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Glucocorticoid Resistance in Premature Pubarche and Adolescent Hyperandrogenism
Molecular Genetics and Metabolism 66, 137–141 (1999) Article ID mgme.1998.2796, available online at http://www.idealibrary.com on
BRIEF COMMUNICATION Glucocorticoid Resistance in Premature Pubarche and Adolescent Hyperandrogenism 1 occurs. Clinical manifestations, if conspicuous, include hirsutism, irregular menses, hypertension, and hypokalemia. Inheritance is autosomal dominant, but the phenotype can vary even within the same family (3,4). Mutations in the glucocorticoid receptor have been identified in several families with generalized glucocorticoid resistance (5,6). To determine if glucocorticoid resistance due to GRL mutations is associated with premature pubarche or adolescent hyperandrogenism, we performed mutation detection analysis of the GRL in genomic DNA samples from 41 patients.
To determine whether glucocorticoid resistance due to mutations in the glucocorticoid receptor (GRL) gene is associated with premature pubarche, hirsutism, or oligo/amenorrhea, we performed single-strand conformational polymorphism analysis of genomic DNA obtained from 25 children and 16 adolescent girls referred for the evaluation of premature pubarche, hirsutism, or oligo/amenorrhea. A missense mutation, N363S, and a presumed polymorphism in the 3*-UTR of exon 9a were identified. We conclude that glucocorticoid resistance due to GRL mutations is an infrequent cause of mild hyperandrogenism. © 1999 Academic Press Key Words: glucocorticoid resistance; hyperandrogenism; glucocorticoid receptor; premature pubarche.
MATERIALS AND METHODS Subjects
Familial glucocorticoid resistance is characterized by elevated cortisol concentrations in the absence of symptoms typical for Cushing’s syndrome (1,2). In this disorder, impaired functioning of the glucocorticoid receptor (GRL) leads to a compensatory increased set point for ambient cortisol concentrations. Increased ACTH and cortisol secretion ensue. The elevated cortisol production can be accompanied by increased mineralocorticoid and adrenal androgen production. Consistent with decreased sensitivity to endogenous glucocorticoids, resistance to the hypothalamic–pituitary–adrenal axis to suppression with exogenous glucocorticoid treatment also
Forty-one children and adolescent girls from 40 unrelated families were referred to the Children’s Hospital of Pittsburgh for the evaluation of symptoms of androgen excess (Table 1). Twenty-five children (21 girls and 4 boys) were evaluated for the development of premature pubic hair. Sixteen adolescent girls were evaluated for hirsutism and/or irregular menses. Two siblings from one family, a 16 7/12-year-old adolescent girl with hirsutism and oligomenorrhea and an 8 10/12-year-old boy with premature pubic hair, were studied. Patients with congenital adrenal hyperplasia or Cushing’s syndrome were excluded. Genomic DNA samples were obtained from 65 healthy control subjects; all healthy women had regular menses. This protocol is approved by the Human Rights Committee of the Children’s Hospital of Pittsburgh. Informed parental consent and patient assent were obtained prior to participation in these studies.
1
Supported in part by grants awarded by the National Institutes of Health to S.F.W. (HD34808) and to the General Clinical Research Center at the Children’s Hospital of Pittsburgh (M01RR00084). Also supported in part by grants from the Pennsylvania Chapter of the American Heart Association (S.F.W.), Research Advisory Committee of the Children’s Hospital of Pittsburgh (S.F.W.), Genentech Foundation (S.F.W.), and Serono Laboratories, Inc, Norwell, MA (S.F.W.). 137
1096-7192/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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TABLE 1 Clinical and Hormonal Characteristics (Mean 6 SD) in Patients with Premature Pubarche (PP) and Adolescent Hyperandrogenism (HA) PP Number Sex CA (years) BA (years) BA/CA BMI (kg/m 2) F-09 (mg/dl) F-309 (mg/dl)
25 21 F 7.1 6 1.8 8.6 6 2.5 1.2 6 0.2 19.3 6 4.6 16.8 6 8.2 31.8 6 4.2
HA 16 16 F 16.0 6 1.2 ND ND 29.4 6 8.1 14.0 6 6.3 32.4 6 11.0
1
2
3
4
F 15.6 ND ND 22.3 11.5 27.4
F 17.6 ND ND 22.9 5.7 23.5
M 9.5 13 1.37 29.1 11.0 29.5
F 15.9 ND ND 40.8 21.3 32.9
Note. Bone age assessments were not obtained in the HA patients (ND). Clinical and hormonal features of the four patients heterozygous for N363S are listed individually (F, female; M, male) in the four columns on the right side of the table.
