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Female Pseudohermaphroditism Caused by a Novel Homozygous Missense Mutation of the GR Gene

Unidade de Endocrinologia do Desenvolvimento e Laboratório de Hormônios e Genética Molecular, Disciplina de Endocrinologia, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo (B.B.M., T.A.S.B., A.E.C.B., I.J.P.A., A.C.L.), and Departamento de Fisiologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo (M.C., L.L.K.E.), 05403-900 São Paulo, Brazil; Universidade Federal do Rio Grande do Norte (M.V.L.), 59022-020 Natal, Brazil; and Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health (T.K., G.P.C.), Bethesda, Maryland 20814

Address all correspondence and requests for reprints to: Berenice B. Mendonca, M.D., Hospital das Clínicas, Laboratório de Hormônios, Avenue Dr. Eneas de Carvalho Aguiar 155 PAMB, 2 andar Bloco 6, 05403-900, Sao Paulo Brazil. E-mail: . beremen{at}usp.br

Abstract

Familial glucocorticoid resistance is characterized by increased cortisol secretion without clinical evidence of hypercortisolism, but with manifestations of androgen and mineralocorticoid excess. This condition is mainly caused by mutations of the GR gene that cause inadequate transduction of the glucocorticoid signal in glucocorticoid target tissues. The clinical features of glucocorticoid resistance in females include hirsutism, acne, male pattern baldness, oligomenorrhea, and oligoanovulation. We describe here a new phenotype, female pseudohermaphroditism and severe hypokalemia, caused by a homozygous inactivating mutation of the GR gene. The proband was born with ambiguous genitalia from consanguineous parents and was mistreated as a 21-hydroxylase deficiency case since the age of 5 yr. She had very high levels of plasma ACTH (759 pg/ml or 167 pmol/liter) and high levels of cortisol (28–54 µg/dl or 772-1490 nmol/liter), androstenedione (5–14 ng/ml or 17–48 nmol/liter), T (174–235 ng/dl or 7–8 nmol/liter), and 17-hydroxyprogesterone (8–12 ng/ml or 24–36 nmol/liter). Her cortisol and 17-hydroxyprogesterone levels were not compatible with the diagnosis of classic congenital adrenal hyperplasia; furthermore, cortisol was not properly suppressed after dexamethasone administration (28 µg/d or 772 nmol/liter). Her laboratory evaluation indicated a diagnosis of glucocorticoid resistance. To investigate this puzzling clinical and biochemical picture, we analyzed both GR and CYP21 genes. Indeed, a homozygous T to C substitution at nucleotide 1844 in exon 5 of the GR gene was identified in the patient that caused a valine to alanine substitution at amino acid 571 in the ligand domain of the receptor. Her parents and an older sister were heterozygous for this mutation. A whole Epstein-Barr virus-transformed cell dexamethasone-binding assay revealed that this Ala⁵⁷¹ mutant had a 6-fold reduction in binding affinity compared with the wild-type receptor. In a functional assay using mouse mammary tumor virus promoter-driven luciferase reporter gene, the mutant receptor displayed 10- to 50-fold less trans-activation activity than the wild-type receptor. In addition, a large heterozygous CYP21 conversion was identified in the patient and her father.

In conclusion, we described the first case of female pseudohermaphroditism caused by a novel homozygous GR gene mutation. This phenotype indicates that pre- and postnatal virilization can occur in females with the glucocorticoid resistance syndrome.

GLUCOCORTICOID HORMONES exert their cellular actions through their cytoplasmic/nuclear receptor, which belongs to the nuclear receptor superfamily (1, 2, 3). Characterization of human GR cDNA was performed by Hollenberg et al. in 1985 (2). Later, two GR isoforms were identified from alternative splicing, a ligand-binding isoform, GR, and a dominant negative nonligand-binding isoform, GRß (4, 5). The GR isoform is a 777-amino acid protein with a molecular structure carrying multiple functional domains (1, 2, 3). Upon appropriate ligand binding, GR is liberated from associated chaperone proteins, its nuclear localization signals are exposed, and the ligand-bound molecule translocates into the nucleus, where it interacts with specific DNA sequences in the promoters of glucocorticoid-responsive genes and/or other transcription factors (2, 3).

