ABSTRACT

Primary generalized glucocorticoid resistance syndrome is a rare genetic disorder characterized by resistance of entire tissues to glucocorticoids. Affected subjects demonstrate elevation of serum cortisol without Cushingoid manifestations, as the hypothalamic-pituitary-adrenal (HPA) axis is upregulated to compensate for the reduced action of this steroid in local tissues. Instead, these patients develop hypertension and/or signs of hyperandrogenism, because hyper-secreted adrenocorticotropic hormone (ACTH) stimulates production of adrenal mineralocorticoids and/or androgens in addition to the glucocorticoid cortisol. At the molecular level, this syndrome is caused by inactivating mutations in the NR3C1 gene that encodes the human glucocorticoid receptor (hGR) protein. Biochemical, molecular and structural exploration on pathologic mutant receptors revealed a variety of functional defects, such as reduced affinity to glucocorticoids or target DNA, inability to transactivate glucocorticoid-responsive genes, and slowing of the cytoplasmic to nuclear translocation. The clinical spectrum of this syndrome is thus broad, ranging from asymptomatic to severe cases of mineralocorticoid and/or androgen excess depending on the severity of genetic defects and resulting dysfunction of the mutated receptors. When this syndrome is suspected, a detailed personal and family history should be obtained. Physical examination should include an assessment for signs of mineralocorticoid and/or androgen excess. In neonates and young children, severe hypoglycemia and loss of consciousness due to reduced actions of glucocorticoids in the liver may be present as initial manifestations in addition to hypertension and/or genital abnormalities. Suspected subjects should undergo a detailed endocrinologic evaluation with particular emphasis on the measurement of diurnal serum cortisol and plasma ACTH concentrations and determination of the 24-hour urinary free cortisol excretion to identify upregulation of the HPA axis with preservation of the normal circadian rhythmicity. The diagnosis of this syndrome should be confirmed by sequencing of the NR3C1 gene including exon/intron junctions and subsequent validation of functional defects of the mutated receptors. Treatment involves administration of high doses of mineralocorticoid activity-sparing pure glucocorticoids like dexamethasone, which stimulate the mutant and/or the wild-type hGR, and suppress the endogenous secretion of ACTH and adrenal steroids in the affected subjects. For complete coverage of all related areas of Endocrinology, please visit our on-line FREE web-text, WWW.ENDOTEXT.ORG.

INTRODUCTION

Organisms are exposed continuously to internal and external stressors, and live through them by maintaining the internal equilibrium called homeostasis (1). In order to respond adequately to such stressors through coordinating various body activities, we humans are equipped with a highly sophisticated stress responsive system, the hypothalamic-pituitary-adrenal (HPA) axis, which consists of the brain hypothalamus, the anterior pituitary gland, and the adrenal cortex, and employs glucocorticoids as its end-effector hormones. Actions of glucocorticoids, which are essential for life, can be determined by a balance between circulating levels of these hormones and local tissue sensitivity (2, 3). Exceeding appropriate ranges of tissue sensitivity to glucocorticoids may present either as glucocorticoid resistance or glucocorticoid hypersensitivity with their specific manifestations (3, 4). Such alterations in tissue glucocorticoid actions can occur in general (that is, throughout the body) or in tissue-specific manner (restricted in some organs and tissues; e.g., immune organs/cells, central nervous system (CNS), liver and fat tissues) (3). They are caused primarily by genetic defects of the molecules involved in the glucocorticoid signaling pathway or secondary through modulation of this pathway by other pathologic conditions, such as infectious, inflammatory and autoimmune diseases, obesity, and insulin resistance/overt diabetes mellitus. One such condition is the primary generalized glucocorticoid resistance syndrome, which is caused by inactivating mutations in the glucocorticoid receptor gene (5). Affected subjects develop partial glucocorticoid resistance observed in entire organs and tissues of the affected subjects (5). In recognition of Professor George P. Chrousos' novel and extensive research work in this field, the term “Chrousos Syndrome” may be used for this syndrome (6, 7).

GLUCOCORTICOIDS

Glucocorticoids (cortisol in humans and corticosterone in rodents) are produced from cholesterol through multiple enzymatic reactions in the zona fasciculata of the adrenal cortex in response to the adrenocorticotropic hormone (ACTH) released from the pituitary gland (1). Glucocorticoids regulate a broad spectrum of physiologic functions essential for life, such as growth, reproduction, immunity, intermediary metabolism, cardiovascular tone, and CNS functions, playing essential and indispensable roles in the maintenance of resting and stress-related homeostasis (1, 7, 8). In addition, glucocorticoids exert potent anti-inflammatory and immunomodulatory effects particularly with their stress-equivalent or pharmacologic doses, thus they are widely used in the treatment of inflammatory, autoimmune, and lymphoproliferative diseases (8).

GLUCOCORTICOID RECEPTOR PROTEINS, ISOFORMS AND ITS ENCODING GENE, NR3C1

Circulating cortisol freely passes through the cytoplasmic membrane and enters into the cytoplasm of its target cells, and binds to an intracellular protein, the glucocorticoid receptor (GR) (9, 10). The human (h) GR is one of the steroid/thyroid/retinoic acid nuclear hormone receptor superfamily proteins, which consist of over 600 members in the animal kingdom (11). Many of them mediate extracellular signals transduced mainly by lipophilic hormones/compounds into the cell nucleus by binding them as ligands and by acting as ligand-dependent transcription factors (12, 13). hGR influences transcription rates of numerous glucocorticoid-responsive genes (up to 3~5% of the entire protein-coding genes) in a positive or a negative fashion by interacting directly or indirectly with promoter/enhancer regions of these genes (14). The hGR gene (NR3C1: nuclear receptor subfamily 3, group C, member 1) consists of 9 exons and is located at chromosome 5q31.3. Exons 2-9 constitute the protein-coding sequence, whereas exon 1 encodes an untranslated region (12, 14, 15). The human NR3C1 gene has multiple exon 1s (see below) that harbor specific promoters containing a respective transcription start site for conferring tissue-specific expression of the receptor protein (15). Alternative splicing of the NR3C1 gene in exon 9s generates two highly homologous receptor isoforms, the hGRα and the hGRβ (16). They share amino (N)-terminal 727 common amino acids, but then diverge, with hGRα having an additional 50 amino acids and hGRβ having an additional, nonhomologous 15 amino acids at their carboxyl (C)-termini (17). hGRα resides primarily in the cytoplasm of cells and represents the classic GR that binds natural and synthetic glucocorticoids and mediates most of the actions of these hormones (15). On the other hand, hGRβ does not bind glucocorticoids, has intrinsic, gene-specific transcriptional activity, and exerts a dominant negative effect on the transcriptional activity of hGRα (18). Although physiologic and pathologic roles of hGRβ are still largely unknown (19, 20), recent studies demonstrated that this isoform is implicated in modulation of the insulin signaling and participates in the pathogenesis of brain gliomas (21-23).

The hGRα mRNA expresses not only the classic, full-length hGRα, but also multiple translational isoforms by using at least eight alternative amino-terminal translation initiation sites (24). All these hGRα isoforms are differentially distributed in the cytoplasm and/or the nucleus in the absence of ligand, have different transcriptional activity, and display distinct transactivating or transrepressing activities on various glucocorticoid-responsive genes (24). Since hGRβ shares with hGRα a common amino-terminal domain that contains the same translation initiation sites, the hGRβ variant mRNA might also be translated through the same translation initiation sites to a similar host of hGRβ isoforms (14).

The human NR3C1 has 11 different promoters with their alternative first exons (1A1, 1A2, 1A3, 1B, 1C, 1D, 1E, 1F, 1H, 1I and 1J) (25, 26). Therefore, it can produce 11 different hGRα mRNA transcripts from different promoters that encode the same hGRα protein, as these transcripts share common exon 2 to exon 9α that contains the same translation initiation codon. 1A1, 1A2, 1A3 and 1I are located in the distal promoter region spanning ~32,000-36,000 bps upstream of the translation initiation site, while 1B, 1C, 1D, 1E, 1F, 1H and 1J position in the proximal promoter region located up to ~5,000 bps upstream of this site (25). Through differential use of these promoters, expression levels of hGRα can vary among tissues in different physiologic and pathologic conditions, as each tissue has specific expression profiles of local transcription factors and epigenetic modification of chromatin-associated molecules bound on these exon 1-associated promoters (25, 27, 28). Again, differential tissue-specific expression of hGRβ through the use of these promoters appears to be present. The above-indicated marked complexity in transcription/translation of the human NR3C1 gene enables target tissues to respond differently to circulating cortisol and accounts for stochastic, but still a highly organized nature of tissue glucocorticoid actions, in order to fulfil specific local needs of glucocorticoid hormonal effects (14). Such complexity of the glucocorticoid signaling at the receptor level also indicates that the proper biologic action of glucocorticoids in every target tissue is extremely important.