Hormone Studies ACTH stimulation tests were performed as previously described (7). For one adolescent girl, an ACTH stimulation test had been performed elsewhere prior to referral and her cortisol values were excluded from statistical analysis. Genetic Studies Blood samples were obtained from all 41 subjects. Genomic DNA was extracted from peripheral blood lymphocytes. Using nucleotide sequence data generously provided by Dr. Sevilla Detera-Wadleigh and primer sequences kindly provided by Dr. Michael Karl (6), PCR primers were designed to amplify the coding and intron/exon boundary regions of the glucocorticoid receptor (GRL) gene. Exons 1 and 3– 8 were individually amplified. Exon 2 was amplified using four overlapping sets of primers. Exon 9 encoding both the a and the b isoforms of the glucocorticoid receptor was amplified using 15 overlapping sets of primer pairs. The PCR reactions consisted of 0.15 mg genomic DNA, 3 pmol of each primer, 1.25 ml 103 PCR buffer (Perkin–Elmer Cetus, Norwalk, CT), 200 mM dNTP, 0.09 ml [ 32P]dATP (10 mCi/ml, NEN, Boston, MA), 0.3 units of Taq polymerase (Perkin–Elmer Cetus), and 8.3 ml of sterile water in a total volume of 12.5 ml (PCR thermocycler conditions and primer sequences are available upon request). Single-strand conformational polymorphism (SSCP) analysis was performed using three distinct gel conditions as previously described (8). Following electrophoresis, gels were dried and autoradiography was performed. Unique conformers were excised from the gel and eluted into 100 ml 0.13 TE overnight. Using
these three gel conditions, we detect 85–90% of mutations (9,10). Eluted conformers were reamplified using the original primer pair and thermocycling conditions. The PCR product was sequenced in both sense and anti-sense orientations using manual sequencing (SequiTherm, Epicentre Technologies, Madison, WI) or an ABI automated sequencer 373A according to the manufacturer’s protocol (Applied BioSystems, Foster City, CA). The presence of sequence variants was confirmed by restriction fragment digestion of PCR-amplified genomic DNA. To determine the relative sizes of the PCR product obtained using primers 9D1F and 9D1R, a denaturing gel (8% Long Ranger, FMC BioProducts, Rockland, ME) was electrophoresed at 50 W room temperature for 2.5 h. Statistical Analysis Statistical analysis was performed using AbSTAT statistical software (Anderson-Bell, Arvada, CO). Data in the text are expressed as means 6 1 SD. RESULTS ACTH Stimulation Tests The 41 patients selected for molecular genotype analysis of the GRL showed stimulated cortisol responses greater than 21 mg/dl. Twenty-four (59%) had stimulated cortisol responses $30 mg/dl. The median stimulated cortisol response was 30.8 mg/dl. Mutation Screening For exons 1, 3, 4, 5, 6, 7, and 8, a single mobility pattern was observed for the 41 patients with all three gel conditions. SSCP analysis of the 39 portion
