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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-2212
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 2 694-701
Copyright © 2006 by The Endocrine Society

Strategies for the Characterization of Disorders in Cortisol Sensitivity

Henk Russcher1, Pauline Smit1, Elisabeth F. C. van Rossum, Erica L. T. van den Akker, Albert O. Brinkmann, Loek J. M. de Heide, Frank H. de Jong, Jan W. Koper and Steven W. J. Lamberts

Departments of Internal Medicine (H.R., P.S., E.F.C.v.R., E.L.T.v.d.A., F.H.d.J., J.W.K., S.W.J.L.) and Reproduction and Development (A.O.B.), Erasmus MC, University Medical Center Rotterdam, 3000 CA Rotterdam, The Netherlands; and Department of Internal Medicine (L.J.M.d.H.), Medical Center Leeuwarden, 8901 BR Leeuwarden, The Netherlands

Address all correspondence and requests for reprints to: Jan W. Koper, Erasmus MC, Department of Internal Medicine, Room Ee585b, Dr. Molewaterplein 40, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands. E-mail: f.koper{at}erasmusmc.nl.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: The clinical presentation of abnormalities in glucocorticoid (GC) sensitivity is diverse, and therefore it is difficult to diagnose this condition.

Objective and Design: The objective of the study was to develop strategies for the characterization of GC sensitivity disorders.

Setting: The study was conducted in an outpatient clinic.

Patients: Nine patients with GC sensitivity disorders participated.

Interventions: Sequence analysis of the GC receptor (GR), determination of GR number per cell, GR ligand-binding affinity, and GR splice regulation were performed in freshly prepared peripheral blood mononuclear lymphocytes and Epstein-Barr virus-transformed lymphoblasts. Cellular GC sensitivity was determined ex vivo by measuring the effect of dexamethasone on GC-induced leucine-zipper and IL-2 mRNA levels and on cell proliferation.

Results: Differences in GR number per cell, GR affinity, GR splice variants, and effects on transactivation or transrepression of GC-sensitive genes were observed between patients and controls. Epstein-Barr virus transformation of lymphoblasts had no influence on GR affinity but increased the GR number 5-fold in healthy controls. In patients diagnosed as cortisol resistant, however, GR number after transformation was increased significantly less than 5-fold, whereas a higher GR number was observed in a patient suspected of cortisol hypersensitivity.

Conclusion: This study illustrates several strategies to define abnormalities in GC sensitivity by describing nine patients with affected GC sensitivity, all with a unique clinical course and background.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
GLUCOCORTICOIDS (GCs) ARE key hormones in metabolic and immunological homeostasis and regulate many physiological processes (1). Cortisol concentration is tightly regulated by the hypothalamic-pituitary-adrenal (HPA)-axis feedback system and depends on neural and other stimuli (2, 3).

The extremes of variability in cortisol sensitivity can be divided in cortisol resistance (CR) and hypersensitivity (CH). So far, only one case of CH has been reported, diagnosed in a patient with Cushingoid manifestations, despite persistent hypocortisolemia (4). CR was first described (5) as an inherited disorder characterized by hypercortisolism without Cushingoid features. The negative feedback on the HPA axis is reduced, due to diminished GC sensitivity, resulting in higher cortisol secretion by the adrenal glands to keep balance between need and production. However, adrenal production of androgens and mineralocorticoids (MCs) is also increased, causing the symptoms of CR: hypertension, hypokalemia, disturbed spermatogenesis, and infertility in men and acne, hirsutism, male pattern of baldness, oligomenorrhea, and infertility in women (5, 6). In children, premature adrenarche was reported (7).

Decreased GC sensitivity is often caused by abnormalities in the GC receptor (GR) including decreased affinity for GCs (6, 7, 8), decreased receptor number (6, 8, 9), decreased receptor DNA binding (6, 10), receptor thermolability (11), impaired receptor translocation to the nucleus (12), or altered protein-protein interaction with coactivators (13). An increased concentration of the GR-ß splice variant, a dominant-negative inhibitor of active GR-{alpha}, has also been reported to cause CR, but always as acquired rather than inherited (14, 15). A GR-P splice variant is thought to increase GR-{alpha} activity (15).

The molecular basis of CR has been elucidated in six kindreds and three sporadic cases as caused by mutations in the DNA- or hormone-binding domain of the GR gene. However, several years ago, we reported five patients diagnosed with clinical and/or biochemical CR, each with very diverse clinical presentations, without GR gene alterations (16).

For the present study, we invited nine patients with abnormal GC sensitivity. One patient hyperreacted to GC medication, whereas the others were diagnosed as CR. Of the latter group, three patients had been previously reported with mutations in the GR gene (17, 18, 19) and two patients without genetic GR alterations (16); the other three patients were recently diagnosed and have not been described previously. The aim of our study was to develop a strategy for the diagnosis of (inherited) disorders in GC sensitivity. This should also include techniques using materials from patients in whom current GC therapy cannot be interrupted, as well as opportunities to study cells more intensively, without the need for freshly isolated cells.


