90th percentile for age) and
weighed 52.3 kg (90th percentile for age). She was normotensive and had
no hirsutism, acne, or Cushingoid features. Breasts were small Tanner
stage V, and pubic hair was Tanner stage V. Her external genitalia were
that of a normal female without clitoromegaly.
Family history (Fig. 2). There
was no family history of hypertension. The mother was a member of the
Cayuga tribe of the Iroquois nation. The paternal grandfather was a
member of the Mohawk tribe of the Iroquois nation, and the paternal
grandmother was a Caucasian. The Caucasian grandmother died in her
thirties after giving birth to her ninth child. The patients’ elder
brother is healthy and has a normal biochemical profile.
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Informed written consent was obtained before the studies for all subjects. In minors, the consent was obtained from the guardian.
Hormone assays. Steroid hormone assays were performed by standard RIA as previously described (16, 17, 18, 19).
Cell culture and cytosol preparation. A lymphoid cell line (P2) was established by Epstein-Barr virus transformation of peripheral blood lymphocytes obtained from patient 2 as previously described (20). Briefly, 2 x 106 peripheral blood mononuclear cells were added to a 12-well plate containing 1 mL filtered (0.45-µm pore size filter) culture supernatant from an Epstein-Barr virus-infected B95–8 marmoset cell line with 30 mL cyclosporin A (0.1 mg/mL; final concentration, 1 ng/mL). Cells were cultured at 37 C for 10 days. Growing colonies were transferred and expanded into T25 flasks. In addition, human Epstein-Barr virus-transformed B lymphocyte cell lines (C1 and C2) established from peripheral blood lymphocytes from normal individuals were used as controls. Cell lines were grown in RPMI 1640 culture medium [RPMI 1640 containing 25 mmol/L HEPES buffer (Life Technologies, Grand Island, NY) supplemented with 10% FBS (Sigma Chemical Co., St. Louis, MO) and 1% antibiotic-antimycotic solution (Sigma Chemical Co.)] at 37 C in a 5% CO2 atmosphere. Cells were harvested after 4 days of growth by centrifugation (1500 x g, 10 min) and washed three times with phosphate-buffered saline, pH 7.3, at room temperature to remove growth medium. Cell viability was determined by trypan blue dye exclusion. Cytosol was prepared from cells by sonication in 4 vol ice-cold HEPES buffer (10 mmol/L HEPES, 0.5 mmol/L ethylenediamine tetraacetate, and 0.5 mmol/L dithiothreitol, pH 7.4), followed by centrifugation at 100,000 x g at 4 C for 1 h.
Cytosol mixing studies. Cytosol prepared from Epstein-Barr virus-transformed lymphoid cell lines established from patient 2 or human controls C1 and C2 were used to conduct mixing studies. To determine total binding, cytosol of equivalent protein concentration was mixed in a 1:1 ratio (vol/vol), and 100 µL were incubated with 100 µL [3H]dexamethasone (final concentration, 2 nmol/L) for 18 h at 4 C in duplicate. Nonspecific binding was assessed by the addition of a 200-fold excess of unlabeled dexamethasone in parallel tubes. After incubation, bound steroid was separated from free using dextran-coated charcoal, and bound radioactivity was determined in an aliquot as described above.
Western blot studies. Rabbit polyclonal IgG glucocorticoid
receptor antibody P20 (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA) was used for Western blot detection of glucocorticoid
receptor as previously described (21). This antibody is specific for
the 
-isoform of the glucocorticoid receptor and is not
cross-reactive with the ß-isoform. It recognizes a conserved epitope
in the carboxyl-terminus of the receptor and is mouse, rat, and human
reactive.
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Results |
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and 2)
The baseline laboratory results of patients 1 and 2, including
complete blood counts, biochemistry profiles, and urinary analyses,
were within normal ranges. Serum potassium in patient 1 ranged from
2.6–3.1 mmol/L (normal, 3.2–5.2 mmol/L), and serum CO2
ranged from 28–32 mmol/L (normal, 22–32). Serum electrolytes were
normal in patient 2. In patients 1 and 2 there was elevation of many
serum steroid concentrations, including
4-androstenedione, testosterone, deoxycorticosterone,
corticosterone, and cortisol. Estradiol levels fell within the normal
range for the follicular phase. The 24-h urinary free cortisol,
17-ketosteroid, and 17-hydroxycorticosteroid levels were markedly
elevated in patient 1 and to a lesser extent in patient 2. The serum
ACTH concentration was markedly elevated. The serum and urinary
aldosterone levels were low to undetectable in patient 1.