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of exon 2 showed 4 patients to have a distinct conformer. In addition, all 4 showed the wild-type conformer pattern suggesting that they were heterozygotes for the wild-type and a sequence variant. SSCP analysis of the PCR products amplified from the coding region of exon 9a showed identical conformers in all patients. The PCR product obtained with primers 9DF and 9DR, amplifed from the 39UTR of exon 9a, showed a unique pattern in one patient. SSCP analysis of 32 healthy controls using the same primers and electrophoretic conditions showed several distinct conformers in a pattern suggestive of a polymorphism. DNA Sequence Analysis of Exon 2 Wild-type and discrete conformers were eluted from the gels, reamplified, and sequenced. Nucleotide sequence analysis of the exon 2 conformer showed a point mutation, A 3 G at cDNA position 1220 causing a missense mutation, N363S. This mutation also causes the loss of a Tsp509I restriction site (Fig. 1). Restriction fragment analysis confirmed that all four patients were heterozygous for N363S. DNA Sequence Analysis of Exon 9 Nucleotide sequence analysis of the exon 9D conformer from 1 girl with premature pubarche showed a loss of 3 adenine nucleotides at positions 3218 – 3235 of the cDNA sequence. Sequence analysis of DNA samples from 3 healthy controls confirmed that the conformers were due to variation in the number of adenine nucleotides. To determine the relative size of this exon 9a 39-UTR region, a new primer pair, internal to the original primer pair, was used to amplify genomic DNA from the propositus, her family, and 32 healthy controls. Electrophoresis of the radiolabeled PCR product on a denaturing gel with a sequencing ladder verified that almost all control subjects were heterozygous for variants differing in the number of adenine nucleotides with the most common variants showing 15 or 18 adenine nucleotides. Hormone Concentrations vs Genotype Mean basal cortisol concentrations were 16.2 6 7.7 mg/dl among the subjects homozygous for N363 (wild type) and 12.4 6 6.5 mg/dl among those heterozygous for S363, P . 0.05. Mean stimulated cortisol concentrations were 32.4 6 7.6 mg/dl among those homozygous for N363 and 28.3 6 3.9 among
FIG. 1. Restriction digest analysis of N363S. The top portion of the figure shows the results of Tsp509I digestion on a 4% agarose gel. The A 3 G mutation at cDNA position 1220 causes the loss of a Tsp509I site. When the wild-type allele is digested with Tsp509I, four DNA fragments, 136, 135, 56, and 18 bp, are observed. For the mutant allele, three fragments, 154, 135, and 56 bp, are generated. The anticipated results of digestion are illustrated in the cartoon on the lower portion of the figure for homozygous N363 (WT), heterozygous (HET), and homozygous S363 (MUT). Lanes 1, 2, 3, 4, and 8 are heterozygous for the N363S variant, whereas lanes 5, 6, 7, 9, and 10 are homozygous for the wild-type, N363, allele. Lanes 1 and 2 contain samples from hyperandrogenic patients and lanes 3–10 contain samples from healthy controls. The molecular weight marker is in the lane on the right. The 18-bp fragment is difficult to visualize and not shown on the gel image. SSCP analyses confirmed heterozygosity for all individuals.
those heterozygous for S363, P . 0.05. Basal and stimulated cortisol concentrations are listed individually in Table 1 for the four heterozygotic carriers. Frequency of N363S Variant Genomic DNA samples from 65 healthy controls were assayed by SSCP and restriction digest analyses for the N363S variant. Five of 65 healthy controls (7.7%) were found to be heterozygous carriers of the N363S variant. Frequency of the N363S allele was comparable for the hyperandrogenic patients and the healthy adult controls, 4.9 vs 3.8%.