The hGRα protein consists of three major domains and one region, namely the N-terminal (NTD), DNA-binding (DBD) and ligand-binding domain (LBD), and the hinge region (HR) (15). Exact amino acid location of these domains/region in the hGRα protein explained below is based on the data retrieved from the Pfam source of the Ensembl database (www.ensembl.org). NTD is encoded by exon 2 and represents the largest domain of the receptor, spanning over amino acids 1 to 401. It contains an unstructured acidic transactivation surface called activation function (AF) -1, which is used as a molecular platform for modulating the transcription of glucocorticoid-responsive genes (10). This domain also undergoes several post-translational modifications particularly at AF-1. DBD is expressed from exons 3 and 4, and lies between amino acids 417 and 494. This domain consists of two 4C (cysteine)-type zinc fingers and support the interaction between the receptor and its target DNA sequences known as glucocorticoid response elements (GREs) (10, 29). LBD is encoded by exons 5-9 and positions at the C-terminal end of the receptor corresponding to amino acids 531 to 777. This domain is structurally formed with 12 α-helices and four β-sheets, and contains two functional structures, the ligand-binding pocket (LBP) and the second transactivation surface called AF-2, as well as several other molecular platforms including the one responsible for nuclear translocation of the receptor (29, 30). Most of the protein surfaces of LBD that mediate these LBD-specific functions are formed upon binding of the receptor to a ligand and following conformational changes of this domain (15). Finally, HR lies between DBD and LBD, is encoded by 5’ part of exon 5, and spans between amino acids 495 and 530. This region provides appropriate structural flexibility to the receptor and allows the dimerized receptors to interact with different classic/alternative tandem GREs with various length of spacing nucleotides (the classic tandem GREs has three spacing nucleotides) (15).

MOLECULAR ACTIONS OF hGRα

Intracellular Shuttling of hGRα and its Regulators

At target cells, hGRα in the absence of glucocorticoids resides primarily in the cytoplasm as part of the hetero-oligomeric complex consisting of chaperone heat shock proteins (HSPs) 90, 70 and 50, immunophilins (e.g., FK506-binding protein (FKBP)), and possibly other proteins (31) (Figure 1). Binding of HSP90 to hGRα induces a conformational change in receptor’s LBD, and confers its ligand-friendly state, exposing the LBP to glucocorticoids and masking two nuclear localization signals (NLS), NL1 and NL2. Upon binding to a ligand, hGRα dissociates from the complex, exposes NL1 and NL2 to their counterpart molecular machinery, and translocates into the nucleus through the nuclear pore. NL1 harbors a classic NLS and is located between the C-terminal portion of DBD and the N-terminal part of HR (32). The function of NL1 is dependent on the importin α, a protein component of the nuclear pore-associated nuclear import system, which transports a liganded GRα as a cargo from the cytoplasm to the nucleus through the nuclear pore in an ATP-dependent fashion (33). NL2 spans over most of the LBD whose molecular mechanism(s) for supporting nuclear translocation of the receptor has(ve) not yet been elucidated (31, 34). Inside the nucleus, ligand-bound hGRα dimerizes and modulates transcription rates of glucocorticoid-responsive genes by associating with promoter/enhancer regions of their encoding genes (15) (Figure 1). The receptor subsequently liberates the ligand and is dissociated from its target genes and slowly translocates back to the cytoplasm with the molecular mechanisms described below (15). The ubiquitin-proteasomal pathway degrades some of the liganded hGRα in the nucleus, facilitating clearance of the receptor from GREs; thus this system negatively regulates the transcriptional activity of hGRα (35).

In addition to translocating into the nucleus, some liganded hGRαs migrate to the cytoplasmic membrane where they modulate the activity of cell surface receptors by associating with their intracellular signaling molecules, such as classic and small GTP-binding (G) proteins, and several serine/threonine and tyrosine kinases (36-38). The ligand-bound hGRα is also known to translocate into the mitochondria and to modulate the activity of this intracellular organelle (39). After modulating transcription rates of glucocorticoid-responsive genes in the nucleus, ligand-liberated hGRα is exported back to the cytoplasm and is re-incorporated into the HSP-containing multiprotein complex to function again as a ligand-binding competent receptor (31, 40) (Figure 1). Several mechanisms are postulated for mediating the GRα export from the nucleus to the cytoplasm. The Ca²⁺-binding protein calreticulin plays a role in this process, directly binding to DBD of the receptor (41-43). The chromosomal maintenance 1 (CRM1, also known as exportin 1)- and the classic nuclear export signal (NES)-mediated nuclear export machinery does not appear to function directly on hGRα (32, 42). Rather, NES-harboring and phospho-serine/threonine-binding proteins 14-3-3s can bind hGRα, and shift its intracellular localization toward the cytoplasm (44, 45). This action of 14-3-3s on hGRα appears to be independent to the ligand-induced nuclear translocation of the receptor, which is mediated in part by the NL1/importin α-associated nuclear pore complex. Numbers of serine and threonine residues of hGRα are phosphorylated by several serine/threonine kinases at their specific target residues, some of which function as phosphorylation-dependent binding sites of 14-3-3 proteins (46). For example, the v-akt murine thymoma viral oncogene homolog 1 (AKT1) (or the protein kinase B α) phosphorylates serine (S) 134 of the hGRα, and 14-3-3 binds to phosphorylated S134. Binding of 14-3-3 on hGRα at this site shifts subcellular localization of the latter to the cytoplasm and downregulates its transcriptional activity inside the nucleus (45, 47). The misshapen-like kinase 1 (MINK1) and the Rho-associated protein kinase (ROCK) respectively phosphorylate threonine (T) 524 and S617 (48). 14-3-3s bind phosphorylated forms of these residues as a dimer (48), possibly modulating subcellular localization and transcriptional activity of the hGRα.

Figure 1:

Intracellular circulation and actions of hGRα. hGRα resides in the cytoplasm in the absence of ligand by forming a heterocomplex with several heat shock proteins (HSPs), immunophilins (e.g., FKBP), and some other proteins. Upon binding to ligand cortisol, hGRα dissociates from the complex and translocates into the nucleus through the nuclear pore. Inside the nucleus, hGRα binds directly to glucocorticoid response elements (GREs) located in promoter/enhancer regions of glucocorticoid-responsive genes. DNA-bound hGRα then stimulates transcription rates of glucocorticoid-responsive genes by attracting the regulatory regions the transcription regulatory complex including the RNA polymerase II (RNPII) and its ancillary components through bridging coactivators, such as p300/CBP and p160 proteins. Promoter/enhancer-bound hGRα also recruits in collaboration with these coactivators various chromatin remodeling molecules, including the DRIP/TRAP complex (DRIP/TRAP), the SWI/SNF chromatin modulator (SWI/SNF), and the Mediator complex (MED). In addition to binding directly to DNA and regulating transcription, hGRα interacts indirectly with regulatory regions of glucocorticoid-responsive genes via protein-protein interaction with other transcription factors (TFs) and/or attracted cofactor molecules, ultimately modulating positively and negatively the transcriptional activity of GRE- and non-GRE-containing glucocorticoid-responsive genes. hGRα then moves back to the cytoplasm to re-form a heterocomplex with HSPs for regaining a ligand-friendly status or is cleared from DNA by proteasomal degradation. Further, hGRα can influence the action of cell surface receptors by associating with their intracellular signaling molecules, such as classic and small G-proteins, and several serine/threonine and tyrosine kinases (known as non-genomic actions of glucocorticoids). Accumulating evidence suggests that liganded hGRα also influences the transcription of mitochondrial genes by translocating into this intracellular organelle. CBP: cAMP-responsive element-binding protein (CREB)-binding protein; DRIP/TRAP: vitamin D receptor-interacting protein/thyroid hormone receptor-associated protein complex; FKBPs: FK506-binding proteins; GREs: glucocorticoid response elements; GR: glucocorticoid receptor; HSPs: heat shock proteins; MED: Mediator complex; p160: p160-type nuclear receptor coactivator; RNPII: RNA polymerase II; SWI/SNF: switching/sucrose non-fermenting complex; TFs: transcription factors; TREs: transcription factor response elements.

PRIMARY GENERALIZED GLUCOCORTICOID RESISTANCE SYNDROME

Pathophysiology and Clinical Manifestations

This syndrome is a condition first described by Chrousos, et.al., as a rare, familial or sporadic, genetic disorder characterized by generalized, partial target tissue insensitivity to glucocorticoids (80). Because of glucocorticoid insensitivity in the central components of the HPA axis, glucocorticoid-mediated negative feedback inhibition on the brain hypothalamus and the anterior pituitary gland is decreased (5, 81) (Figure 2). These changes result in compensatory elevation of the corticotropin-releasing hormone (CRH) and the arginine-vasopressin (AVP) at the hypothalamus and systemic release of the ACTH from the anterior pituitary gland. Excess ACTH secretion then causes bilateral adrenocortical hyperplasia and increased production/secretion of cortisol, which compensates for its reduced actions in target tissues. However, elevated circulating ACTH also stimulates production of other adrenal steroids, such as mineralocorticoids (e.g., deoxycorticosterone (DOC) and corticosterone) and/or adrenal androgens (e.g., androstenedione, dehydroepiandrosterone (DHEA), and DHEA-sulfate (DHEA-S)), leading to the development of excess manifestations of these hormones, because tissue sensitivity to these steroids is not altered. Increased mineralocorticoids may cause hypertension and/or hypokalemic alkalosis, whereas elevated adrenal androgens may develop manifestations (see below) through their direct effects on target tissues and/or indirect actions via modulation of the hypothalamic-pituitary-gonadal axis.

