Familial/Sporadic Glucocorticoid Resistance

Authors: Dr Evangelia Charmandari1, Dr Tomoshige Kino and Prof George P. Chrousos Creation date: February 2004

Scientific editor: Prof Sebastiano Filetti

1Pediatric and Reproductive Endocrinology Branch, National Institutes of Health, Building 10, Room 9D42, 10 Center Drive MSC 1583, Bethesda, MD 20892-1583, U.S.A. [email protected]

Abstract Keywords Introduction Background : Molecular Mechanisms of Glucocorticoid Action Definition Clinical Manifestations Diagnosis Differential diagnosis Treatment Etiology Conclusion References

Abstract Glucocorticoids regulate a variety of biologic processes and exert profound influences on many physiologic functions. Their actions are mediated by the glucocorticoid receptor (GR), a ligand-dependent transcription factor. Glucocorticoid resistance is a rare condition characterized by generalized, partial, target tissue resistance to glucocorticoids. Compensatory elevations in circulating adrenocorticotropic hormone (ACTH) concentrations lead to increased production of adrenal steroids with mineralocorticoid and/or androgenic activity, and increased urinary free cortisol excretion without any clinical evidence of hypercortisolism. The clinical spectrum of the condition is broad, ranging from asymptomatic to severe cases of hyperandrogenism, fatigue and/or mineralocorticoid excess. The molecular basis of glucocorticoid resistance has been ascribed to mutations in the GR gene, which impair glucocorticoid signal transduction and alter tissue sensitivity to glucocorticoids. The study of functional defects of natural hGR mutants enhances our understanding of the molecular mechanisms of hGR action and highlights the importance of integrated cellular and molecular signalling mechanisms for maintaining homeostasis and preserving normal physiology.

Keywords Glucocorticoid receptor (GR); Glucocorticoid resistance; Tissue sensitivity to glucocorticoids; Mutations in the hGR gene

GR action, and the clinical manifestations and Introduction molecular mechanisms of familial/sporadic Glucocorticoids are steroid hormones glucocorticoid resistance. synthesized and secreted by the adrenal cortex. They regulate a variety of physiologic functions Background : Molecular Mechanisms of and play an important role in maintaining basal Glucocorticoid Action and stress-related homeostasis (1-4). At the cellular level, the actions of glucocorticoids are Definition mediated by the glucocorticoid receptor (GR), The clinical syndrome of familial/ sporadic which belongs to the nuclear receptor family of glucocorticoid resistance is characterized by ligand-dependent transcription factors. The increased cortisol secretion without clinical present review focuses on the mechanisms of evidence of hyper- or hypocortisolism, and

Charmandari E, Dr Kino T, Chrousos GP. Familial/Sporadic Glucocorticoid Resistance. Orphanet encyclopedia. February 2004:.http://www.orpha.net/data/patho/GB/uk-glucocorticoidresistance.pdf 1