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

Patients 1–5 have been reported previously. In summary, patients were diagnosed with compensated CR characterized by increased cortisol secretion without Cushingoid features. They showed insufficient suppression of cortisol in a 1-mg dexamethasone (DEX) suppression test. Patient 1 presented with hypertension and oligospermia (17), and his CR was attributed to a heterozygous I559N mutation. The clinical symptoms of patient 2 were hypertension and hypokalemia (17, 18), caused by a heterozygous D641V mutation. Patient 3 presented with symptoms of hyperandrogenism, attributed to a 4-bp deletion ({Delta}4) identified at the 3' boundary of exon 6 and intron 6, removing a donor splice site in one allele, resulting in the transcription of unstable mRNA, consequently decreasing the amount of GRs by 50% (19). Patient 4 presented with hirsutism and menstrual irregularities, and patient 5 also developed acne, fatigue, and mood disorders, but no GR gene alterations were found explaining the clinical and biochemical CR in these two patients (16).

Patient 6, a 36-yr-old female patient, presented with fatigue, hypertension (systolic blood pressure, 225 mm Hg; diastolic blood pressure, 125 mm Hg), and a slight male pattern of baldness, without signs and symptoms of Cushing’s syndrome, hirsutism, or menstrual irregularities [height, 172 cm; 0 SD score (SDS); weight, 66 kg].

In two overnight 1-mg DEX suppression tests, early morning cortisol was insufficiently suppressed [360 and 530 nmol/liter; normal range (N), <145 nmol/liter]. Urinary free cortisol [250–340 nmol/24 h (N, 40–200 nmol/24 h)], as well as early morning cortisol [1280 nmol/liter (N, <850 nmol/liter)], was elevated, accompanied by a slightly elevated plasma ACTH of 120 ng/ml (N, 30–100 ng/ml). Cortisol diurnal rhythm was present, albeit at a higher level. Plasma testosterone varied between 6.7 and 8.4 nmol/liter (N, 1–3 nmol/liter); dehydroepiandrosterone sulfate was 37–43 µmol/liter (N, 3–13 µmol/liter). Bone mineral density of the lumbar spine and hip were normal. The clinical presentation of the patient indicated elevated activity of the HPA axis without signs of Cushing’s disease and was typical for CR.

Patient 7 developed renal insufficiency at the age of 40 yr after an unexplained glomerulonephritis. He was one of the first patients undergoing a postmortem donor kidney transplant in The Netherlands in 1972 at the age of 43 yr. Despite low immunosuppressive medication (prednisone, 7.5 mg/d; azathioprine, 100 mg/d), his renal function remained normal and is only slightly impaired today (creatine, 202–263 µmol/d). The 33 yr after transplantation were clinically largely uneventful. He has no other specific diseases. Blood pressure is normal.

Because of this extraordinary clinical course, we suspected abnormal cortisol sensitivity. Despite long-term prednisolone medication, which could not be stopped, substantial serum concentrations of adrenal androgens were detected, which might indicate decreased GC sensitivity of the HPA-axis feedback system.

Patient 8, a 20-yr-old male patient, was diagnosed at birth with congenital adrenal hyperplasia, and the underlying defect in his 21-hydroxylase gene was recently identified (Timmermans, M. A., F. H. de Jong, unpublished results). He was treated with GCs and MCs (final height, 167 cm; –2.4 SDS; weight, 63 kg). After puberty, he was admitted several times for an Addisonian crisis in relation to intermittent infections. He needed exceptionally high doses of GCs to overcome adrenal insufficiency, indicating GC resistance.

Currently, 20 mg of hydrocortisone three times per day (N, 8–15 mg/m2·d) or 0.5 mg of DEX four times per day are still insufficient to fully normalize serum ACTH, androstenedione, 17-OH-progesterone, and testosterone levels. Serum LH and FSH levels were fully suppressed, whereas serum TSH, free T3, and free T4 were normal. He is also treated with 0.625 mg of 9{alpha}-fludrocortisone three times per day (N, 0.05–0.2 mg/d) to reach a normal blood pressure (systolic, 120 mm Hg; diastolic, 70 mm Hg), without orthostasis or peripheral edema. Recently, bone mineral density of the lumbar spine and hip were found to be within normal values.

Patient 9, a 13-yr-old patient, presented with progressive obesity, some nausea, and tiredness. For asthma, she used low-dose inhalation GCs (budesonide 200 µg/d). Growth retardation was noticed (height 142 cm; –3.2 SDS) in combination with general obesity (weight 64.5 kg; +2.8 SDS) and striae. Blood pressure was normal (systolic, 95 mm Hg; diastolic, 63 mm Hg). Serum fasting cortisol level of less than 30 nmol/liter was too low (N, 200–600 nmol/liter) as well as the urinary free cortisol of less than 3 nmol/24 h (N, <500 nmol/24 h). Bone age was 3.5 yr retarded. Bone mineral density of the lumbar spine showed osteopenia (z-score, –2.5 SDS). These clinical features of Cushing’s syndrome on low-dose steroid treatment in combination with the suppressed cortisol levels in blood and urine were considered typical for CH.