Figure 2.

Pathophysiologic mechanisms and clinical manifestations of primary generalized glucocorticoid resistance syndrome (PGGRS). The HPA axis consists of the brain hypothalamus, the anterior pituitary gland, and the adrenal cortex with their secreting hormones/peptides, CRH/AVP, ACTH and cortisol, respectively. In patients with this syndrome, their HPA axis is re-set to upward with preservation of circadian rhythmicity due to generalized, partial insensitivity to glucocorticoids in entire tissues. Thus, hypothalamic CRH/AVP, pituitary ACTH and adrenal cortisol are all hyper-secreted in order to compensate for the reduced actions of cortisol in both CNS and peripheral tissues. In addition to augmenting production of cortisol in the adrenal glands, elevated ACTH stimulates secretion of mineralocorticoids (e.g., deoxycorticosterone and corticosterone) and androgens (e.g., androstenedione, dehydroepiandrosterone (DHEA) and DHEA-sulfate(S)), which in turn cause a variety of manifestations associated with excess secretion of these hormones. In contrast, manifestations associated with overproduction of cortisol are rare in adult patients but neonates/young children may develop hypoglycemia and associated seizures due to reduced actions of cortisol in the liver. Elevated CRH/AVP in CNS may precipitate anxiety and depression in some patients. Solid lines indicate positive effects, whereas dashed lines show negative effects. Manifestations associated with elevation of the indicated molecules/compounds are shown with red letters. ACTH: adrenocorticotropic hormone; AVP: arginine vasopressin; CNS: central nervous system; CRH: corticotropin-releasing hormone; DHEA: dehydroepiandrosterone; DHEA-S: DHEA-sulfate; PGGRS; primary generalized glucocorticoid resistance syndrome.

Manifestations associated with excess adrenal androgens observed in patients with this syndrome include acne, hirsutism (more common in females), decreased fertility in both sexes, male-pattern hair loss, menstrual irregularities and oligo-anovulation in females, and oligospermia in males. Affected children may develop advanced bone age and subsequent short stature in their adulthood. In female new born babies, clitoromegaly/ambiguous genitalia may be seen (5, 82, 83).

Clinical manifestations of glucocorticoid deficiency are rare in adult patients but are reported in neonates/young children as severe hypoglycemia and associated seizures/coma, because gluconeogenesis depends on the proper action of glucocorticoids in the liver during early childhood (84-86). Some adult patients develop anxiety and/or chronic fatigue, which appear to be caused by elevated hypothalamic CRH and/or AVP (87-92). Increased circulating ACTH may cause bilateral adrenal hyperplasia (5). Some patients harbor adrenal incidentalomas (93, 94). Although this adrenal neoplasm is very common in general population (95), elevated circulating ACTH may facilitate tumor development and/or its growth. Further, one patient with this syndrome harbored an ACTH-producing pituitary adenoma, which might have been caused/facilitated by the elevated CRH/AVP (96).

Finally, the clinical spectrum of this syndrome is broad, ranging from severe to mild forms, and a number of patients may even be asymptomatic, displaying biochemical alterations only (5, 93, 97, 98). This heterogeneity is mainly due to variable impact of the patients’ genetic changes in the receptor protein, but other factors, such as their genetic backgrounds and/or epigenetic and biochemical changes, for example, associated with their ageing and lifestyles, may also contribute to variability of disease expression.

NR3C1 Gene Mutations That Cause Primary Generalized Glucocorticoid Resistance Syndrome

The molecular basis of this syndrome is ascribed to inactivating mutations in the NR3C1 gene, which impair molecular actions of hGRα and hence decrease tissue sensitivity to glucocorticoids. Currently, 36 pathologic mutations that cause this syndrome have been reported (Table 1 and Figure 3). Chrousos, et. al., reported the first family of this syndrome who carried a homozygous miss-sense mutation, which replaces adenine by thymine at nucleotide position 1,922 (80). The NR3C1 gene harboring this mutation expresses the hGRαD641V mutant receptor, which has valine (V) instead of aspartic acid (D) at amino acid position 641 in the LBD (80). Since then, numbers of patients were reported whose pathologic mutations were identified mostly as heterozygous in the coding sequence of LBD (90, 96, 99-102). Most of these patients demonstrated characteristic manifestations, such as those of mineralocorticoid and androgen excess, similar to the original case of Chrousos, et. al., thus they may be considered as “classic cases”. More recently, technological progress in the genome sequencing including the use of capillary or high through-put next generation sequencers enabled clinical researchers to conduct large studies with recruitment of the subjects with conventional/unconventional manifestations, (e.g., obesity and bilateral adrenal incidentalomas, as evident in the French Muta-GR study (ClinicalTrials.gov Identifier: NCT02810496) (97)). Clinicians are now able to obtain much easier and faster than before the data of patients’ genome sequence around the NR3C1 gene. Together with growing acknowledgement of this syndrome among clinicians and clinical researchers, such technological progress appears to have facilitated the discovery of new cases with classic symptoms, as well as those with much milder and/or alternative manifestations or even with biochemical changes only. Further, the identified mutations tend to distribute over the entire NR3C1 gene including coding areas of all three major domains and intronic sequences (Table 1 and Figure3).

Among 36 pathologic NR3C1 mutations, only three are homozygous mutations, while the other 33 are heterozygous (Table 1 and Figure 3). One patient harbors two different NR3C1 mutations each of which are identified in different alleles (thus, compound heterozygous) (86). Among 34 mutations found in the NR3C1 coding sequence, 24 are miss-sense mutations, which replace one amino acid with another (thus, point mutations), five are non-sense mutations, which introduce a stop codon and generate truncated receptor proteins, and another five are frame-shift mutations, which also develop truncated receptors but with additional unrelated amino acids after the mutation point. At the receptor protein level, 22 mutations are located in LBD, two in HR, seven in DBD, and four are in NTD (Figure 3). In addition to these coding sequence mutations, two mutations are identified in the intronic sequence, located in intron F (between exon 5 and 6) and in intron I (between exon 7 and 8), respectively (91, 103).

Table 1.

The NR3C1 Gene Mutations that Cause Primary Generalized Glucocorticoid Resistance Syndrome^†.