manifestations of androgen and/or Table 1: Clinical Manifestations and mineralocorticoid excess i.e absence of stigmata Diagnostic Evaluation of Syndromes of of Cushing's syndrome. This condition results Glucocorticoid Resistance from partial failure of the glucocorticoid receptor (GR) to modulate transcription of its target genes. Clinical Presentation Apparently normal glucocorticoid function Clinical Manifestations Asymptomatic Glucocorticoid resistance is a rare, familial or Chronic fatigue (glucocorticoid deficiency?) sporadic condition characterized by generalized, Mineralocorticoid excess partial end-organ insensitivity to glucocorticoids Hypertension (5-12). Affected subjects have compensatory Hypokalemic alkalosis elevations in circulating cortisol and Androgen excess adrenocorticotropic hormone (ACTH) Females: Ambiguous genitalia at birth, acne, concentrations, but no clinical evidence of hirsutism, male-pattern hair loss, menstrual hypercortisolism. However, the excess ACTH irregularities, oligo-anovulation, infertility, secretion results in increased production of precocious puberty adrenal steroids with mineralocorticoid activity, Males: Acne, hirsutism, oligospermia, infertility, such as cortisol, deoxycorticosterone and precocious puberty corticosterone, and/or androgenic activity, such as androstenedione, dehydroepiandrosterone Diagnostic Evaluation (DHEA) and DHEA-sulfate (DHEAS) (5-12). The Absence of clinical features of Cushing former accounts for symptoms and signs of syndrome mineralocorticoid excess, such as hypertension Normal or elevated plasma ACTH and/or hypokalemic alkalosis. The latter concentrations accounts for manifestations of androgen excess, Elevated plasma cortisol concentrations, such as acne, hirsutism and infertility in both increased 24-hour urinary free cortisol excretion sexes, male-pattern hair-loss, menstrual Maintenance of a normal circadian and stress- irregularities (oligo-amenorrhea) and oligo- induced pattern of cortisol and ACTH secretion anovulation in females, and oligospermia and Resistance of the HPA axis* to dexamethasone infertility in males. In children, the early and suppression excessive prepubertal adrenal androgen Thymidine incorporation assays secretion has been associated with ambiguous Dexamethasone binding assays genitalia at birth and precocious puberty. The Molecular studies clinical spectrum of the disease is broad, ranging * HPA =Hypothalamic-pituitary-adrenal from completely asymptomatic to severe cases Adapted from: Chrousos GP, Detera-Wadleigh SD, Karl M. of hyperandrogenism, fatigue and/or 1993. Syndromes of glucocorticoid resistance. Ann Intern Med. 119(11): 1113-24. mineralocorticoid excess (7-12) (Table 1). Fatigue has been considered a result of cortisol Differential diagnosis deficiency in tissues such as the central nervous The differential diagnosis includes: system and the skeletal muscles. 1- mild forms of Cushing's syndrome, in which hypercortisolism is accompanied by normal or Diagnosis mildly elevated ACTH concentrations, preserved Increased serum cortisol concentrations and 24- circadian pattern of ACTH and cortisol secretion, hour urinary free cortisol excretion in the and lack of cortisol suppression by absence of clinical features of hypercortisolism dexamethasone; are highly suggestive of the condition. The 2- pseudocushing’s states, such as generalized plasma concentrations of ACTH may be normal anxiety disorder and melancholic depression; or high. The circadian pattern of ACTH and 3- conditions associated with elevated serum cortisol secretion and their responsiveness to concentrations of cortisol-binding globulin; stressors are preserved, albeit at higher 4- other causes of mineralocorticoid-induced concentrations, and there is resistance of the hypertension; HPA axis to dexamethasone suppression. 5- other causes of hyperandrogenism or Thymidine incorporation and dexamethasone virilization, such as idiopathic hirsutism, binding assays on peripheral blood mononuclear polycystic ovarian syndrome and congenital cells or cultured skin fibroblasts, as well as adrenal hyperplasia. sequencing of genomic DNA or complementary DNA may be necessary to confirm the diagnosis Treatment (Table 1). The aim of treatment in glucocorticoid resistance is to suppress the excessive secretion of ACTH and, therefore, the increased production of

Charmandari E, Dr Kino T, Chrousos GP. Familial/Sporadic Glucocorticoid Resistance. Orphanet encyclopedia. February 2004:.http://www.orpha.net/data/patho/GB/uk-glucocorticoidresistance.pdf 2