From all patients, informed consent was obtained, and the Medical Ethics Committee of Erasmus MC, The Netherlands, approved this study.

Whole cell DEX binding, [3H]thymidine incorporation, and mRNA expression of GC-induced leucine-zipper, IL-2, and GR

Blood (70 ml) was drawn into heparinized tubes by venipuncture. Peripheral blood mononuclear leukocytes (PBMLs) were isolated, and the number of GRs per cell (n), their dissociation constant (KD), and the sensitivity of PBMLs to the inhibition of phytohemagglutinin (PHA)-stimulated incorporation of [3H]thymidine by 100 nM DEX were determined, as described previously (6, 20). Expression of GC-induced leucine-zipper (GILZ) and IL-2 mRNA levels in response to 100 nM DEX and expression of GR-{alpha}, GR-ß, and GR-P splice variants were measured in a real-time quantitative PCR (Q-PCR), as described previously (21, 22).

Epstein-Barr virus transformation of B lymphocytes

Epstein-Barr virus (EBV)-transformed lymphoblast cell lines were established from PBMLs (23, 24). Cells were grown in RPMI 1640 medium supplemented with 15% fetal calf serum, 100 µg/ml penicillin, and streptomycin at standard culture conditions.

Sequence analysis

The coding sequence of the GR gene including intron/exon boundaries was sequenced in all patients using primers as described previously (25).


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Analysis of GR characteristics and expression

Sequence analysis was performed on the nine exons and intronic flanking sequences of the GR in all patients. We have previously reported on the heterozygous mutations in patients 1 (I559N), 2 (D641V), and 3 ({Delta}4 bp). In patient 3 also, the earlier reported N363S single-nucleotide polymorphism (SNP) was found, enhancing GC sensitivity (26). In the other patients, several different SNPs were found, but only ER22/23EK, heterozygously present in patient 4, has been reported to decrease GC sensitivity (25, 26). Patient 7 had two heterozygous nucleotide changes in intron 8, 81 bases downstream of exon 8 (G to A) and 9 bases upstream of exon 9 (C to G). Codons 750 and 588 in, respectively, patients 7 and 8 were heterozygously mutated from, respectively, CCC to CCT and CAC to CAT, but this did not cause amino acid changes.

Subsequently, we performed radioligand-binding studies to determine the GR number per cell (n) and the KD in PBMLs (Fig. 1AGo) of patients and 14 healthy controls. Patient 3 ({Delta}4 bp) showed only half of the normal receptor number per cell, whereas patient 9, suspected of CH, showed an increased receptor number. Only patient 1, carrying the heterozygous I559N mutation, showed decreased affinity for DEX. The data of patients 5 and 7 were not included in Fig. 1AGo because GC medication could not be stopped and subsequently interfered in these binding studies.


Figure 1
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  		FIG. 1. GR characteristics and GR mRNA copy numbers in PBMLs of patients and healthy controls. GR number per cell (n) and KD (A) and relative copy numbers of GR-{alpha}, GR-ß, and GR-P splice variants (B) in PBMLs of patients with affected GC sensitivity (circled numbers) and controls ({blacksquare}). From patients 5 and 7, no n or KD could be obtained due to interference of DEX medication (indicated as #). Copy numbers were calculated relative to the levels of the housekeeping gene hypoxanthine phosphoribosyl transferase (HPRT) by applying the formula 2[CT (HPRT) – CT (GR)]. For further details, see Livak and Schmittgen (32 ). Data represent means ± SEM, and the assay was performed in duplicate with duplicate measurements or as indicated (m). *, P ≤ 0.05; ***, P ≤ 0.001 by Student’s t test.

 
Expression of the GR was further analyzed by quantification of mRNA copies of the GR-{alpha}, GR-ß, and GR-P splice variants using real-time Q-PCR. Figure 1BGo shows these levels measured in PBMLs of patients and controls. Patient 3 ({Delta}4 bp) shows approximately 50% of the normal amount of GR-{alpha}, GR-ß, and GR-P mRNA copies. In patient 9, 70% more GR-{alpha} mRNA expression was measured, corresponding with GR number measured in PBMLs in the ligand-binding assay. Patient 7 showed 3-fold higher GR-ß expression levels, although no differences for expression of GR-{alpha} and GR-P mRNA splice variants were found.