View in own window

Amino Acid Change	Nucleotide Change	Zygosity	Mutation Type	Proband’s Gender and Age	Clinical Manifestations	Molecular Defects	References
NTD Mutations
P9R	26C>G	Heterozygous	Point Mutation	M, 33	Hypertension	N.D.	(104)
Q123X	367G>T	Heterozygous	Point Mutation	F, 31	Fatigue, Anxiety, Hirsutism, Irregular menstruation, Infertility	N.D.	(87)
E198X	592G>T	Compound heterozygous with 2141G>A mutation	Point Mutation	F, 3	Hypoglycemia Hypertension	Also harbors R714Q expressed from a different allele	(86)
D401H†	1201G>T	Heterozygous	Point Mutation	F, 43	Hypertension Hyperglycemia	Increased transcriptional activity	(105)
DBD Mutations
V423A	1268T>C	Heterozygous	Point Mutation	M, 9	Fatigue Anxiety Hypertension	Decreased DNA-binding activity	(88)
R469X	1405C>T	Heterozygous	Point Mutation	M, 46	Adrenal hyperplasia Hypertension Hypokalemia	No GR mRNA and protein expression from the affected allele	(106)
R477C	1429C>T	Heterozygous	Point Mutation	F, 12	Mild hirsutism Elevated cortisol	N.D.	(92)
R477H	1430G>A	Heterozygous	Point Mutation	F, 41	Hypertension, Hirsutism, Fatigue	No DNA-binding activity	(107)
R477S	1429C>A	Heterozygous	Point Mutation	F, 30	Hypertension Elevated serum cortisol	No DNA-binding activity	(93)
Y478C	1433A>G	Heterozygous	Point Mutation	M, 49	Adrenal incidentaloma No symptoms	Decreased DNA-binding activity	(93)
HR Mutations
R491X	1471C>T	Heterozygous	Point Mutation	M, 44	Bilateral adrenal hyperplasia Elevation of ACTH and cortisol	Decreased transcriptional activity	(97)
Q501H	1503G>T	Heterozygous	Point Mutation	F, 60	No symptoms Mild elevation of urinary free cortisol	Decreased transcriptional activity	(97)
LBD Mutations
S551Y	1652C>A	Heterozygous	Point Mutation	M, 14	Fatigue Hypokalemia Hypertension Polyuria	Decreased affinity to ligand Decreased transcriptional activity	(108)
T556I	1667C>T	Heterozygous	Point Mutation	M, 56	Adrenal incidentaloma Increased UFC	N.D.	(94)
I559N	1676T>A	Heterozygous	Point Mutation	M, 33	Hypertension, Oligospermia, Infertility	No ligand-binding activity	(96, 99)
V571A	1724T>C	Heterozygous	Point Mutation	F, 9	Ambiguous genitalia*, Hypertension, Hypokalemic Alkalosis Hyperandrogenism	Highly decreased ligand-binding activity	(82, 100)
V575G	1724T>G	Heterozygous	Point Mutation	M, 70	Bilateral adrenal hyperplasia (His daughters have mild hirsutism)	Decreased affinity to ligand Decreased transcriptional activity	(98)
H588LfsX5	1762-1765insTTAC>G	Heterozygous	Frame Shift	F, 41	Hirsutism Anxiety Fatigue	N.D.	(92)
L595V	1915C>G	Heterozygous	Point Mutation	F, 16	No symptoms	Decreased affinity to ligand Decreased transcriptional activity	(98)
S612YfsX15	1835delC	Heterozygous	Frame Shift	F, 20	Fatigue Hirsutism	No ligand-binding activity	(109)
D641V	1922A>T	Homozygous	Point Mutation	M, 48	Hypertension, Hypokalemic alkalosis	Reduced affinity to ligand Reduced transcriptional activity	(80)
Y660X	1992A>T	Heterozygous	Point Mutation	F, 70	Hypokalemia Hypertension	No transcription activity	(110)
L672P	2015T>C	Heterozygous	Point Mutation	M, 46	No symptom Mild elevation of urinary free cortisol Adrenal incidentaloma	No ligand-binding activity No transcriptional activity	(93)
G679S	2035G>A	Heterozygous	Point Mutation	F, 19	Hirsutism Fatigue Hypertension	Decreased affinity to ligand Decreased transcriptional activity	(111)
R714Q	2141G>A	Heterozygous	Point Mutation	F, 2	Hypertension Mild clitoromegaly Advanced bone age Precocious puberty Hypokalemia	Decreased affinity to ligand Decreased transcriptional activity	(84)
R714Q	2141G>A	Heterozygous	Point Mutation	F, 31	Unsuccessful attempts for pregnancy for 2.5 years	Decreased affinity to ligand Decreased transcriptional activity	(112)
R714Q	2141G>A	Compound heterozygous with 592G>T mutation	Point Mutation	F, 3	Hypoglycemia Hypertension	Also harbors E198X expressed from the other allele	(86)
H726R	2177A>G	Heterozygous	Point Mutation	F, 30	Hirsutism Acne Alopecia Anxiety Fatigue Irregular menstrual cycles	Decreased affinity to ligand Decreased transcriptional activity	(89)
V729I	2185G>A	Homozygous	Point Mutation	M, 6	Precocious puberty Hyperandrogenism	Reduced affinity to ligand Reduced transcriptional activity	(101)
F737L	2209T>C	Heterozygous	Point Mutation	M, 7	Hypertension Hypokalemia	Decreased affinity to ligand Decreased transcriptional activity	(7)
I747M	2241T>G	Heterozygous	Point Mutation	F, 18	Hirsutism Oligo/amenorrhea	Decreased affinity to ligand Decreased transcriptional activity	(102)
I757V	2269A>G	Heterozygous	Point Mutation	F, 23	No symptoms	Decreased affinity to ligand Decreased transcriptional activity	(97)
L773P	2318T>C	Heterozygous	Point Mutation	F, 29	Hypertension Hirsutism Fatigue Anxiety	Decreased affinity to ligand Decreased transcriptional activity	(90)
L773VfsX25	2317-2318delCT	Heterozygous	Frame Shift	M, 27	Hypoglycemia Fatigability with feeding Hypertension	No ligand-binding activity	(113)
F774SfsX24	2318-2319delTG	Homozygous	Frame Shift	M, 1	Hypokalemia Hypoglycemia Hypertension	No ligand-binding activity	(85)
Intronic Mutations
NR (No protein expression)	1891-1894delGAGT	Heterozygous	Destruction of the splice donor site	F, 26	Hirsutism, Menstrual Irregularities	No GR mRNA and protein expression from the affected allele	(103)
N.D. Predicted to generate V675GfsX10	2024G > T	Heterozygous	Predicted to skip exon 8	F, 49	Hirsutism, Menstrual Irregularities, Anxiety	N.D.	(91)

*

: The case also harbors a heterozygous mutation in the 21-hydroxylase gene.

^†

: The 1201G>T D401H mutation causes mild glucocorticoid hypersensitivity.

N.D.; not determined。

Figure 3.

Location of the NR3C1 gene mutations that cause primary generalized glucocorticoid resistance syndrome^†. Currently, 36 independent mutations are reported. The mutations identified in the coding sequence of LBD, HR, DBD and NTD are shown in a light green, green, yellow and red box, respectively. Miss-sense mutations, non-sense mutations and frame-shift mutations are shown with black, purple and blue letters, respectively. Two mutations identified in the intronic sequence are shown with red letters. Homozygous mutations are shown with underlines. ^†: The 1201G>T D410H mutation causes mild glucocorticoid hypersensitivity; *: The same miss-sense mutation but found in unrelated subjects/families; ^$: Prediction only (the mutated hGRα protein was not biologically identified); ^#: These two mutations were found as compound heterozygous in one affected subject. Numbers of nucleotides and amino acids are based on the transcription initiation site and the first methionine of the hGRα protein, respectively. DBD: DNA-binding domain; HR: hinge region; LBD: ligand-binding domain; NTD: N-terminal domain.

CONCLUSIVE REMARKS AND FUTURE PERSPECTIVES

Primary generalized glucocorticoid resistance syndrome is characterized by hypercortisolism without Cushingoid features but with manifestations caused by upregulation of the HPA axis, such as hypertension (by mineralocorticoid excess) and signs of hyperandrogenism (by adrenal androgen excess) (81). The pathologic cause of this syndrome is ascribed to mutations in the NR3C1 gene, which decrease the action of its encoding protein hGRα, a ligand-dependent transcription factor (15, 81). In honor to Professor George P. Chrousos who discovered the first case and significantly contributed to the progress of this field, this syndrome may be called “Chrousos syndrome”, particularly for the cases who demonstrate classic and characteristic manifestations of this syndrome (6, 80). Recent progress in genome technology including high through-put sequencing has enabled clinical researchers to handle large patient cohorts and clinicians can get access to the NR3C1 gene sequencing much easier and faster than before. Consequently, 35 cases/families of this syndrome are currently reported world-wide who harbor pathologic mutations in the NR3C1 gene. It is of note that some of the recent cases tend to demonstrate much milder manifestations compared to the classic cases of Chrousos syndrome (97, 110). Further, some of them even lack obvious manifestations but show biochemical or imaging abnormalities only (93, 97). For these cases with very mild or no manifestations, their genetic changes may be considered as rare polymorphisms rather than pathologic mutations. Further discussion is needed for distinguishing pathologic mutations and mildly functional polymorphisms based on their clinical manifestations and allele frequency of the nucleotide changes.

In some reported cases, molecular defects of the mutated receptors were not evaluated. Testing them in tandem with the wild-type receptor is crucial for avoiding false-diagnosis, because the NR3C1 gene harbor substantial numbers of biologically silent polymorphisms (13). On the other hand, there are patients who demonstrate characteristic manifestations of Chrousos syndrome but do not harbor pathologic mutations in the NR3C1 gene. These “mutation-silent” subjects might carry their genetic defects not in NR3C1 but in other genes whose encoding proteins function in the glucocorticoid signaling pathway. For example, there was a boy who demonstrated manifestations compatible with multiple steroid hormone resistance (127). He harbored a small gene segmental deletion around one zinc finger protein (ZNF) gene, and Its encoding protein ZNF764 turned out to function as a coactivator of several steroid hormone receptors including the hGRα (127). As our knowledge of the glucocorticoid signaling pathway increases, including new players like long non-coding RNAs (15, 128, 129), we hope that genetic cause(s) of undiagnosed cases with Chrousos syndrome will soon be identified, by employing classic genetic methods (e.g., the linkage analysis) as well as cutting-edge genome-related methodologies including the whole genome/exome sequencing and sophisticated bioinformatical/statistical analysis tools.

REFERENCES

1.

Chrousos GP. Stress and disorders of the stress system. Nat Rev Endocrinology. 2009;5(7):374-381. [PubMed: 19488073]

2.

Chrousos GP, Charmandari E, and Kino T. Glucocorticoid action networks -an introduction to systems biology. J Clin Endocrinol Metab. 2004;89(2):563-564. [PubMed: 14764762]

3.