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The unaffected parents and elder brother had normal serum electrolytes and unsuppressed PRA.
Hypothalamic-pituitary-adrenal axis (HPA)
ACTH stimulation test. The ACTH stimulation test (standard
dose of Cortrosyn, 0.25 mg, iv) revealed a rise in the levels of all
serum steroids that were elevated in the baseline state. There was
minimal stimulation of all serum steroid concentrations (Table 1).
Each parent had a normal baseline and response to ACTH stimulation
(Table 2). In the elder brother, baseline corticosterone was elevated,
but the stimulated level was normal.
Metyrapone and corticotropin-releasing factor (CRF)
tests. The serum cortisol concentrations in both patients were
elevated and did not demonstrate diurnal variation (Table 3). In patient 1, an overnight single
dose metyrapone test resulted in the elevation of ACTH 6.5-fold, with
simultaneous adequate suppression of serum cortisol (Table 4). In the CRF test (Table 5), serum cortisol and ACTH
concentrations were high and rose to higher levels with CRF
administration (ACTH rose 4.5-fold).
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, and Table 6b).
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LH-releasing hormone test. The LH-releasing hormone test
demonstrated normal FSH and LH responses in patient 1, but in patient 2
the responses were blunted. The steroid concentrations of androgens and
estrogen were elevated in both sisters (Table 7a, and Table 7b).
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-Fludrocortisone acetate challenge test. With continued
dexamethasone administration, 9-fludrocortisone acetate was added at
high doses (0.6 mg/day) in patient 1. In patient 2,
9-fludrocortisone acetate was administered without dexamethasone. No
salt retention was demonstrated in either sister (Fig. 4a and Fig. 4b).
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A computed tomography scan of patient 1 showed massively enlarged adrenal glands with diffused and increased nodularity. The uterus was structurally normal, as imaged by a magnetic resonance scan. The magnetic resonance scan of the head was normal. Bone mineral density was normal for age-matched controls, and the patient’s pelvic sonogram demonstrated normal ovaries. The bone age was advanced to 17 yr at the chronological age of 14 yr.
The bone age of patient 2 was 15 yr at a chronological age of 11.5 yr. She had normal adrenal glands, as observed by computed tomography scan. A pelvic sonogram also produced normal results. Her bone mineral density was normal for age-appropriate standards.
Western blot detection of molecular chaperones
Studies have indicated that steroid receptors remain inactive in a
complex with heat shock protein-90 (hsp90) and other stress family
proteins before binding with hormones. Hormone binding induces critical
conformational changes in the steroid receptor that cause them to
dissociate from the inhibitory complex, thus modulating gene
transcription (39). Western blotting for chaperonin proteins indicated
no alteration in the size or expression levels of hsp70, hsp90 and
ß isoforms, hsp70, protein phosphatase 5, FKBP52, or
cyclophilin 40 in the P2 cell line compared to those in the C2 control
cell line (data not shown). However, Western analysis does not detect
alterations in the primary structure of a given protein. Therefore,
involvement of chaperonin proteins cannot be completely rule out.
Cytosol mixing studies
Mixing studies using cytosol prepared from a New World primate
cell line mixed with cytosol derived from human tissues showed
inhibited binding of dexamethasone to the glucocorticoid receptor (22).
This inhibitory activity may be involved in the steroid resistance of
New World primates. When cytosol of equivalent protein concentration
was prepared from the control cell line (C1) and mixed 1:1 with cytosol
prepared from the patient cell line (P2), no evidence for inhibition of
[3H]dexamethasone binding was found (Fig. 5). However, the concentration of an
inhibitor may be too low for the detection limits of this assay.
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To test for a coactivator deficiency, deletion analysis of several coactivators [SRC-1, SRC-2 (TIF-2/GRIP-1), SRC-3 (pCIP), E6AP, SRA (unpublished), TRIP-230, P/CAF, and TIF-1, whose function is not yet known] was performed. Southern analysis of genomic DNA was carried out, along with multiple restriction enzyme digests of genomic fragments (23). No deletions or major rearrangements were found. However, deletion analysis only detects large gene rearrangements; therefore, other defects in these coactivators cannot be ruled out.