Cellular GC sensitivity

Liganded GR acts together with several cofactor complexes to regulate transcription of GC-responsive genes. Affected GC sensitivity was further investigated by measuring the expression of GILZ and IL-2, two endogenous GC-sensitive genes, which could be strongly up- and down-regulated by GCs, respectively. Figure 2Go shows the increase of GILZ (A) and decrease of IL-2 (B) in PBMLs of our patients when stimulated with PHA and 100 nM DEX, relative to levels in the absence of DEX. The average increase/decrease of GILZ/IL-2 mRNA in PBMLs of healthy controls was set to 100%. PHA, necessary to induce IL-2 gene transcription, did not affect expression of GILZ or GR mRNA levels. PHA induction, as well as basal expression levels of GILZ and IL-2 in the absence of DEX, was comparable between patients and controls (data not shown). Patients 2 (D641V), 3 ({Delta}4 bp), 4, and 7 showed less up-regulation of GILZ mRNA than the controls (50 ± 3%, 52 ± 5%, 52 ± 3%, and 53 ± 9%, respectively), whereas transrepression of the IL-2 gene was mainly unaffected. In patients 1 (I559N) and 8, transactivation of the GILZ, as well as transrepression of the IL-2 gene, was reduced (GILZ up-regulation and IL-2 repression compared with controls in patient 1, 35 ± 5% and 49 ± 9%, respectively; in patient 8, 62 ± 10% and 85 ± 6%, respectively). The same trend was observed in patients 5 and 6 (in patient 5, 66 ± 6% and 84 ± 5%, respectively; in patient 6, 88 ± 9% and 75 ± 6%, respectively). In patient 9, who overreacted to GC medication, more transcriptional regulation of the GILZ and IL-2 gene seemed to occur, but this was not significantly different from controls. In Fig. 2CGo, the GILZ response is plotted against the IL-2 response. Patients 1, 5, 6, 8, and 9 lie close to the diagonal, indicating defects that equally affect transactivation and transrepression, whereas the marked GILZ defect without substantial alterations in IL-2 response puts patients 2, 3, 4, and 7 off the diagonal in the lower right section, clearly demonstrating that transactivation and transrepression are separable entities.


Figure 2
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  		FIG. 2. GILZ and IL-2 mRNA expression levels and repression of PHA-stimulated proliferation in PBMLs. Relative increase of GILZ (A) and decrease of IL-2 (B) mRNA levels induced by 100 nM DEX in PBMLs of patients (circled numbers) and controls (©). Cells were incubated for 4 h with PHA and with or without 100 nM DEX, followed by mRNA isolation and quantitation by real-time RT-PCR. Data are presented as the increase of GILZ (A) and decrease of IL-2 (B) mRNA relative to the values in the absence of DEX, which are also presented in C as GILZ vs. IL-2 response. DEX incubations were performed in duplicate, and duplicate real-time Q-PCR was performed for every sample. Levels for controls in arbitrary units were: 100 ± 8 (without DEX) and 1577 ± 115 (100 nM DEX) in the GILZ assay and 100 ± 5 (without DEX) and 28 ± 4 (100 nM DEX) in the IL-2 assay. These response levels were set to 100%. No systematic differences between patients and healthy controls were observed in the absence of DEX (data not shown). For all subjects, PHA treatment in the absence of DEX equally stimulated IL-2 mRNA levels 12- to 18-fold but did not affect GILZ or GR mRNA levels (data not shown). D, Relative inhibition of PHA-induced [3H]thymidine incorporation by 100 nM DEX in PBMLs of patients and controls. The average response in the healthy controls was 100 ± 6 (without DEX) and 18 ± 3 (100 nM DEX) and was set to 100%. Data represent means ± SEM, and the assay was performed in duplicate with incubations in triplicate. The average response in the healthy controls was set to 100%. *, P < 0.05 by Student’s t test.

 
The PBMLs were also tested in a PHA-stimulated proliferation assay. The decrease in proliferation induced by 100 nM DEX is shown in Fig. 2DGo and is related to the average decrease in the healthy control group set to 100%. Less suppression of proliferation was observed for patients 1 (23 ± 3%), 4 (60 ± 12%), and 8 (70 ± 15%), whereas more suppression was observed for patient 9 (115 ± 2%). Approximately the same trend was shown compared with the results of the IL-2-transrepression assay, only the outcomes for patients 4 and 6 did not correspond. In this proliferation assay, IL-2 gene repression certainly plays a role, but it is carried out over a much longer time scale (4 d, rather than 4 h), and the outcome is formed by the integration of many processes, including apoptosis.

EBV-transformed B lymphocytes

To obtain permanent cell lines, native B lymphocytes of patients and controls were transformed with EBV to obtain immortalized lymphoblast cell lines, and then GR number and ligand KD were measured (Fig. 3AGo). Ligand affinity was not influenced by viral transformation (Spearman’s correlation: r = 0.73; P = 0.06). Patient 1 (I559N) showed decreased affinity for DEX. GR numbers in lymphoblasts were approximately five times higher than those measured in lymphocytes, even after correction for cell volume (data not shown). The increase in receptor number after EBV transformation in patients diagnosed as CR, however, was significantly less, whereas the receptor number in the patient who overreacted to GC treatment was significantly higher, than in controls (Fig. 3AGo). Quantifying GR-{alpha} and GR-P mRNA expression levels also showed that GR expression is affected after viral transformation (Fig. 3BGo). Figure 4Go shows the correlation in lymphoblasts between GR number determined in the radioligand-binding assay and the number of GR-{alpha} mRNA copies, further indicating that GR concentrations in affected patients were different from those measured in controls. GR-ß levels, however, seemed not to be affected by viral transformation, but this might be obscured by the higher variability in quantifying these very low expression levels. Interestingly, GR-ß expression in patient 7 was more than 4-fold higher than in the other subjects, which was also shown, although to a lesser extent, in PBMLs (Fig. 1BGo).