Kino T, De Martino MU, Charmandari E, Mirani M, and Chrousos GP. Tissue glucocorticoid resistance/hypersensitivity syndromes. J Steroid Biochem Mol Biol. 2003;85(2-5):457-467. [PubMed: 12943736]

4.

Chrousos GP. The hypothalamic-pituitary-adrenal axis and immune-mediated inflammation. N Engl J Med. 1995;332(20):1351-1362. [PubMed: 7715646]

5.

Charmandari E, Kino T, Ichijo T, and Chrousos GP. Generalized glucocorticoid resistance: clinical aspects, molecular mechanisms, and implications of a rare genetic disorder. J Clin Endocrinol Metab. 2008;93(5):1563-1572. [PMC free article: PMC2386273] [PubMed: 18319312]

6.

Charmandari E, and Kino T. Chrousos syndrome: a seminal report, a phylogenetic enigma and the clinical implications of glucocorticoid signalling changes. Eur J Clin Invest. 2010;40(10):932-942. [PMC free article: PMC2948853] [PubMed: 20649902]

7.

Charmandari E, Kino T, Ichijo T, Jubiz W, Mejia L, Zachman K, et al. A novel point mutation in helix 11 of the ligand-binding domain of the human glucocorticoid receptor gene causing generalized glucocorticoid resistance. J Clin Endocrinol Metab. 2007;92(10):3986-3990. [PubMed: 17635946]

8.

Kino T, and Chrousos GP. In: Steckler T, Kalin NH, and Reul JMHM eds. Handbook on Stress and the Brain. Amsterdam: Elsevier BV; 2005:295-312.

9.

Chrousos GP. The glucocorticoid receptor gene, longevity, and the complex disorders of Western societies. Am J Med. 2004;117(3):204-207. [PubMed: 15300973]

10.

Nicolaides NC, Galata Z, Kino T, Chrousos GP, and Charmandari E. The human glucocorticoid receptor: molecular basis of biologic function. Steroids. 2010;75(1):1-12. [PMC free article: PMC2813911] [PubMed: 19818358]

11.

Nuclear Receptors Nomenclature Committee. A unified nomenclature system for the nuclear receptor superfamily. Cell. 1999;97(2):161-163. [PubMed: 10219237]

12.

Germain P, Staels B, Dacquet C, Spedding M, and Laudet V. Overview of nomenclature of nuclear receptors. Pharmacol Rev. 2006;58(4):685-704. [PubMed: 17132848]

13.

Mackeh R, Marr AK, Dargham SR, Syed N, Fakhro KA, and Kino T. Single-nucleotide variations of the human nuclear hormone receptor genes in 60,000 individuals. J Endocr Soc. 2018;2(1):77-90. [PMC free article: PMC5779106] [PubMed: 29379896]

14.

Chrousos GP, and Kino T. Intracellular glucocorticoid signaling: a formerly simple system turns stochastic. Sci STKE. 2005;2005(304):pe48. [PubMed: 16204701]

15.

Nicolaides NC, Chrousos G, and Kino T. Glucocorticoid Receptor. In: Feingold KR, Anawalt B, Blackman MR, Boyce A, Chrousos G, Corpas E, et al. eds. Endotext. South Dartmouth (MA): MDText.com, Inc. Copyright © 2000-2024, MDText.com, Inc.; 2000.

16.

Bamberger CM, Bamberger AM, de Castro M, and Chrousos GP. Glucocorticoid receptor β, a potential endogenous inhibitor of glucocorticoid action in humans. J Clin Invest. 1995;95(6):2435-2441. [PMC free article: PMC295915] [PubMed: 7769088]

17.

Lewis-Tuffin LJ, and Cidlowski JA. The physiology of human glucocorticoid receptor β (hGRβ) and glucocorticoid resistance. Ann N Y Acad Sci. 2006;1069:1-9. [PubMed: 16855130]

18.

Oakley RH, Jewell CM, Yudt MR, Bofetiado DM, and Cidlowski JA. The dominant negative activity of the human glucocorticoid receptor β isoform. Specificity and mechanisms of action. J Biol Chem. 1999;274(39):27857-27866. [PubMed: 10488132]

19.

Kino T, Su YA, and Chrousos GP. Human glucocorticoid receptor isoform β: Recent understanding of its potential implications in physiology and pathophysiology. Cell Mol Life Sci. 2009;66(21):3435-3448. [PMC free article: PMC2796272] [PubMed: 19633971]

20.

Ramos-Ramírez P, and Tliba O. Glucocorticoid receptor β (GRβ): Beyond its dominant-negative function. Int J Mol Sci. 2021;22(7);3649. [PMC free article: PMC8036319] [PubMed: 33807481]

21.

Stechschulte LA, Wuescher L, Marino JS, Hill JW, Eng C, and Hinds TD, Jr. Glucocorticoid receptor β stimulates Akt1 growth pathway by attenuation of PTEN. J Biol Chem. 2014;289(25):17885-17894. [PMC free article: PMC4067219] [PubMed: 24817119]

22.

Yin Y, Zhang X, Li Z, Deng L, Jiao G, Zhang B, et al. Glucocorticoid receptor β regulates injury-mediated astrocyte activation and contributes to glioma pathogenesis via modulation of β-catenin/TCF transcriptional activity. Neurobiol Dis. 2013;59:165-176. [PubMed: 23906498]

23.

Wang Q, Lu PH, Shi ZF, Xu YJ, Xiang J, Wang YX, et al. Glucocorticoid receptor β acts as a co-activator of T-cell factor 4 and enhances glioma cell proliferation. Mol Neurobiol. 2015;52(3):1106-1118. [PubMed: 25301232]

24.

Lu NZ, and Cidlowski JA. Translational regulatory mechanisms generate N-terminal glucocorticoid receptor isoforms with unique transcriptional target genes. Mol Cell. 2005;18(3):331-342. [PubMed: 15866175]

25.

Presul E, Schmidt S, Kofler R, and Helmberg A. Identification, tissue expression, and glucocorticoid responsiveness of alternative first exons of the human glucocorticoid receptor. J Mol Endocrinol. 2007;38(1-2):79-90. [PubMed: 17242171]

26.

Turner JD, and Muller CP. Structure of the glucocorticoid receptor (NR3C1) gene 5' untranslated region: Identification, and tissue distribution of multiple new human exon 1. J Mol Endocrinol. 2005;35(2):283-292. [PubMed: 16216909]

27.

Sinclair D, Fullerton JM, Webster MJ, and Shannon Weickert C. Glucocorticoid receptor 1B and 1C mRNA transcript alterations in schizophrenia and bipolar disorder, and their possible regulation by GR gene variants. PLoS One. 2012;7(3):e31720. [PMC free article: PMC3302776] [PubMed: 22427805]

28.

Sinclair D, Webster MJ, Fullerton JM, and Weickert CS. Glucocorticoid receptor mRNA and protein isoform alterations in the orbitofrontal cortex in schizophrenia and bipolar disorder. BMC Psychiatry. 2012;12:84. [PMC free article: PMC3496870] [PubMed: 22812453]

29.

Frank F, Ortlund EA, and Liu X. Structural insights into glucocorticoid receptor function. Biochem Soc Trans. 2021;49(5):2333-2343. [PMC free article: PMC9274455] [PubMed: 34709368]

30.

Bledsoe RK, Montana VG, Stanley TB, Delves CJ, Apolito CJ, McKee DD, et al. Crystal structure of the glucocorticoid receptor ligand binding domain reveals a novel mode of receptor dimerization and coactivator recognition. Cell. 2002;110(1):93-105. [PubMed: 12151000]

31.

Pratt WB. The role of heat shock proteins in regulating the function, folding, and trafficking of the glucocorticoid receptor. J Biol Chem. 1993;268(29):21455-21458. [PubMed: 8407992]

32.

Savory JG, Hsu B, Laquian IR, Giffin W, Reich T, Haché RJ, et al. Discrimination between NL1- and NL2-mediated nuclear localization of the glucocorticoid receptor. Mol Cell Biol. 1999;19(2):1025-1037. [PMC free article: PMC116033] [PubMed: 9891038]

33.

Hoelz A, Debler EW, and Blobel G. The structure of the nuclear pore complex. Annu Rev Biochem. 2011;80:613-643. [PubMed: 21495847]

34.

Terry LJ, Shows EB, and Wente SR. Crossing the nuclear envelope: hierarchical regulation of nucleocytoplasmic transport. Science. 2007;318(5855):1412-1416. [PubMed: 18048681]

35.

Kinyamu HK, Chen J, and Archer TK. Linking the ubiquitin-proteasome pathway to chromatin remodeling/modification by nuclear receptors. J Mol Endocrinol. 2005;34(2):281-297. [PubMed: 15821097]

36.

Kino T, Tiulpakov A, Ichijo T, Chheng L, Kozasa T, and Chrousos GP. G protein β interacts with the glucocorticoid receptor and suppresses its transcriptional activity in the nucleus. J Cell Biol. 2005;169(6):885-896. [PMC free article: PMC2171637] [PubMed: 15955845]

37.