Miscellaneous tests
The thyroid evaluation, PTH, calcium, phosphate, alkaline
phosphatase, and vitamin D metabolites were normal for both patients
(Table 8). Sex hormone-binding globulin
levels in both patients 1 and 2 were within the normal range. A vaginal
smear showed low estrogen effects for both sisters despite normal
levels of estradiol.
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Discussion |
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Glucocorticoids
Despite high cortisol serum concentrations and the absence of diurnal rhythm, there were no Cushingoid features. The degree of ACTH compensation in these two patients is strikingly different compared to that of their serum cortisol and urinary cortisol metabolites. The cause of this discrepancy is unknown. Further, prolonged administration of large doses of dexamethasone did not produce Cushingoid signs.
Androgens
The serum androgen concentrations were high and in the male range, yet the sisters were not masculinized. They did not manifest acne, hirsutism, or clitoromegaly. However, their bone ages were advanced, indicating only partial resistance.
Estrogens
Estrogen levels were normal, but the vaginal epithelium showed poor estrogenization. Puberty (including breast and pubic hair development), although early, was normal, and menses were regular. The sex hormone-binding globulin level was not low. The regular menses, normal secondary sex characteristics, and normal bone mineral density indicate that the resistance to estrogens and androgens is partial.
Mineralocorticoids
Although the serum concentrations of aldosterone were low to
undetectable in patient 1, salt retention did not occur even with
administration of 9-fludrocortisone acetate at 6 times the usual
dose. The low levels of aldosterone and suppressed renin may result
from the high levels of cortisol, which can activate the
mineralocorticoid receptor. This, in turn, would cause sodium retention
and plasma volume expansion and suppress PRA, which would result in
decreased aldosterone secretion. Nevertheless, the failure to retain
sodium with very high doses of 9-fludrocortisone acetate
administration demonstrated partial resistance to
mineralocorticoids.
Because there was no clinical indication of progesterone resistance, there was no histological study of uterine mucosa performed. Nevertheless, the patients could have partial resistance as reported in the case study, in which the patient had a normal menstrual cycle and normal progesterone levels, but abnormal maturation demonstrated by histology (10).
Suppression tests
Suppression of ACTH, which should cause a decrease in steroid concentrations in the serum, occurred at only very high doses of dexamethasone administration. This indicates that the HPA axis is partially resistant to glucocorticoid action.
Theories postulated for the multiple hormone resistance
The mechanism for such resistance to multiple steroid hormones in these patients could be caused by defects at various points in the cascade of the hormone-receptor-DNA interaction. Cellular sensitivity to a steroid hormone is influenced by a number of factors, including the level of receptors, their affinity for ligands, their rate of translocation to the nucleus after activation, and their ability to trans-activate a response. Disruption at each of these levels can lead to decreased sensitivity to one or more steroid hormones, most often leading to a compensatory increase in plasma hormone levels.
1) Resistance to steroid hormones found in New World primates may represent cumulative adaptive changes over a period of some 60 million yr (24, 25, 26, 27, 28, 29, 30, 31, 32). It is also possible that a single mutation in one of these signaling pathways leads to diminished responses to other steroid hormones. In fact, New World primates have adapted to partial resistance to high circulating levels of many steroid hormones (24). Unlike these patients, the squirrel monkey (New World primate) has increased aldosterone concentrations, which may be due to an adaptive mechanism in their mineralocorticoid target tissue (30). In addition, small New World primates have elevated estrogen levels, but large New World primates do not have elevated estrogen levels as found in our patients (28, 33). However, it remains unclear whether a primary alteration in a steroid receptor gene underlies resistance in these species. For example, these primates are profoundly resistant to glucocorticoid, but the glucocorticoid receptor has been cloned from several species of New World primates, and in each case the receptor exhibits normal binding affinity for ligand when measured outside its native cellular milieu despite markedly diminished affinity for ligand when measured in its native cell (22, 34, 35, 36). In addition, the New World primate glucocorticoid receptor can activate ligand-dependent gene transcription from a glucocorticoid response element (36) in a heterologous system similar to the human glucocorticoid receptor. The New World primate cell apparently expresses a soluble protein activity that somehow interferes with binding of ligand to the glucocorticoid receptor, but this activity has no effect on binding of progestin or estrogen to their cognate receptors (22, 36). It remains possible that one or more of the chaperonin proteins involved in steroid-receptor heterocomplex formation may be altered in the New World primate, thereby simultaneously affecting multiple steroid receptor pathways. Historically, the observation of generalized hormone resistance in the New World primates led to speculation that steroid receptors were somehow related, an observation that preceded the cloning of steroid receptors and the attendant realization that these proteins are highly related in structure and function (27). The New World primates offer an important opportunity to examine aspects of the mechanism of the action of steroid hormones.