Figure 3
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  		FIG. 3. GR characteristics and GR mRNA copy numbers in lymphoblast cell lines of patients and healthy controls. GR number per cell (n) and KD (A) and relative copy numbers of GR-{alpha}, GR-ß, and GR-P splice variants (B) in lymphoblasts of patients with affected GC sensitivity (circled numbers) and controls ({blacksquare}). Copy numbers were calculated relative to the levels of the housekeeping gene hypoxanthine phosphoribosyl transferase (HPRT) by applying the formula 2[CT (HPRT) – CT (GR)]. For further details, see Livak and Schmittgen (32 ). Data represent means ± SEM, and assays were performed at least three times in duplicate, exactly 3 months after transfection. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 by Student’s t test.

 

Figure 4
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  		FIG. 4. Correlation analysis (Spearman’s correlation) between GR number and relative GR-{alpha} mRNA copy numbers in lymphoblast cell lines of patients and healthy controls.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
We have used three different approaches to study altered GC sensitivity: 1) analysis of the GR characteristics (n and KD), its coding sequence, and its expression (including mRNA splice variants); 2) examination of GC sensitivity ex vivo by measuring responses of endogenous GC sensitive genes (GILZ and IL-2) and the inhibition of mitogen stimulated proliferation; and 3) obtaining permanent cell lines, free of systemic influences, and preceding therapy by transforming B lymphocytes with EBV. A table describing patients, genotype, clinical presentation, biochemical phenotype, and a summary of the data obtained from this study is published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org.

The process through which the I559N and D641V mutations in patients 1 and 2 impair the physiological mechanisms of GC action at the molecular level is multifactorial and involves impaired ligand-binding ability, decreased intrinsic transcriptional activity, and abnormal interaction with certain coactivators (27, 28). Furthermore, the D641V mutation showed aberrant nucleocytoplasmic trafficking and the I559N mutation exerted a dominant-negative effect on GR-{alpha} activity by hampering nuclear translocation (28). The ex vivo results presented in this paper were in line with the results found in these in vitro studies (27, 28): transactivation and transrepression was affected in patient 1. Due to the dominant-negative effect of the GR (I559N), only 30–40% transactivating activity was measured. Transrepression in patient 2, however, was normal, and only decreased transactivational activity was observed. In a previous in vitro study, we also observed decreased effects on transactivation of GR (D641V), with no effects on transrepression (27). An explanation for this discrepancy between patients 1 and 2 may be that transactivation occurs through a mechanism characterized by GR interaction with specific DNA sequences, the GC response elements (GREs), whereas transrepression involves interaction of GR with other transcription factors in the absence of specific DNA binding (29).

Patient 3, expressing only half of the normal number of GRs, might demonstrate the strong relationship between GR number and GC sensitivity. Transactivation is 50% reduced (Fig. 2AGo), whereas maximal transrepression was unaffected (Fig. 2Go, B and C). This might indicate that GR action through transactivation might be more GR-concentration dependent than through transrepression. Malchoff and Malchoff (30) already speculated that alterations of the promoter region or factors modulating gene expression, leading to fewer GRs, could cause CR.

Patients 4, 5, and 6 had hypercortisolism without Cushingoid features, insufficient suppression of early morning serum cortisol concentrations in reaction to 1-mg DEX, variable degrees of androgen overproduction, and fatigue. Cellular sensitivity at the level of transactivation of GILZ was significantly reduced in patients 4 and 5 (Fig. 2Go) and on transrepression of IL-2 also in patients 5 and 6 (Fig. 2Go). GR expression levels and characteristics were normal (Figs. 1Go and 3Go), suggesting that the condition of the patients was not caused by reduced GR expression, as might be the result of mutations in the promoter region of the GR gene. However, GR mRNA copy numbers and DEX binding after EBV transformation was lower, which might suggest a defect in GR synthesis or regulation that only becomes apparent in these lymphoblasts. Possible pathophysiological bases of CR in these patients could also be formed by alterations in cellular trafficking or in interactions with other nuclear cofactors. The ER22/23EK SNP in patient 4 is reported to slightly decrease GC sensitivity (26) but could not be exclusively responsible for the severe reduced sensitivity as described in this study. At present, hyperactivity of the HPA axis was normalized by low doses of DEX.