Kino T, Souvatzoglou E, Charmandari E, Ichijo T, Driggers P, Mayers C, et al. Rho family guanine nucleotide exchange factor Brx couples extracellular signals to the glucocorticoid signaling system. J Biol Chem. 2006;281(14):9118-9126. [PMC free article: PMC4152920] [PubMed: 16469733]

38.

Boldizsar F, Szabo M, Kvell K, Czompoly T, Talaber G, Bjorkan J, et al. ZAP-70 tyrosines 315 and 492 transmit non-genomic glucocorticoid (GC) effects in T cells. Mol Immunol. 2013;53(1-2):111-117. [PubMed: 22898186]

39.

Kokkinopoulou I, and Moutsatsou P. Mitochondrial glucocorticoid receptors and their actions. Int J Mol Sci. 2021;22(11):6054. [PMC free article: PMC8200016] [PubMed: 34205227]

40.

Haché RJ, Tse R, Reich T, Savory JG, and Lefebvre YA. Nucleocytoplasmic trafficking of steroid-free glucocorticoid receptor. J Biol Chem. 1999;274(3):1432-1439. [PubMed: 9880517]

41.

Black BE, Holaska JM, Rastinejad F, and Paschal BM. DNA binding domains in diverse nuclear receptors function as nuclear export signals. Curr Biol. 2001;11(22):1749-1758. [PubMed: 11719216]

42.

Holaska JM, Black BE, Love DC, Hanover JA, Leszyk J, and Paschal BM. Calreticulin is a receptor for nuclear export. J Cell Biol. 2001;152(1):127-140. [PMC free article: PMC2193655] [PubMed: 11149926]

43.

Holaska JM, Black BE, Rastinejad F, and Paschal BM. Ca²⁺-dependent nuclear export mediated by calreticulin. Mol Cell Biol. 2002;22(17):6286-6297. [PMC free article: PMC133999] [PubMed: 12167720]

44.

Kino T, Souvatzoglou E, De Martino MU, Tsopanomihalu M, Wan Y, and Chrousos GP. Protein 14-3-3σ interacts with and favors cytoplasmic subcellular localization of the glucocorticoid receptor, acting as a negative regulator of the glucocorticoid signaling pathway. J Biol Chem. 2003;278(28):25651-25656. [PubMed: 12730237]

45.

Habib T, Sadoun A, Nader N, Suzuki S, Liu W, Jithesh PV, et al. AKT1 has dual actions on the glucocorticoid receptor by cooperating with 14-3-3. Mol Cell Endocrinol. 2017;439:431-443. [PubMed: 27717743]

46.

Kino T. GR-regulating serine/threonine kinases: New physiologic and pathologic implications. Trends Endocrinol Metab. 2018;29(4):260-270. [PubMed: 29501228]

47.

Piovan E, Yu J, Tosello V, Herranz D, Ambesi-Impiombato A, Da Silva AC, et al. Direct reversal of glucocorticoid resistance by AKT inhibition in acute lymphoblastic leukemia. Cancer Cell. 2013;24(6):766-776. [PMC free article: PMC3878658] [PubMed: 24291004]

48.

Munier CC, De Maria L, Edman K, Gunnarsson A, Longo M, MacKintosh C, et al. Glucocorticoid receptor Thr524 phosphorylation by MINK1 induces interactions with 14-3-3 protein regulators. J Biol Chem. 2021;296:100551. [PMC free article: PMC8080530] [PubMed: 33744286]

49.

Ramamoorthy S, and Cidlowski JA. Exploring the molecular mechanisms of glucocorticoid receptor action from sensitivity to resistance. Endocr Dev. 2013;24:41-56. [PMC free article: PMC4770453] [PubMed: 23392094]

50.

Schaaf MJ, and Cidlowski JA. Molecular mechanisms of glucocorticoid action and resistance. J Steroid Biochem Mol Biol. 2002;83(1-5):37-48. [PubMed: 12650700]

51.

Beato M, and Sánchez-Pacheco A. Interaction of steroid hormone receptors with the transcription initiation complex. Endocr Rev. 1996;17(6):587-609. [PubMed: 8969970]

52.

McKenna NJ, and O'Malley BW. Combinatorial control of gene expression by nuclear receptors and coregulators. Cell. 2002;108(4):465-474. [PubMed: 11909518]

53.

Osz J, Brélivet Y, Peluso-Iltis C, Cura V, Eiler S, Ruff M, et al. Structural basis for a molecular allosteric control mechanism of cofactor binding to nuclear receptors. Proc Natl Acad Sci U S A. 2012;109(10):E588-594. [PMC free article: PMC3309777] [PubMed: 22355136]

54.

Bulynko YA, and O'Malley BW. Nuclear receptor coactivators: Structural and functional biochemistry. Biochemistry. 2011;50(3):313-328. [PMC free article: PMC3647688] [PubMed: 21141906]

55.

Mahajan MA, and Samuels HH. Nuclear hormone receptor coregulator: Role in hormone action, metabolism, growth, and development. Endocr Rev. 2005;26(4):583-597. [PubMed: 15561801]

56.

York B, and O'Malley BW. Steroid receptor coactivator (SRC) family: Masters of systems biology. J Biol Chem. 2010;285(50):38743-38750. [PMC free article: PMC2998129] [PubMed: 20956538]

57.

Heery DM, Kalkhoven E, Hoare S, and Parker MG. A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature. 1997;387(6634):733-736. [PubMed: 9192902]

58.

Darimont BD, Wagner RL, Apriletti JW, Stallcup MR, Kushner PJ, Baxter JD, et al. Structure and specificity of nuclear receptor-coactivator interactions. Genes Dev. 1998;12(21):3343-3356. [PMC free article: PMC317236] [PubMed: 9808622]

59.

Chinenov Y, Gupte R, Dobrovolna J, Flammer JR, Liu B, Michelassi FE, et al. Role of transcriptional coregulator GRIP1 in the anti-inflammatory actions of glucocorticoids. Proc Natl Acad Sci U S A. 2012;109(29):11776-11781. [PMC free article: PMC3406827] [PubMed: 22753499]

60.

Sheppard KA, Rose DW, Haque ZK, Kurokawa R, McInerney E, Westin S, et al. Transcriptional activation by NF-κB requires multiple coactivators. Mol Cell Biol. 1999;19(9):6367-6378. [PMC free article: PMC84607] [PubMed: 10454583]

61.

Richter WF, Nayak S, Iwasa J, and Taatjes DJ. The Mediator complex as a master regulator of transcription by RNA polymerase II. Nat Rev Mol Cell Biol. 2022;23(11):732-749. [PMC free article: PMC9207880] [PubMed: 35725906]

62.

Colley SM, and Leedman PJ. SRA and its binding partners: an expanding role for RNA-binding coregulators in nuclear receptor-mediated gene regulation. Crit Rev Biochem Mol Biol. 2009;44(1):25-33. [PubMed: 19280430]

63.

Colley SM, and Leedman PJ. Steroid Receptor RNA Activator - A nuclear receptor coregulator with multiple partners: Insights and challenges. Biochimie. 2011;93(11):1966-1972. [PubMed: 21807064]

64.

Kino T, Hurt DE, Ichijo T, Nader N, and Chrousos GP. Noncoding RNA gas5 is a growth arrest- and starvation-associated repressor of the glucocorticoid receptor. Sci Signal. 2010;3(107):ra8. [PMC free article: PMC2819218] [PubMed: 20124551]

65.

Gao Y, Liu C, Wu T, Liu R, Mao W, Gan X, et al. Current status and perspectives of non-coding RNA and phase separation interactions. Biosci Trends. 2022;16(5):330-345. [PubMed: 36273890]

66.

Johnson TA, Chereji RV, Stavreva DA, Morris SA, Hager GL, and Clark DJ. Conventional and pioneer modes of glucocorticoid receptor interaction with enhancer chromatin in vivo. Nucleic Acids Res. 2018;46(1):203-214. [PMC free article: PMC5758879] [PubMed: 29126175]

67.

Reichardt HM, Kaestner KH, Tuckermann J, Kretz O, Wessely O, Bock R, et al. DNA binding of the glucocorticoid receptor is not essential for survival. Cell. 1998;93(4):531-541. [PubMed: 9604929]

68.

Reichardt HM, Tuckermann JP, Göttlicher M, Vujic M, Weih F, Angel P, et al. Repression of inflammatory responses in the absence of DNA binding by the glucocorticoid receptor. EMBO J. 2001;20(24):7168-7173. [PMC free article: PMC125338] [PubMed: 11742993]

69.

Cruz-Topete D, and Cidlowski JA. One hormone, two actions: anti- and pro-inflammatory effects of glucocorticoids. Neuroimmunomodulation. 2015;22(1-2):20-32. [PMC free article: PMC4243162] [PubMed: 25227506]

70.