Glucocorticoid resistance in humans is characterized by high levels of circulating cortisol without Cushing syndrome stigmata, resistance of the hypothalamic-pituitary-adrenal axis to dexamethasone suppression, normal diurnal rhythm of cortisol, and normal hypothalamic-pituitary-adrenal axis response to stress (6). Different GR abnormalities have been detected in affected families with generalized glucocorticoid resistance, including decreased ligand binding affinity and/or receptor concentration and increased thermolability (6). The molecular basis of glucocorticoid resistance in several kindreds has been ascribed to mutations in the GR, mainly in the ligand-binding domain (3, 6). The spectrum of clinical manifestations in patients with glucocorticoid resistance is quite broad. Most of these patients present no clinical manifestations of cortisol deficiency due to the compensation for glucocorticoid insensitivity by the elevated cortisol levels. However, excess ACTH secretion results in an increased production of adrenal steroidogenesis and intermediate steroids with salt-retaining and/or androgenic activity (6). The glucocorticoid resistance phenotype can include chronic fatigue and/or hypertension with or without hypokalemia in both sexes; females can also present hirsutism, acne, male-pattern baldness, oligomenorrhea, and oligoanovulation. Precocious puberty and infertility have been described in males (6). Biochemically, patients have elevated plasma and free urinary cortisol, corticosterone, and deoxycorticosterone levels and increased ACTH and androgen secretion with or without hypokalemia and/or metabolic alkalosis. We describe here a new phenotype, female pseudohermaphroditism and severe hypokalemia, caused by a novel homozygous mutation in the GR gene.

Case Report

This study was approved by the ethics committee of Hospital das Clinicas da Faculdade de Medicina da Universidade de Sao Paulo (Sao Paulo, Brazil).

A Brazilian Mulatto girl was born with ambiguous genitalia from consanguineous parents (second cousins). Her genitalia were characterized by an enlarged clitoris, posterior labial fusion, and a urogenital sinus (Fig. 1). She had been treated as a case of simple virilizing 21-hydroxylase deficiency, with cortisone acetate (20 mg/m²/d) since age 5 yr. She was referred to our hospital at 9 yr of age after a gastroenteritis episode associated with severe dehydration and acute renal failure. At that time she presented with no breast development, Tanner V stage pubic hair, and advanced bone age (14 yr). She had elevated blood pressure (150/100 mm Hg) and very low serum potassium levels (1.1–1.9 mmol/liter). She subsequently underwent clitoroplasty and opening of the urogenital sinus. A review of her previous laboratory data showed that she had very high levels of ACTH, cortisol, androstenedione, and T and slightly elevated levels of compound S (Table 1). She also had increased 17-hydroxyprogesterone levels, which, however, were not compatible with the diagnosis of classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Moreover, cortisol levels were not suppressed (28 µg/dl or 772 nmol/liter) after a dexamethasone test (2 mg/d for 4 d).

View larger version (184K): [in this window] [in a new window]   Figure 1. Patient’s external genitalia, showing clitoral enlargement, labial fusion, and a single perineal opening.

Materials and Methods

Laboratory evaluation

Serum androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, aldosterone, and cortisol were determined by commercial ¹²⁵I immunoassays. Serum T and compound S were determined by the method of Abraham (7) without prior chromatography after demonstrating that the antisera used were adequately specific (8). Plasma ACTH was measured by an immunoradiometric kit, and PRA was measured by RIA. Interassay variation was less than 14% for all hormones measured.

Molecular studies

After obtaining informed consent of parents, DNA samples were obtained from peripheral blood leukocytes by standard procedures.

The entire coding region of the GR gene were amplified by PCR and sequenced in an automatic sequencer (ABI Prism 310, Perkin-Elmer Corp.). The primers (Table 2) were designed using the exon/intron junction sequences described previously (5).

View this table: [in this window] [in a new window]   Table 2. Intronic primers used in the study of the GR gene

Trans-activation activity of GRV571A

pRShGR was a gift from Dr. R. M. Evans (The Salk Institute, La Jolla, CA). pMMTV-luc, which contains luciferase cDNA under control of the glucocorticoid-responsive mouse mammary tumor virus (MMTV) promoter, was a gift from Dr. G. L. Hager (NIH, Bethesda, MD). pSV40-ß-Gal was purchased from Promega Corp. (Madison, WI).

Mutagenesis

pRShGRV571A was constructed using a PCR site-directed mutagenesis kit (QuikChange Site-Directed Mutagenesis, Stratagene, La Jolla, CA) to replace the thymidine (T) residue at position 1844 of the normal hGR cDNA with a cytosine (C) residue. Sequencing of the full length of the hGR cDNA of the pRShGRV571A plasmid confirmed the presence of the mutation without any other base changes.