Patient 7, who only needed low immunosuppressive medication for a postmortem donor kidney transplant, was suspected of increased immunosuppressive function of the HPA axis. Sequence analysis of the GR gene revealed (among other mutations) a heterozygous C to G mutation in the pyrimidine tract of the exon 9{alpha} splice acceptor. Splice site analysis (https://splice.cmh.edu) predicted that the strength of the acceptor splice site is slightly weakened, possibly resulting in skipping of exon 9{alpha} in favor of exon 9ß. However, this is not an absolute effect, because at the level of the mRNA, another heterozygous mutation in this patient (P750P in exon 9{alpha}) was also found to be present. But quantitative RT-PCR did show that the GR-ß expression was three to four times higher than in controls, both in PBMLs and in lymphoblasts (Figs. 1BGo and 3BGo). In the GILZ assay, cells from this patient showed a significantly reduced response, indicating reduced transactivating capacity, whereas in the IL-2 assay, the response was similar to that measured in controls. Our hypothesis is that in this patient, the reduction of transactivating capacity, possibly due to increased expression of GR-ß, results in CR at the level of GRE-mediated GR action (also involved in the feedback sensitivity of the HPA axis), whereas the immunosuppressive function (not GRE mediated) is not affected. As a result, the immune system is exposed to higher compensatory cortisol concentrations and is subsequently relatively suppressed. Increased GR-ß levels have frequently been associated with acquired GC resistance in various disease states (e.g. asthma, rheumatoid arthritis); however, increased GR-ß levels in this particular patient may have resulted in positive effects.

In patient 8, transactivation and transrepression activities were decreased (Fig. 2Go), whereas GR characteristics were normal (Fig. 1Go). Extraordinarily high doses of both GCs and MCs were needed to overcome his 21-hydroxylase deficiency. However, this was still insufficient to fully compensate and normalize adrenal function because 17-OH-progesterone, androstenedione, testosterone, and ACTH plasma levels remained elevated. Cofactors influencing both the GR and the MC receptor (MR) activity could be involved, but then, androgen and thyroid receptor function might also be impaired because many coactivators are involved in the functioning of more than one nuclear receptor. Plasma TSH and T3 were within the reference range, indicating normal thyroid function. Furthermore, plasma LH and FSH were fully suppressed by the elevated testosterone, indicating that androgen receptor function is not impaired either. It is not clear whether cofactors exist that specifically interact with the GR and MR, without influencing other nuclear receptors. Recently, differences between splice variants of steroid receptor coactivator-1 have been reported that strongly interact with GRs and MRs in a promoter-, receptor-, and ligand-dependent way (31). Disturbances in splicing regulation or tissue-specific expression of these cofactors could have dramatic influences on the cellular GC sensitivity, whereas other nuclear receptor activities might hardly be affected.

The increased sensitivity in patient 9 is mainly due to an increased receptor number (Figs. 1Go and 3Go) and slightly increasing cellular sensitivity (Fig. 2Go). Cofactors inducing GR expression or alterations in the promoter region of the GR could be responsible for this. After stopping steroid medication that was used to treat her asthma, the Cushingoid features disappeared.

Viral transformation had no influence on GR quality because similar receptor affinities (KD) were found for native and transformed cells (Figs. 1Go and 3Go) but increased GR number 5-fold. Interestingly, in lymphoblasts of patients diagnosed as CR, induction was less, whereas in the patient diagnosed as CH, a higher GR number than in controls was measured (Fig. 3Go). Tomita et al. (24) already reported that CR patients (from the D641V kindred) showed diminished induction during viral transformation. The molecular mechanism explaining this phenomenon, however, is still unknown. We hypothesize that during viral transformation, autoregulation of the GR occurs that might be impaired or enhanced by CR or CH, respectively. Although this phenomenon limits our possibilities to study the GR in its signaling context, due to noncomparable GR concentrations, the abnormalities in GR up-regulation in lymphoblasts of patients might be an additional indicator of altered GC sensitivity. Plotting GR number per lymphoblast against GR mRNA copy number (Fig. 4Go) grouped all patients diagnosed as CR into the lower left sector, regardless of the molecular basis of the defect. Compared with measuring GR characteristics and GC response in freshly prepared cells, EBV transformation is more laborious. However, measuring GR up-regulation during EBV transformation seemed to be the most powerful tool to differentiate CR and CH from controls. The other markers (GILZ, IL-2, and proliferation) are easier to obtain, but as individual markers, they are less powerful because they sample distinct aspects of GC sensitivity (transactivating, transrepressing capacity but also proliferation processes, including apoptosis), which can differ strongly between patients.

In conclusion, for the appropriate diagnosis of CR or CH, a careful interpretation of clinical presentation is essential, but it is subsequently also important to quantify these syndromes biochemically. To do this, we have investigated GR characteristics and GC response using freshly isolated PBMLs and permanent cell lines. The results of these approaches are illustrated in this study by describing nine patients with suspected abnormalities in GC sensitivity, all with a unique clinical course and background.


   Acknowledgments
 
We express our gratitude to Prof. Dr. W. Weimar, Dr. A. W. L. van den Wall Bake, and M. J. Affourtit for their contributions.


   Footnotes
 
This work was supported by The Netherlands Organization for Scientific Research (NWO) Grant 903-43-093.