Oakley RH, and Cidlowski JA. The biology of the glucocorticoid receptor: new signaling mechanisms in health and disease. J Allergy Clin Immunol. 2013;132(5):1033-1044. [PMC free article: PMC4084612] [PubMed: 24084075]

71.

Hinz B, and Hirschelmann R. Rapid non-genomic feedback effects of glucocorticoids on CRF-induced ACTH secretion in rats. Pharm Res. 2000;17(10):1273-1277. [PubMed: 11145234]

72.

Karst H, Berger S, Turiault M, Tronche F, Schütz G, and Joëls M. Mineralocorticoid receptors are indispensable for nongenomic modulation of hippocampal glutamate transmission by corticosterone. Proc Natl Acad Sci U S A. 2005;102(52):19204-19207. [PMC free article: PMC1323174] [PubMed: 16361444]

73.

Hafezi-Moghadam A, Simoncini T, Yang Z, Limbourg FP, Plumier JC, Rebsamen MC, et al. Acute cardiovascular protective effects of corticosteroids are mediated by non-transcriptional activation of endothelial nitric oxide synthase. Nat Med. 2002;8(5):473-479. [PMC free article: PMC2668717] [PubMed: 11984591]

74.

Löwenberg M, Verhaar AP, Bilderbeek J, Marle J, Buttgereit F, Peppelenbosch MP, et al. Glucocorticoids cause rapid dissociation of a T-cell-receptor-associated protein complex containing LCK and FYN. EMBO Rep. 2006;7(10):1023-1029. [PMC free article: PMC1618362] [PubMed: 16888650]

75.

Ayroldi E, Cannarile L, Migliorati G, Nocentini G, Delfino DV, and Riccardi C. Mechanisms of the anti-inflammatory effects of glucocorticoids: Genomic and nongenomic interference with MAPK signaling pathways. FASEB J. 2012;26(12):4805-4820. [PubMed: 22954589]

76.

Demonacos C, Djordjevic-Markovic R, Tsawdaroglou N, and Sekeris CE. The mitochondrion as a primary site of action of glucocorticoids: the interaction of the glucocorticoid receptor with mitochondrial DNA sequences showing partial similarity to the nuclear glucocorticoid responsive elements. J Steroid Biochem Mol Biol. 1995;55(1):43-55. [PubMed: 7577720]

77.

Demonacos C, Tsawdaroglou NC, Djordjevic-Markovic R, Papalopoulou M, Galanopoulos V, Papadogeorgaki S, et al. Import of the glucocorticoid receptor into rat liver mitochondria in vivo and in vitro. J Steroid Biochem Mol Biol. 1993;46(3):401-413. [PubMed: 9831490]

78.

Psarra AM, and Sekeris CE. Glucocorticoids induce mitochondrial gene transcription in HepG2 cells: role of the mitochondrial glucocorticoid receptor. Biochim Biophys Acta. 2011;1813(10):1814-1821. [PubMed: 21664385]

79.

Lee SR, Kim HK, Song IS, Youm J, Dizon LA, Jeong SH, et al. Glucocorticoids and their receptors: insights into specific roles in mitochondria. Prog Biophys Mol Biol. 2013;112(1-2):44-54. [PubMed: 23603102]

80.

Chrousos GP, Vingerhoeds A, Brandon D, Eil C, Pugeat M, DeVroede M, et al. Primary cortisol resistance in man. A glucocorticoid receptor-mediated disease. J Clin Invest. 1982;69(6):1261-1269. [PMC free article: PMC370198] [PubMed: 6282933]

81.

Chrousos G. Q&A: primary generalized glucocorticoid resistance. BMC Med. 2011;9:27. [PMC free article: PMC3086527] [PubMed: 21429213]

82.

Mendonca BB, Leite MV, de Castro M, Kino T, Elias LL, Bachega TA, et al. Female pseudohermaphroditism caused by a novel homozygous missense mutation of the GR gene. J Clin Endocrinol Metab. 2002;87(4):1805-1809. [PubMed: 11932321]

83.

Nicolaides NC, and Charmandari E. Chrousos syndrome: from molecular pathogenesis to therapeutic management. Eur J Clin Invest. 2015;45(5):504-514. [PubMed: 25715669]

84.

Nader N, Bachrach BE, Hurt DE, Gajula S, Pittman A, Lescher R, et al. A novel point mutation in helix 10 of the human glucocorticoid receptor causes generalized glucocorticoid resistance by disrupting the structure of the ligand-binding domain. J Clin Endocrinol Metab. 2010;95(5):2281-2285. [PMC free article: PMC2869551] [PubMed: 20335448]

85.

McMahon SK, Pretorius CJ, Ungerer JP, Salmon NJ, Conwell LS, Pearen MA, et al. Neonatal complete generalized glucocorticoid resistance and growth hormone deficiency caused by a novel homozygous mutation in Helix 12 of the ligand binding domain of the glucocorticoid receptor gene (NR3C1). J Clin Endocrinol Metab. 2010;95(1):297-302. [PubMed: 19933394]

86.

Tatsi C, Xekouki P, Nioti O, Bachrach B, Belyavskaya E, Lyssikatos C, et al. A novel mutation in the glucocorticoid receptor gene as a cause of severe glucocorticoid resistance complicated by hypertensive encephalopathy. J Hypertens. 2019;37(7):1475-1481. [PMC free article: PMC10913058] [PubMed: 31145715]

87.

Paragliola RM, Costella A, Corsello A, Urbani A, and Concolino P. A novel pathogenic variant in the N-terminal domain of the glucocorticoid receptor, causing glucocorticoid resistance. Mol Diagn Ther. 2020;24(4):473-485. [PubMed: 32607951]

88.

Roberts ML, Kino T, Nicolaides NC, Hurt DE, Katsantoni E, Sertedaki A, et al. A novel point mutation in the DNA-binding domain (DBD) of the human glucocorticoid receptor causes primary generalized glucocorticoid resistance by disrupting the hydrophobic structure of its DBD. J Clin Endocrinol Metab. 2013;98(4):E790-795. [PMC free article: PMC3615201] [PubMed: 23426617]

89.

Nicolaides NC, Geer EB, Vlachakis D, Roberts ML, Psarra AM, Moutsatsou P, et al. A novel mutation of the hGR gene causing Chrousos syndrome. Eur J Clin Invest. 2015;45(8):782-791. [PubMed: 26031419]

90.

Charmandari E, Raji A, Kino T, Ichijo T, Tiulpakov A, Zachman K, et al. A novel point mutation in the ligand-binding domain (LBD) of the human glucocorticoid receptor (hGR) causing generalized glucocorticoid resistance: the importance of the C terminus of hGR LBD in conferring transactivational activity. J Clin Endocrinol Metab. 2005;90(6):3696-3705. [PubMed: 15769988]

91.

Mauri S, Nieto-Moragas J, Obón M, and Oriola J. The glucocorticoid resistance syndrome. Two cases of a novel pathogenic variant in the glucocorticoid receptor gene. JCEM Case Rep. 2024;2(1):luad153. [PMC free article: PMC10759794] [PubMed: 38170043]

92.

Velayos T, Grau G, Rica I, Pérez-Nanclares G, and Gaztambide S. Glucocorticoid resistance syndrome caused by two novel mutations in the NR3C1 gene. Endocrinol Nutr. 2016;63(7):369-371. [PubMed: 27211791]

93.

Vitellius G, Fagart J, Delemer B, Amazit L, Ramos N, Bouligand J, et al. Three novel heterozygous point mutations of NR3C1 causing glucocorticoid resistance. Hum Mutat. 2016;37(8):794-803. [PubMed: 27120390]

94.

Zhu HJ, Dai YF, Wang O, Li M, Lu L, Zhao WG, et al. Generalized glucocorticoid resistance accompanied with an adrenocortical adenoma and caused by a novel point mutation of human glucocorticoid receptor gene. Chin Med J (Engl). 2011;124(4):551-555. [PubMed: 21362280]

95.

Sherlock M, Scarsbrook A, Abbas A, Fraser S, Limumpornpetch P, Dineen R, et al. Adrenal Incidentaloma. Endocr Rev. 2020;41(6):775-820. [PMC free article: PMC7431180] [PubMed: 32266384]

96.

Karl M, Lamberts SW, Koper JW, Katz DA, Huizenga NE, Kino T, et al. Cushing's disease preceded by generalized glucocorticoid resistance: clinical consequences of a novel, dominant-negative glucocorticoid receptor mutation. Proc Assoc Am Physicians. 1996;108(4):296-307. [PubMed: 8863343]

97.

Vitellius G, Trabado S, Hoeffel C, Bouligand J, Bennet A, Castinetti F, et al. Significant prevalence of NR3C1 mutations in incidentally discovered bilateral adrenal hyperplasia: Results of the French MUTA-GR Study. Eur J Endocrinol. 2018;178(4):411-423. [PubMed: 29444898]

98.