mineralocorticoids and androgens from the removed a donor splice site in one allele, adrenal cortex. Treatment involves affecting the last two bases of the exon 6 and administration of high doses of the first two bases of the intron 6. This resulted mineralocorticoid-sparing synthetic in complete ablation of the expression of one of glucocorticoids, such as dexamethasone (1–3 the hGRα alleles and a decrease in GRα protein mg/day), which activate the mutated and/or wild- by 50% in affected members of the family (16). type hGRα, and suppress the endogenous The propositus of the third kindred was secretion of ACTH (7-12). Adequate suppression homozygous for a point mutation at nucleotide of the HPA axis is of particular importance in position 2317, which results in substitution of cases of severe impairment of hGRα function, valine for isoleucine at amino acid 729 of the because long-standing corticotroph ligand-binding domain of hGRα (17). This hyperstimulation in association with decreased mutation resulted in decreased transcriptional glucocorticoid negative feedback may lead to the activity of the receptor and a four-fold reduction development of an ACTH-secreting adenoma in the affinity for dexamethasone (17). The (13). Long-term dexamethasone treatment mutant receptor was localized primarily in the should be carefully titrated based on the clinical nucleus of cells in the absence of ligand, while manifestations and biochemical profile (5). further translocation from the cytoplasm into the Asymptomatic, normotensive subjects with nucleus required longer (120 min) exposure to primary glucocorticoid resistance do not require dexamethasone (10-6 M), and demonstrated a any treatment. weak, ligand-dependent interaction with the full- length and carboxyl-terminal fragment but not Etiology with the amino-terminal fragment of GRIP1 in Molecular Mechanisms of Glucocorticoid vitro (22). Resistance The first sporadic case of glucocorticoid The molecular basis of glucocorticoid resistance resistance was due to a de novo, germ-line, has been ascribed to mutations in the hGRα heterozygous mutation at nucleotide position gene, which impair one or more of the molecular 1808, resulting in substitution of isoleucine for mechanisms of glucocorticoid receptor function, asparagine at amino acid 559 in the hormone- altering tissue sensitivity to glucocorticoids. binding domain of hGRα. This mutation reduced Abnormalities of several hGRα characteristics, the transcriptional activity of hGRα significantly such as cell concentration, affinity for ligand and and was associated with the development of an translocation into the nucleus, have been ACTH-secreting adenoma (13). Although the associated with this condition (5,14-22). The affinity for ligand was preserved in the patient molecular defects elucidated in the reported studied, there was a 50% decrease in the hGR cases are summarized in Table 2, while the binding sites (13). Furthermore, the mutant corresponding mutations in the hGRα gene are receptor had a markedly delayed nuclear shown in both Table 2 and Figure 5. translocation (180 min) and a dominant negative The propositus of the original kindred was activity upon the wild-type receptor, i.e. it homozygous for a single point mutation at decreased the transcriptional activity of hGRα in nucleotide position 2054, which resulted in a a dose-dependent manner (19). The latter may nonconservative amino acid substitution at account for manifestation of the disease at the position 641, replacing aspartic acid with valine heterozygotic state. There was no interaction (15). Compared to the wild-type receptor, this between the mutant receptor and the p160 mutant receptor exerted decreased coactivator GRIP1 (22). transactivation effects on the glucocorticoid- The fifth and sixth cases of glucocorticoid responsive mouse mammary tumor virus resistance were due to heterozygous mutations (MMTV) promoter and had a three-fold reduction at nucleotide positions 1430 and 2035, resulting, in the affinity for dexamethasone (15). In the respectively, in substitution of arginine for absence of ligand, the mutant receptor was histidine at amino acid 477 and glycine for serine primarily localized in the cytoplasm of cells. at amino acid 679 (18). The former mutation is -6 Exposure to dexamethasone (10 M) induced a located in the second zinc finger of the DNA- slow translocation into the nucleus, which took binding domain. This mutant receptor had no 22 min as opposed to the 12 min required for transactivation activity due to impaired binding to nuclear translocation of the wild-type receptor GREs but had the same affinity for ligand as the (22). Finally, the mutant receptor interacted with wild-type receptor. The latter mutation is located the amino-terminal but not with the carboxyl- in the ligand-binding domain, outside the ligand- terminal fragment or full-length GRIP1 in vitro binding pocket, and resulted in a 50% reduction (22). both in the transcriptional activity and the ligand- The proposita of the second family was binding affinity of the receptor (18). heterozygous for a 4-base deletion at the 3’- The proposita of the seventh case was boundary of exon and intron 6. The deletion homozygous for a point mutation at nucleotide