First Published Online November 29, 2005

1 H.R. and P.S. contributed equally to this study. Back

Abbreviations: CH, Cortisol hypersensitivity; CR, cortisol resistance; EBV, Ebstein-Barr virus; GC, glucocorticoid; GILZ, GC-induced leucine-zipper; GR, GC receptor; GRE, GC response element; HPA, hypothalamic-pituitary-adrenal; KD, dissociation constant; MC, mineralocorticoid; MR, MC receptor; PBML, peripheral blood mononuclear leukocyte; PHA, phytohemagglutinin; Q-PCR, quantitative PCR; SDS, SD score; SNP, single-nucleotide polymorphism.

Received October 6, 2005.

Accepted November 21, 2005.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

  1. Drucker D, Shandling M 1985 Variable adrenocortical function in acute medical illness. Crit Care Med 13:477–479[Medline]
  2. Chrousos GP 1995 The hypothalamic-pituitary-adrenal axis and immune-mediated inflammation. N Engl J Med 332:1351–1362[Free Full Text]
  3. Antoni FA 1986 Hypothalamic control of adrenocorticotropin secretion: advances since the discovery of 41-residue corticotropin-releasing factor. Endocr Rev 7:351–378[Medline]
  4. Iida S, Nakamura Y, Fujii H, Nishimura J, Tsugawa M, Gomi M, Fukata J, Tarui S, Moriwaki K, Kitani T 1990 A patient with hypocortisolism and Cushing’s syndrome-like manifestations: cortisol hyperreactive syndrome. J Clin Endocrinol Metab 70:729–737[Abstract]
  5. Vingerhoeds AC, Thijssen JH, Schwarz F 1976 Spontaneous hypercortisolism without Cushing’s syndrome. J Clin Endocrinol Metab 43:1128–1133[Abstract]
  6. Lamberts SW, Koper JW, Biemond P, den Holder FH, de Jong FH 1992 Cortisol receptor resistance: the variability of its clinical presentation and response to treatment. J Clin Endocrinol Metab 74:313–321[Abstract]
  7. Malchoff CD, Javier EC, Malchoff DM, Martin T, Rogol A, Brandon D, Loriaux DL, Reardon GE 1990 Primary cortisol resistance presenting as isosexual precocity. J Clin Endocrinol Metab 70:503–507[Abstract]
  8. Werner S, Thoren M, Gustafsson JA, Bronnegard M 1992 Glucocorticoid receptor abnormalities in fibroblasts from patients with idiopathic resistance to dexamethasone diagnosed when evaluated for adrenocortical disorders. J Clin Endocrinol Metab 75:1005–1009[Abstract]
  9. Iida S, Gomi M, Moriwaki K, Itoh Y, Hirobe K, Matsuzawa Y, Katagiri S, Yonezawa T, Tarui S 1985 Primary cortisol resistance accompanied by a reduction in glucocorticoid receptors in two members of the same family. J Clin Endocrinol Metab 60:967–971[Abstract]
  10. Nawata H, Sekiya K, Higuchi K, Kato K, Ibayashi H 1987 Decreased deoxyribonucleic acid binding of glucocorticoid-receptor complex in cultured skin fibroblasts from a patient with the glucocorticoid resistance syndrome. J Clin Endocrinol Metab 65:219–226[Abstract]
  11. Bronnegard M, Werner S, Gustafsson JA 1986 Primary cortisol resistance associated with a thermolabile glucocorticoid receptor in a patient with fatigue as the only symptom. J Clin Invest 78:1270–1278
  12. Kino T, Stauber RH, Resau JH, Pavlakis GN, Chrousos GP 2001 Pathologic human GR mutant has a transdominant negative effect on the wild-type GR by inhibiting its translocation into the nucleus: importance of the ligand-binding domain for intracellular GR trafficking. J Clin Endocrinol Metab 86:5600–5608[Abstract/Free Full Text]
  13. Vottero A, Kino T, Combe H, Lecomte P, Chrousos GP 2002 A novel, C-terminal dominant negative mutation of the GR causes familial glucocorticoid resistance through abnormal interactions with p160 steroid receptor coactivators. J Clin Endocrinol Metab 87:2658–2667[Abstract/Free Full Text]
  14. Leung DY, Hamid Q, Vottero A, Szefler SJ, Surs W, Minshall E, Chrousos GP, Klemm DJ 1997 Association of glucocorticoid insensitivity with increased expression of glucocorticoid receptor beta. J Exp Med 186:1567–1574[Abstract/Free Full Text]
  15. de Lange P, Segeren CM, Koper JW, Wiemer E, Sonneveld P, Brinkmann AO White A, Brogan IJ, de Jong FH Lamberts SW 2001 Expression in hematological malignancies of a glucocorticoid receptor splice variant that augments glucocorticoid receptor-mediated effects in transfected cells. Cancer Res 61:3937–3941[Abstract/Free Full Text]