Nicolaides NC, Roberts ML, Kino T, Braatvedt G, Hurt DE, Katsantoni E, et al. A novel point mutation of the human glucocorticoid receptor gene causes primary generalized glucocorticoid resistance through impaired interaction with the LXXLL motif of the p160 coactivators: dissociation of the transactivating and transreppressive activities. J Clin Endocrinol Metab. 2014;99(5):E902-907. [PMC free article: PMC4010692] [PubMed: 24483153]

99.

Kino T, Stauber RH, Resau JH, Pavlakis GN, and Chrousos GP. 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. 2001;86(11):5600-5608. [PubMed: 11701741]

100.

Charmandari E, Kino T, Souvatzoglou E, Vottero A, Bhattacharyya N, and Chrousos GP. Natural glucocorticoid receptor mutants causing generalized glucocorticoid resistance: molecular genotype, genetic transmission, and clinical phenotype. J Clin Endocrinol Metab. 2004;89(4):1939-1949. [PubMed: 15070967]

101.

Malchoff CD, Javier EC, Malchoff DM, Martin T, Rogol A, Brandon D, et al. Primary cortisol resistance presenting as isosexual precocity. J Clin Endocrinol Metab. 1990;70(2):503-507. [PubMed: 2105334]

102.

Vottero A, Kino T, Combe H, Lecomte P, and Chrousos GP. 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. 2002;87(6):2658-2667. [PubMed: 12050230]

103.

Karl M, Lamberts SW, Detera-Wadleigh SD, Encio IJ, Stratakis CA, Hurley DM, et al. Familial glucocorticoid resistance caused by a splice site deletion in the human glucocorticoid receptor gene. J Clin Endocrinol Metab. 1993;76(3):683-689. [PubMed: 8445027]

104.

Lin L, Wu X, Hou Y, Zheng F, and Xu R. A novel mutation in the glucocorticoid receptor gene causing resistant hypertension: A case report. Am J Hypertens. 2019;32(11):1126-1128. [PubMed: 31414133]

105.

Charmandari E, Ichijo T, Jubiz W, Baid S, Zachman K, Chrousos GP, et al. A novel point mutation in the amino terminal domain of the human glucocorticoid receptor (hGR) gene enhancing hGR-mediated gene expression. J Clin Endocrinol Metab. 2008;93(12):4963-4968. [PMC free article: PMC2626453] [PubMed: 18827003]

106.

Bouligand J, Delemer B, Hecart AC, Meduri G, Viengchareun S, Amazit L, et al. Familial glucocorticoid receptor haploinsufficiency by non-sense mediated mRNA decay, adrenal hyperplasia and apparent mineralocorticoid excess. PLoS One. 2010;5(10):e13563. [PMC free article: PMC2962642] [PubMed: 21042587]

107.

Ruiz M, Lind U, Gåfvels M, Eggertsen G, Carlstedt-Duke J, Nilsson L, et al. Characterization of two novel mutations in the glucocorticoid receptor gene in patients with primary cortisol resistance. Clin Endocrinol (Oxf). 2001;55(3):363-371. [PubMed: 11589680]

108.

Ma L, Tan X, Li J, Long Y, Xiao Z, De J, et al. A novel glucocorticoid receptor mutation in primary generalized glucocorticoid resistance disease. Endocr Pract. 2020;26(6):651-659. [PubMed: 32045292]

109.

Trebble P, Matthews L, Blaikley J, Wayte AW, Black GC, Wilton A, et al. Familial glucocorticoid resistance caused by a novel frameshift glucocorticoid receptor mutation. J Clin Endocrinol Metab. 2010;95(12):E490-499. [PMC free article: PMC4110505] [PubMed: 20861124]

110.

Vitellius G, Delemer B, Caron P, Chabre O, Bouligand J, Pussard E, et al. Impaired 11β-hydroxysteroid dehydrogenase type 2 in glucocorticoid-resistant patients. J Clin Endocrinol Metab. 2019;104(11):5205-5216. [PubMed: 31225872]

111.

Raef H, Baitei EY, Zou M, and Shi Y. Genotype-phenotype correlation in a family with primary cortisol resistance: possible modulating effect of the ER22/23EK polymorphism. Eur J Endocrinol. 2008;158(4):577-582. [PubMed: 18362306]

112.

Molnár Á, Patócs A, Likó I, Nyírő G, Rácz K, Tóth M, et al. An unexpected, mild phenotype of glucocorticoid resistance associated with glucocorticoid receptor gene mutation case report and review of the literature. BMC Med Genet. 2018;19(1):37. [PMC free article: PMC5840839] [PubMed: 29510671]

113.

Donner KM, Hiltunen TP, Jänne OA, Sane T, and Kontula K. Generalized glucocorticoid resistance caused by a novel two-nucleotide deletion in the hormone-binding domain of the glucocorticoid receptor gene NR3C1. Eur J Endocrinol. 2013;168(1):K9-k18. [PubMed: 23076843]

114.

Hurt DE, Suzuki S, Mayama T, Charmandari E, and Kino T. Structural analysis on the pathologic mutant glucocorticoid receptor ligand-binding domains. Mol Endocrinol. 2016;30(2):173-188. [PMC free article: PMC4792232] [PubMed: 26745667]

115.

Cole TJ, Blendy JA, Monaghan AP, Krieglstein K, Schmid W, Aguzzi A, et al. Targeted disruption of the glucocorticoid receptor gene blocks adrenergic chromaffin cell development and severely retards lung maturation. Genes Dev. 1995;9(13):1608-1621. [PubMed: 7628695]

116.

Nicolaides NC, and Charmandari E. Novel insights into the molecular mechanisms underlying generalized glucocorticoid resistance and hypersensitivity syndromes. Hormones (Athens). 2017;16(2):124-138. [PubMed: 28742501]

117.

Kino T, Liou SH, Charmandari E, and Chrousos GP. Glucocorticoid receptor mutants demonstrate increased motility inside the nucleus of living cells: time of fluorescence recovery after photobleaching (FRAP) is an integrated measure of receptor function. Mol Med. 2004;10(7-12):80-88. [PMC free article: PMC1431369] [PubMed: 16307173]

118.

Kaziales A, Rührnößl F, and Richter K. Glucocorticoid resistance conferring mutation in the C-terminus of GR alters the receptor conformational dynamics. Sci Rep. 2021;11(1):12515. [PMC free article: PMC8206104] [PubMed: 34131228]

119.

Dahlman-Wright K, Wright A, Gustafsson JA, and Carlstedt-Duke J. Interaction of the glucocorticoid receptor DNA-binding domain with DNA as a dimer is mediated by a short segment of five amino acids. J Biol Chem. 1991;266(5):3107-3112. [PubMed: 1993683]

120.

van Rossum EF, and Lamberts SW. Polymorphisms in the glucocorticoid receptor gene and their associations with metabolic parameters and body composition. Recent Prog Horm Res. 2004;59:333-357. [PubMed: 14749509]

121.

Vitellius G, Trabado S, Bouligand J, Delemer B, and Lombès M. Pathophysiology of glucocorticoid signaling. Ann Endocrinol (Paris). 2018;79(3):98-106. [PubMed: 29685454]

122.

Wester VL, Koper JW, van den Akker EL, Franco OH, Stolk RP, and van Rossum EF. Glucocorticoid receptor haplotype and metabolic syndrome: the Lifelines cohort study. Eur J Endocrinol. 2016;175(6):645-651. [PubMed: 27634941]

123.

Ferriman D, and Gallwey JD. Clinical assessment of body hair growth in women. J Clin Endocrinol Metab. 1961;21:1440-1447. [PubMed: 13892577]

124.

Marshall WA, and Tanner JM. Variations in pattern of pubertal changes in girls. Arch Dis Child. 1969;44(235):291-303. [PMC free article: PMC2020314] [PubMed: 5785179]

125.

Marshall WA, and Tanner JM. Variations in the pattern of pubertal changes in boys. Arch Dis Child. 1970;45(239):13-23. [PMC free article: PMC2020414] [PubMed: 5440182]

126.

Chrousos GP, Kino T, and Charmandari E. Evaluation of the hypothalamic-pituitary-adrenal axis function in childhood and adolescence. Neuroimmunomodulation. 2009;16(5):272-283. [PMC free article: PMC2790806] [PubMed: 19571588]

127.

Kino T, Pavlatou MG, Moraitis AG, Nemery RL, Raygada M, and Stratakis CA. ZNF764 haploinsufficiency may explain partial glucocorticoid, androgen, and thyroid hormone resistance associated with 16p11.2 microdeletion. J Clin Endocrinol Metab. 2012;97(8):E1557-1566. [PMC free article: PMC3410270] [PubMed: 22577170]

128.

Kadmiel M, and Cidlowski JA. Glucocorticoid receptor signaling in health and disease. Trends Pharmacol Sci. 2013;34(9):518-530. [PMC free article: PMC3951203] [PubMed: 23953592]

129.

Chrousos GP, and Kino T. Glucocorticoid action networks and complex psychiatric and/or somatic disorders. Stress (Amsterdam, Netherlands). 2007;10(2):213-219. [PubMed: 17514590]