Charmandari E, Dr Kino T, Chrousos GP. Familial/Sporadic Glucocorticoid Resistance. Orphanet encyclopedia. February 2004:.http://www.orpha.net/data/patho/GB/uk-glucocorticoidresistance.pdf 3

position 1844, which results in a valine to alanine caused up to 50-fold decrease in the substitution at amino acid 571 in the ligand- transcriptional activity of the receptor and a six- binding domain of hGRα (20). This mutation fold reduction in the affinity for ligand (20). The caused up to 50-fold decrease in the nuclear translocation of the mutant receptor was transcriptional activity of the receptor and a six- delayed (25 min), while its interaction with the fold reduction in the affinity for ligand (20). The GRIP1 coactivator occurred mostly via its AF-1 nuclear translocation of the mutant receptor was domain (22). delayed (25 min), while its interaction with the The eighth case of glucocorticoid resistance was GRIP1 coactivator occurred mostly via its AF-1 due to a heterozygous mutation at nucleotide domain (22). position 2373, which causes an isoleucine to methionine substitution at amino acid 747 in the ligand-binding domain of the receptor (21). This Figure 5: Location of the known mutations of the mutation is located at the carboxyl-terminus of human glucocorticoid receptor gene (upper the ligand-binding domain, close to helix 12, panel) and protein (lower panel). which plays a pivotal role in the formation of AF- 2, a domain that interacts with p160 and other coactivators. The mutant receptor had a 20- to 30-fold decrease in the transactivation of the MMTV promoter, two-fold reduction in the affinity for dexamethasone and delayed nuclear translocation. It also exerted a dominant negative effect upon the wild-type hGRα and interacted with the GRIP1 coactivator in vitro only through its intact AF-1 domain. Overexpression of GRIP1 restored the transcriptional activity and reversed the dominant negative activity of the mutant upon the wild-type receptor (21). We have demonstrated that the mutant receptors hGRαI559N, hGRαV571A, hGRαD641V, hGRαV729I and hGRαI747M preserve their ability to bind to DNA, and that their ligand-binding domains have decreased intrinsic transcriptional activity (22). Therefore, The latter may account for manifestation of the disease at the heterozygotic state. There was no the process through which hGRα mutant interaction between the mutant receptor and the receptors studied thus far impair the physiologic p160 coactivator GRIP1 (22). mechanisms of glucocorticoid action at the The fifth and sixth cases of glucocorticoid molecular level is multifactorial, and involves resistance were due to heterozygous mutations impaired ability to bind ligand, aberrant at nucleotide positions 1430 and 2035, resulting, nucleocytoplasmic trafficking, and abnormal respectively, in substitution of arginine for interaction with the p160 coactivators. These histidine at amino acid 477 and glycine for serine variable functional defects of the mutant at amino acid 679 (18). The former mutation is receptors upon the glucocorticoid signaling located in the second zinc finger of the DNA- pathway may explain the genetic transmission binding domain. This mutant receptor had no and the variable clinical phenotype of transactivation activity due to impaired binding to glucocorticoid resistance. GREs but had the same affinity for ligand as the In addition to mutations in the hGRα gene, wild-type receptor. The latter mutation is located steroid receptor coactivator defects may also in the ligand-binding domain, outside the ligand- account for generalized glucocorticoid resistance binding pocket, and resulted in a 50% reduction and/or resistance to other steroid hormones (23- both in the transcriptional activity and the ligand- 24). Affected subjects present with the clinical binding affinity of the receptor (18). and biochemical manifestations of glucocorticoid The proposita of the seventh case was resistance, however, no defect of the hGR gene homozygous for a point mutation at nucleotide has been identified. position 1844, which results in a valine to alanine substitution at amino acid 571 in the ligand- binding domain of hGRα (20). This mutation

Charmandari E, Dr Kino T, Chrousos GP. Familial/Sporadic Glucocorticoid Resistance. Orphanet encyclopedia. February 2004:.http://www.orpha.net/data/patho/GB/uk-glucocorticoidresistance.pdf 4

Table 2: Mutations of the human glucocorticoid receptor gene causing glucocorticoid resistance