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DISCUSSION Phenotypes described for glucocorticoid resistance associated with mutations in the glucocorticoid receptor include premature pubarche, hirsutism, oligo/amenorrhea, and elevated cortisol concentrations (1– 6). None of 41 hyperandrogenic patients in whom mutation screening of the GRL was performed were found to have GRL mutations leading to glucocorticoid resistance. We speculate that the 9a 39-UTR variant involving a variable number of adenine nucleotides among patients and control subjects is a common polymorphism. Thus, glucocorticoid resistance due to GRL mutations appears to be an infrequent cause of premature pubarche and adolescent hyperandrogenism. However, we cannot exclude the possibility that some of our patients have glucocorticoid resistance due to other factors affecting glucocorticoid signal transduction such as intracellular hormone availability (11), ligand-binding affinity (12), intracellular trafficking, and glucocorticoid receptor phosphorylation (13). False-negative mutation screening analysis cannot be entirely excluded. Nonetheless, since our protocol ascertains 85–90% of sequence variants (9,10), the expenditure of limited resources, i.e., time and money, to sequence the GRL from all 41 patients is difficult to justify. As part of the Rotterdam population-based study on glucocorticoid sensitivity, SSCP analysis identified six sequence variants. Only two mutations led to amino acid changes: R23K and N363S (14). Among our patient population, we detected the N363S variant. Transient transfection assays showed that the N363S variant exhibits transcriptional activation and repression comparable to the wild type (6,15,16). Interestingly, individuals carrying this variant showed increased glucocorticoid and decreased insulin sensitivity (17). Among our patients, there was a trend for the heterozygotic carriers to have lower basal and stimulated cortisol concentrations as would be anticipated with increased glucocorticoid sensitivity. The frequencies of the N363S allele in our patients and our healthy control subjects, 4.9 and 3.8%, respectively, are comparable to that of the Rotterdam study, 3.0%. Insulin resistance/hyperinsulinemia and hyperandrogenism are characteristic features of polycystic ovary syndrome (PCOS). Premature pubarche appears to be an early manifestation of PCOS (18 – 22). As glucocorticoids induce insulin resistance through a poorly defined mechanism (23,24), it is
interesting that we found 9.8% of our patients to carry a genetic marker which seems to be associated with decreased glucocorticoid and insulin sensitivities in an elderly Dutch population. Thus, glucocorticoid resistance due to GRL mutations is an uncommon cause of mild hyperandrogenism. Additional studies are essential to determine if this variant influences insulin sensitivity in children, adolescents, and women with PCOS. ACKNOWLEDGMENTS We appreciate Peter A. Lee, M.D., Ph.D., for providing DNA samples from his patients. We are indebted to Eric P. Hoffman, Ph.D., and Donald B. DeFranco, Ph.D., for helpful discussions. We thank Amy Jones-Gilliland and Tamara Johnston for nursing assistance. We gratefully acknowledge Debbie Cleary for technical assistance with hormone assays.
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Vingerhoeds ACM, Thijssen JHH, Schwartz F. Spontaneous hypercortisolism without Cushing’s syndrome. J Clin Endocrinol Metab 43:1128 –1133, 1976.
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Lamberts SWJ, Poldermans D, Zweens M, de Jong FH. Familial cortisol resistance: Differential diagnostic and therapeutic aspects. J Clin Endocrinol Metab 63:1328 – 1333, 1986.
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Lamberts SWJ, Koper JW, Biemond P, den Holder FH, de Jong FH. Cortisol receptor resistance: The variability of its clinical presentation and response to treatment. J Clin Endocrinol Metab 74:313–321, 1992.
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Hurley DM, Accili D, Stratakis CA, Karl M, Vamvakopoulos N, Roere E, Constantine K, Taylor SI, Chrousos GP. Point mutation causing a single amino acid substitution in the hormone binding domain of the glucocorticoid receptor in familial glucocorticoid resistance. J Clin Invest 87:680 – 686, 1991.
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Karl M, Lamberts SWJ, Detera-Wadleigh SD, Encio IJ, Stratakis CA, Hurley DM, Accili D, Chrousos GP. Familial glucocorticoid resistance caused by a splice side deletion in the human glucocorticoid receptor gene. J Clin Endocrinol Metab 76:683– 689, 1993.
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Siegel SF, Finegold DN, Murray PJ, Lee PA. Assessment of clinical hyperandrogenism in adolescent girls. Adolesc Pediatr Gynecol 5:13–20, 1991.