  16. Huizenga NA, de Lange P, Koper JW, de Herder WW, Abs R, Kasteren JH, de Jong FH, Lamberts, SW 2000 Five patients with biochemical and/or clinical generalized glucocorticoid resistance without alterations in the glucocorticoid receptor gene. J Clin Endocrinol Metab 85:2076–2081[Abstract/Free Full Text]
  17. Karl M, Lamberts SW, Koper JW, Karl M, Lamberts SW, Koper JW, Katz DA, Huizenga NE, Kino T, Haddad BR, Hughes MR, Chrousos GP 1996 Cushing’s disease preceded by generalized glucocorticoid resistance: clinical consequences of a novel, dominant-negative glucocorticoid receptor mutation. Proc Assoc Am Physicians 108:296–307[Medline]
  18. Hurley DM, Accili D, Stratakis CA, Karl M, Vamvakopoulos N, Rorer E, Constantine K, Taylor SI, Chrousos GP 1991 Point mutation causing a single amino acid substitution in the hormone binding domain of the glucocorticoid receptor in familial glucocorticoid resistance. J Clin Invest 87:680–686
  19. Karl M, Lamberts SW, Detera-Wadleigh SD, Encio IJ, Stratakis CA, Hurley DM, Accili D, Chrousos GP 1993 Familial glucocorticoid resistance caused by a splice site deletion in the human glucocorticoid receptor gene. J Clin Endocrinol Metab 76:683–689[Abstract]
  20. Molijn GJ, Koper JW, van Uffelen CJ, de Jong FH, Brinkmann AO, Bruining HA, Lamberts SW 1995 Temperature-induced down-regulation of the glucocorticoid receptor in peripheral blood mononuclear leucocyte in patients with sepsis or septic shock. Clin Endocrinol (Oxf) 43:197–203[Medline]
  21. Smit P, Russcher H, de Jong FH, Brinkmann AO, Lamberts SW, Koper JW 2005 Differential regulation of synthetic glucocorticoids on gene expression levels of glucocorticoid-induced leucine zipper and interleukin-2. J Clin Endocrinol Metab 90:2994–3000[Abstract/Free Full Text]
  22. ter Borg PC, Hagendorf A, van Buuren HR, Koper JW, Lamberts SW 2004 A pilot study exploring the role of glucocorticoid receptor variants in primary biliary cirrhosis and primary sclerosing cholangitis. Neth J Med 62:326–331[Medline]
  23. Chrousos GP, Loriaux DL, Tomita M, Brandon DD, Renquist D, Albertson B, Lipsett MB 1986 The new world primates as animal models of glucocorticoid resistance. Adv Exp Med Biol 196:129–144[Medline]
  24. Tomita M, Brandon DD, Chrousos GP, Vingerhoeds AC, Foster CM, Fowler D, Loriaux, DL, Lipsett MB 1986 Glucocorticoid receptors in Epstein-Barr virus-transformed lymphocytes from patients with glucocorticoid resistance and a glucocorticoid-resistant New World primate species. J Clin Endocrinol Metab 62:1145–1154[Abstract]
  25. Koper JW, Stolk RP, de Lange P, Huizenga NA, Molijn GJ, Pols HA, Grobbee DE, Karl M, de Jong FH, Brinkmann AO, Lamberts SW 1997 Lack of association between five polymorphisms in the human glucocorticoid receptor gene and glucocorticoid resistance. Hum Genet 99:663–668[CrossRef][Medline]
  26. Russcher H, Smit P, van den Akker EL, van Rossum EF, Brinkmann AO, de Jong FH, Lamberts SW, Koper, JW 2005 Two polymorphisms in the glucocorticoid receptor gene directly affect glucocorticoid-regulated gene expression. J Clin Endocrinol Metab 90:5804–5810[Abstract/Free Full Text]
  27. de Lange P, Koper JW, Huizenga NA, Brinkmann AO, de Jong FH, Karl M, Chrousos GP, Lamberts SW 1997 Differential hormone-dependent transcriptional activation and -repression by naturally occurring human glucocorticoid receptor variants. Mol Endocrinol 11:1156–1164[Abstract/Free Full Text]
  28. Charmandari E, Kino T, Souvatzoglou E, Vottero A, Bhattacharyya N, Chrousos GP 2004 Natural glucocorticoid receptor mutants causing generalized glucocorticoid resistance: molecular genotype, genetic transmission, and clinical phenotype. J Clin Endocrinol Metab 89:1939–1949[Abstract/Free Full Text]
  29. Bamberger CM, Schulte HM, Chrousos GP 1996 Molecular determinants of glucocorticoid receptor function and tissue sensitivity to glucocorticoids. Endocr Rev 17:245–261[Abstract]
  30. Malchoff CD, Malchoff DM 1995 Glucocorticoid resistance in humans. Trends Endocrinol Metab 6:89–95
  31. Meijer OC, Kalkhoven E, van der Laan S, Steenbergen PJ, Houtman SH, Dijkmans TF, Pearce D, de Kloet ER 2005 Steroid receptor coactivator-1 splice variants differentially affect corticosteroid receptor signaling. Endocrinology 146:1438–1448[Abstract/Free Full Text]
  32. Livak KJ, Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408[CrossRef][Medline]




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