Author cDNA Amino Biochemical Present Study Genotype Transmission Phenotype (Reference) acid Phenotype Chrousos et 2054 641 Affinity for ligand↓ Transcriptional Homozygous Autosomal Hypertension al. (5) (A→T) (D→V) Transactivation ↓ activity of LBD ↓ Recessive Hypokalemic Hurley et al. Transdominance(- alkalosis (15) ) Nuclear translocation↓ DNA binding (+) Abnormal interaction with GRIP1 Karl et al. 4 bp hGRα number: Heterozygous Autosomal Hirsutism (16) deletion 50% of control Dominant Male-pattern hair- in Inactivation of the loss exon- affected allele Menstrual intron 6 irregularities Malchoff et 2317 729 Affinity for ligand↓ Transcriptional Homozygous Autosomal Precocious al. (17) (G→A) (V→I) Transactivation↓ activity of LBD↓ Recessive puberty Transdominance(- Hyperandrogenism ) Nuclear translocation↓ DNA binding (+) Abnormal interaction with GRIP1 Karl et al. 1808 559 Affinity for ligand↓ Transcriptional Heterozygous Sporadic Hypertension (13) (T→A) (I→N) Transactivation↓ activity of LBD ↓ Oligospermia, Kino et al. Trandominance(+) DNA binding (+) Infertility (19) Nuclear Abnormal Hyperandrogenism translocation↓ interaction with Hypokalemia GRIP1 Mendonca 1844 571 Affinity for ligand↓ Transcriptional Homozygous Autosomal Ambiguous et al. (20) (C→T) (V→A) Transactivation activity of LBD↓ Recessive genitalia at birth Transdominance(- DNA binding (+) Hypertension )Nuclear Abnormal translocation↓ interaction with GRIP1 Vottero et 2373 747 Affinity for ligand↓ Transcriptional Heterozygous Autosomal Cystic acne, al. (21) (T→G) (I→M) Transactivation↓ activity of LBD ↓ Dominant Hirsutism, Trandominance(+) DNA binding (+) amenorrhoea Nuclear translocation↓ Ruiz et al. 1430 477 Transactivation ↓ Heterozygous Sporadic Hirsutism, Fatigue, (19) (G→A) (R→H) Hypertension

2035 679 Affinity for ligand (G→A) (G→S) ↓ Heterozygous Sporadic Hirsutism, Fatigue, Transactivation ↓ Hypertension Adapted from: Charmandari E, Kino T, Vottero A, Souvatzoglou E, Bhattacharyya N, Chrousos GP. 2004. Natural glucocorticoid receptor mutants causing generalized glucocorticoid resistance: Molecular genotype, genetic transmission and clinical phenotype. J Clin Endocrinol Metab. (in press) ligand-binding, nuclear translocation, DNA binding and interaction with coactivators, and Conclusion highlights the importance of integrated cellular Mutations in the human glucocorticoid receptor and molecular signaling mechanisms for gene impair one or more of the molecular maintaining homeostasis and preserving normal mechanisms of glucocorticoid action, affecting physiology. glucocorticoid signal transduction and altering tissue sensitivity to these hormones. A References subsequent increase in the activity of the HPA 1- Tsai MJ, O'Malley BW. 1994. Molecular axis compensates for the reduced sensitivity of mechanisms of action of steroid/thyroid receptor peripheral tissues to glucocorticoids at the superfamily members. Annu Rev Biochem. 63: expense, however, of ACTH hypersecretion- 451-486. related pathology. The study of the functional 2- Simpson ER, Waterman MR. 1995. Steroid defects of natural hGR mutants sheds light to the biosynthesis in the adrenal cortex and its molecular mechanisms of hGR action, including regulation by adrenocorticotropin. In: DeGroot hGR-mediated transactivation of target genes, LJ, Besser M, Burger HG, Jameson JL, Loriaux

Charmandari E, Dr Kino T, Chrousos GP. Familial/Sporadic Glucocorticoid Resistance. Orphanet encyclopedia. February 2004:.http://www.orpha.net/data/patho/GB/uk-glucocorticoidresistance.pdf 5

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