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Siegel SF, Hoffman EP, Trucco M. Molecular diagnosis of 21-hydroxylase deficiency: Detection of four mutations on a single gel. Biochem Med Metab Biol 51:66 –73, 1994.
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Kobayashi H, Hoffman EP, Marks H. The rumpshaker mutation in spastic paraplegia. Nature Genet 7:351–352, 1994.
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Nayak S, Lee PA, Witchel SF. Variants of the type II 3bhydroxysteroid dehydrogenase gene in children with prema-
BRIEF COMMUNICATION ture pubarche and hyperandrogenic adolescents. Mol Genet Metab 64:184 –192, 1998. 11.
Walker BR, Stewart PM, Shackleton CH, Padfield PL, Edwards CR. Deficient activation of cortisol by 11 beta-hydroxysteroid dehydrogenase in essential hypertension. Clin Endocrinol 39:221–227, 1993.
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Cadepond F, Jibard N, Binart M, Schweizer-Groyer G, Segard-Maurel I, Baulieu EE. Selective deletions in the 90 kDa heat shock protein (hsp90) impede hetero-oligomeric complex formation with the glucocorticoid receptor (GR) or hormone-binding by GR. J Steroid Biochem Mol Biol 48:361– 367, 1994.
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Webster JC, Jewell CM, Bodwell JE, Munck A, Sar M, Cidlowski JA. Mouse glucocorticoid receptor phosphorylation status influences multiple functions of the receptor protein. J Biol Chem 272:9287–9293, 1997.
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Koper JW, Stolk RP, de Lange P, Huizenga NATM, Molijn G-J, Pols HAP, Grobbee DE, Karl M, de Jong FH, Brinkmann AO, Lamberts SWJ. Lack of association between five polymorphisms in the human glucocorticoid receptor gene and glucocorticoid resistance. Hum Genet 99:663– 668, 1997.
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Gaitan D, DeBold CR, Turney MK, Zhou P, Orth DN, Kovacs WJ. Glucocorticoid receptor structure and function in an adrenocorticotropin-secreting small cell lung cancer. Mol Endocrinol 9:1193–1201, 1995.
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de Lange P, Koper JW, Huizenga NATM, Brinkmann AO, de Jong FH, Karl M, Chrousos GP, Lamberts SWJ. Differential hormone-dependent transcriptional activation and repression by naturally occurring human glucocorticoid receptor variants. Mol Endocrinol 11:1156 –1164, 1997.
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Huizenga NATM, Koper JW, de Lange P, Pols HAP, Stolk RP, Burger H, Grobbee DE, Brinkmann AO, de Jong FH, Lamberts SWJ. A polymorphism in the glucocorticoid receptor gene may be associated with an increased sensitivity to glucocorticoids in vivo. J Clin Endocrinol Metab 83:144 – 151, 1998.
18.
Iban˜ez L, Potau N, Virdis R, Zampolli M, Terzi C, Gussinye´ M, Carrascosa A, Vicens-Calvet E. Postpubertal outcome in girls diagnosed of premature pubarche during childhood:
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Increased frequency of functional ovarian hyperandrogenism. J Clin Endocrinol Metab 76:1599 –1603, 1993. Iban˜ez L, Potau N, Zampolli M, Rique´ S, Saenger P, Carrascosa A. Hyperinsulinemia and decreased insulin-like growth factor-binding protein-1 are common features in prepubertal and pubertal girls with a history of premature pubarche. J Clin Endocrinol Metab 82:2283–2288, 1997. Iban˜ez L, Potau N, Zampolli M, Street ME, Carrascosa A. Girls diagnosed with premature pubarche show an exaggerated ovarian androgen synthesis from the early stages of puberty: Evidence from gonadotropin-releasing hormone agonist testing. Fertil Steril 67:49 –55, 1997. Apter D, Bu¨tzow T, Laughlin GA, Yen SSC. Accelerated 24-hour luteinizing hormone pulsatile activity in adolescent girls with ovarian hyperandrogenism: Relevance to the developmental phase of polycystic ovary syndrome. J Clin Endocrinol Metab 79:119 –125, 1994. Apter D, Bu¨tzow T, Laughlin GA, Yen SSC. Metabolic features of polycystic ovary syndrome are found in adolescent girls with hyperandrogenism. J Clin Endocrinol Metab 80: 2966 –2973, 1994. Yasuda K, Hines E III, Kitabchi AE. Hypercortisolism and insulin resistance: Comparative effects of prednisone, hydrocortisone, and dexamethasone on insulin binding of human erythrocytes. J Clin Endocrinol Metab 55:910 –915, 1982. Rizza RA, Mandarino LJ, Gerich JE. Cortisol-induced insulin resistance in man: Impaired suppression of glucose production and stimulation of glucose utilization due to a postreceptor defect of insulin action. J Clin Endocrinol Metab 54:131–138, 1982.