Cell culture and transfections

CV-1 cells were purchased from American Type Culture Collection (Manassas, VA), cultured in DMEM supplemented with 10% FBS, 50 µg/ml streptomycin, and 50 U/ml penicillin, and seeded into six-well plates 24 h before transfection. Cells were transfected by the lipofectin (Life Technologies, Inc., Gaithersburg, MD) method as previously described (11). Cells were cotransfected with 0.5 µg/well pMMTV-luc, 0.1 µg/well simian virus 40 promoter-ß-galactosidase, and 0.05 µg/well pRShGR or pRShGRV571A as previously described (4, 11, 12). Briefly, after 24 h the medium was replaced with either normal medium or medium containing graded concentrations of dexamethasone. Therefore, the cells were incubated for 24 h and harvested for the luciferase and ß-galactosidase assays. For the luciferase activity assay, the cells were washed twice with PBS and incubated for 20 min at 4 C in 350 µl assay buffer solution consisting of 25 mM Gly-Gly, 10 mM ATP, 25 mM MgSO₄, and 1% Triton X-100, pH 8.0 (Analytical Bioluminescence Laboratory, San Diego, CA). Cell lysates were centrifuged for 3 min at 14,000 rpm, and supernatants were analyzed for luciferase activity in a luminometer (LKB Wallac, Inc., Turku, Finland) using a commercially available assay system (Analytical Bioluminescence Laboratory, San Diego, CA). ß-Galactosidase activity was determined in the same sample using a galactosidase assay system (Promega Corp.). Luciferase values were divided by galactosidase values to normalize for variations in transfection efficiency. All experiments were performed in triplicate.

Whole cell dexamethasone binding assay

Transformation of B lymphocytes with Epstein-Barr virus (EBV) was performed as previously described (13, 14). We established transformed lymphocytes from the female proposita and from normal subjects. Cell viability was determined by trypan blue exclusion. The whole EBV-transformed cell dexamethasone-binding assay was performed as previously described (14). Cells were suspended in RPMI medium, adjusted to 1 x 10⁶ cells/tube, and incubated with six concentrations (1.56–50 nM) of [1,2,4,6,7-³H]dexamethasone (Amersham Pharmacia Biotech, Little Chalfont, UK) at 37 C in the presence or absence of a 1000-fold molar excess of unlabeled dexamethasone (Sigma, St. Louis, MO) for 1 h. After incubation, the cells were washed twice to separate bound from free steroid with 1.5 ml cold PBS and centrifuged at 400 x g for 10 min. After the second wash, the pellets were suspended in 100 µl medium and transferred to vials, and the radioactivity was counted in a ß-counter. Specific binding was calculated by subtracting nonspecific binding from total binding. Receptor assay data were analyzed by the method of Scatchard (15), using computerized regression analysis. The binding capacity was expressed as femtomoles of dexamethasone bound per 10⁶ cells, and the ligand affinity (K_d) was expressed in nanomolar concentrations.

Results

Molecular study

A single nucleotide substitution (TC) was identified in the GR gene (accession no. NM 000176) at position 1844 in exon 5, causing a nonconservative valine substitution by alanine at codon 571 in the hormone ligand-binding domain of GR (Fig. 2A). The patient was homozygous for this mutation, whereas her parents and an older sister were heterozygous (Fig. 2B). In addition, a large conversion in the heterozygous state of the CYP21 gene was identified in the patient and her father.

View larger version (36K): [in this window] [in a new window]   Figure 2. A, Automatic sequencing of exon 5 showed a homozygous TC substitution (arrow) converting an alanine into a valine in codon 571 in the ligand-binding domain of GR of the patient. B, Her unaffected sister was heterozygous for this mutation.

Transactivation activity of GR V571A

GR V571A dramatically lost its trans-activation activity on the MMTV promoter compared with the wild-type receptor. The concentration of dexamethasone required for half-maximal stimulation of luciferase activity by the wild-type GR was about 10^-8 M, whereas by the mutant receptor it was about 10^-6.5 M, indicating that the trans-activation activity of the mutant receptor was 10- to 50-fold less than that of the wild-type receptor (Fig. 3).

View larger version (18K): [in this window] [in a new window]   Figure 3. The mutant V571A weakly stimulates glucocorticoid-responsive MMTV promoter in CV-1 cells. , GR wild type; •, GR V571A. Values are the mean ± SE luciferase activity corrected for ß-galactosidase activity.

The linearity of the Scatchard plots indicates a single class of binding site affinity. The number of binding sites of GR in the patient’s cells was 23.4 fmol/10⁶ cells and was similar to that in the control cells (26.3 fmol/10⁶ cells). The GR apparent dissociation constant (K_d) of the patient’s cells was about 6-fold higher (6.9 nM) than that observed in the control (1.2 nM; Fig. 4).

View larger version (17K): [in this window] [in a new window]   Figure 4. Representative saturation curves of [3H]dexamethasone binding to EBV-transformed whole lymphocytes and the Scatchard plots of GR binding studies of the patient with glucocorticoid resistance and a normal subject.

Generalized inherited glucocorticoid resistance is a rare condition characterized by reduced sensitivity of target tissues to glucocorticoids. The presence of a defective receptor is compensated by increased ACTH and cortisol secretion at the expense of concurrent hypermineralocorticism and hyperandrogenism. The latter causes isosexual precocity, abnormal spermatogenesis, and infertility in males and hirsutism, mild virilization, acne, male-pattern baldness, menstrual irregularities, and disturbance of reproductive function in females (6, 16).