2) Adams et al. concluded that vitamin D-resistant and gonadal steroid-resistant New World Primate cells contain proteins that may silence receptor action by interacting directly with responsive elements and interfering with receptor binding (37, 38).
3) Another proposal has arisen with the recent explosion of knowledge regarding proteins involved in the regulation of transcription, that is, coactivators and corepressors. Steroid receptors belong to a superfamily of ligand-inducible transcription factors that regulate hormone-responsive genes. Steroid receptors remain inactive in a complex with hsp-90 and other stress family proteins before binding with hormones. Hormone binding induces critical conformational changes in the steroid receptor that cause them to dissociate from the inhibitory complex, bind as homodimers to specific DNA enhancer elements associated with target genes, and therefore modulate the transcription of the target gene (39). After binding to enhancer elements, transcription factors require transcriptional coactivator proteins to mediate their stimulation of transcription initiation.
The first functional coactivator cloned and identified was SRC-1 (steroid receptor coactivator-1) (40). It was shown to interact with and stimulate nuclear receptor action in a ligand-dependent manner with both type 1 receptors (estrogen, progesterone, glucocorticoid, androgen, and mineralocorticoid) as well as type II receptors (for T3, vitamin D3, retinoic acid, fatty acids, orphans, etc.). This molecule was confirmed to have an in vivo function using SRC-1 knockout mice, in which partial hormone resistance was demonstrated (41). SRC-1 belongs to a family of three coactivators that includes TIF-2 (GRIP-I) and pCIP (ACTR, RAC-3) (40, 42, 43, 44, 45, 46, 47, 48, 49). Subsequently, a number of other coactivators and corepressors have been cloned, but there is no reason to believe that all coregulators have been identified to date.
Results to date from a number of laboratories indicate that coactivators are required for full transcriptional activity of the steroid receptor superfamily. Partial resistance to estrogens, androgens, and progesterone were concomitant in the SRC-1 knockout mouse model. Further increased expression of TIF2 in the face of an inability to produce SRC-1 in these knockout mice indicates a certain degree of redundancy and overlap between different coactivators (41). The phenotypic features of the mouse with knockout of SRC-1 were similar to those of our patients.
Human examples of coactivator defects
Rubinstein-Taybi syndrome (RTS) is the first human disease to be shown to be due to a coactivator defect. Many patients with RTS have been shown to have microdeletion of chromosome 16p13.3. All of these breakpoints are restricted to a region containing the gene for human cAMP response element-binding protein (or cyclic AMP-responsive DNA-binding protein), a nuclear protein participating as a coactivator in cAMP-regulated gene expression. The role of cAMP response element-binding protein as transcriptional coactivator is suggested by a single mutated gene capable of producing a broad range of the symptoms seen in RTS (50).
It is shown that two retinoic acid receptor fusion proteins recruit the nuclear corepressor histone deacetylase complex through the receptor nuclear corepressor box. Recruitment of histone deacetylase is crucial to the transforming potential of acute promyelocytic leukemia fusion proteins, and the different effects of retinoic acid on the stability of the corepressor complexes determine the differential response of acute promyelocytic leukemias to retinoic acid (51, 52, 53).
Summary and conclusion
Genetic mutations in a specific steroid receptor gene have been demonstrated to cause hormonal resistance to the specific receptor ligand. As each individual steroid hormone receptor is encoded by a different gene on separate chromosomes (54), it is unlikely that the simultaneous genetic mutations of each of theses steroid receptors is the cause of the multiple hormone resistance. Involvement of more than one steroid receptor suggests a shared defect involving multiple steroid receptors. The findings from studies of our two patients are consistent with a unique clinical entity of multiple hormone resistance, similar to the New World Primate syndrome. We propose a coactivator defect to be the most likely mechanism underlying this partial multiple hormone resistance. Further studies are necessary to define the role of transcription factors in human disease.
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Acknowledgments |
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Footnotes |
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Received January 15, 1999.
Revised March 5, 1999.
Accepted March 15, 1999.
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References |
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recruit histone
deacetylase in promyelocytic leukaemia. Nature. 391:815–818.[CrossRef][Medline]
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