The present case is the first report of a girl with ambiguous genitalia and inherited glucocorticoid resistance. This patient had a novel homozygous missense mutation in exon 5 (TC), leading to a substitution of alanine into a valine in codon 571 in the ligand-binding domain of the GR. Her parents and an asymptomatic sister were heterozygous for this mutation. CV-1 cells transfected with the V571A mutation showed partial inactivation of GR trans-activation. V571 is located in helix 5 of the GR ligand-binding domain, which is important for the formation of the ligand-binding pocket. Therefore, one of the major causes of the decreased trans-activation of this mutant receptor must be the reduction of its affinity to the ligand. However, the trans-activation activity was much more affected than the ability to bind to the ligand, suggesting an additional ligand-independent defect involving the trans-activation function.

The inheritance of glucocorticoid resistance can be autosomal recessive or autosomal dominant. In this Brazilian family the pattern of inheritance is autosomal recessive, as heterozygous family members are normal. Besides that, the absence of clinical and biochemical abnormalities in these carriers suggests that the mutant receptor does not have a significant dominant negative activity upon the wild-type receptor as previously reported in other natural GR mutants (6).

To date, the molecular pathology has been elucidated in a few unrelated kindreds and individuals with glucocorticoid resistance (6, 17, 18, 19, 20, 21). All of the mutations described are located at the hormone-binding domain of the GR. In the first kindred with glucocorticoid resistance to have its molecular defects elucidated, the propositus, a severely affected 57-yr-old man, was homozygous for a nonconservative Asp⁶⁴¹ to Val substitution in the hormone-binding domain of the GR, leading to a significant reduction in the binding affinity of the GR (17). His mildly affected son and nephew were heterozygous for the same mutation. The second mutation, an IVS6DS 4-bp del, was identified in the heterozygous state in a female with hyperandrogenism and in her two asymptomatic brothers and father (18). The third mutation, a homozygous Val⁷²⁹Ile, was identified in a boy with isosexual pseudoprecocious puberty and led to a decreased affinity for glucocorticoids with normal maximum binding capacity; the boy’s asymptomatic mother was a carrier (19). The fourth mutation, an Ile⁵⁵⁹Asn substitution, which abolished ligand binding completely, was described in heterozygous state in a 33-yr-old man who presented with mild hypertension and infertility (20). This mutation was a de novo germline mutation and conferred a dominant negative activation upon the mutant receptor. The fifth mutation was a heterozygous Ile⁷⁴⁷Met substitution identified in a woman with hyperandrogenism, which diminished the affinity for ligand and its interaction with the p160 type of nuclear receptor coactivators (21).

It is noteworthy that the three cases, all males, bearing GR-inactivating mutations in a homozygous state were more severely affected. To date, only three females with GR mutations, all in the heterozygous state, have been reported (19, 20, 21). These females presented normal external genitalia and mild or moderate signs of hyperandrogenism after puberty. The patient described here was homozygous for a novel GR mutation and had a more severe phenotype characterized by pre- and postnatal virilization resulting in female pseudohermaphroditism and precocious pubarche.

Deficiency of 21-hydroxylase activity is one of the most commonly inherited genetic disorders in humans. Heterozygosity for CYP21 mutations has been estimated to affect approximately 1 in 60 of the general non-Jewish Caucasian population (22). This condition does not appear to increase the risk of clinical hyperandrogenism (23, 24). However, some carriers show higher mean baseline total and free T levels than controls (23). This Brazilian girl with glucocorticoid resistance was also heterozygous for a CYP21 macroconversion. The glucocorticoid resistance, certainly the main diagnosis in this case, represents a state of long-term ACTH stimulation of adrenals. We speculate whether the association of CYP21 and GR gene defects could have increased 17-hydroxyprogesterone and androgen levels in this girl. Therefore, the role of the association of haploinsufficiency for 21-hydroxylase with glucocorticoid resistance in causing this particularly severe phenotype remains debatable.

Acknowledgments

We thank Dr. Luisa Guilherme of the Laboratory of Transplant Immunology, Heart Institute-Incor, School of Medicine, University of Sao Paulo, for providing transformation of B lymphocytes with EBV, and the staff of the Laboratório de Hormônios e Genética Molecular LIM/42 for hormonal measurements and molecular studies. We also thank Sonia Strong for help with English usage.

Footnotes

This work was supported by FAPESP Grant 97/1196-1 and CNPq Grants 300151/96-9 (to A.C.L.) and 301246/95-5 (to B.B.M.).

Abbreviations: EBV, Epstein-Barr virus; MMTV, mouse mammary tumor virus.

Received July 16, 2001.

Accepted November 19, 2001.

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