Selma F. Witchel Rhonda R. Smith Division of Pediatric Endocrinology Children’s Hospital of Pittsburgh University of Pittsburgh 3705 Fifth Avenue Pittsburgh, Pennsylvania 15213 Received December 3, 1998, and in revised form December 28, 1998
BRIEF COMMUNICATION Glucocorticoid Resistance in Premature Pubarche and Adolescent Hyperandrogenism 1 occurs. Clinical manifestations, if conspicuous, include hirsutism, irregular menses, hypertension, and hypokalemia. Inheritance is autosomal dominant, but the phenotype can vary even within the same family (3,4). Mutations in the glucocorticoid receptor have been identified in several families with generalized glucocorticoid resistance (5,6). To determine if glucocorticoid resistance due to GRL mutations is associated with premature pubarche or adolescent hyperandrogenism, we performed mutation detection analysis of the GRL in genomic DNA samples from 41 patients.
To determine whether glucocorticoid resistance due to mutations in the glucocorticoid receptor (GRL) gene is associated with premature pubarche, hirsutism, or oligo/amenorrhea, we performed single-strand conformational polymorphism analysis of genomic DNA obtained from 25 children and 16 adolescent girls referred for the evaluation of premature pubarche, hirsutism, or oligo/amenorrhea. A missense mutation, N363S, and a presumed polymorphism in the 3*-UTR of exon 9a were identified. We conclude that glucocorticoid resistance due to GRL mutations is an infrequent cause of mild hyperandrogenism. © 1999 Academic Press Key Words: glucocorticoid resistance; hyperandrogenism; glucocorticoid receptor; premature pubarche.
MATERIALS AND METHODS Subjects
Familial glucocorticoid resistance is characterized by elevated cortisol concentrations in the absence of symptoms typical for Cushing’s syndrome (1,2). In this disorder, impaired functioning of the glucocorticoid receptor (GRL) leads to a compensatory increased set point for ambient cortisol concentrations. Increased ACTH and cortisol secretion ensue. The elevated cortisol production can be accompanied by increased mineralocorticoid and adrenal androgen production. Consistent with decreased sensitivity to endogenous glucocorticoids, resistance to the hypothalamic–pituitary–adrenal axis to suppression with exogenous glucocorticoid treatment also
Forty-one children and adolescent girls from 40 unrelated families were referred to the Children’s Hospital of Pittsburgh for the evaluation of symptoms of androgen excess (Table 1). Twenty-five children (21 girls and 4 boys) were evaluated for the development of premature pubic hair. Sixteen adolescent girls were evaluated for hirsutism and/or irregular menses. Two siblings from one family, a 16 7/12-year-old adolescent girl with hirsutism and oligomenorrhea and an 8 10/12-year-old boy with premature pubic hair, were studied. Patients with congenital adrenal hyperplasia or Cushing’s syndrome were excluded. Genomic DNA samples were obtained from 65 healthy control subjects; all healthy women had regular menses. This protocol is approved by the Human Rights Committee of the Children’s Hospital of Pittsburgh. Informed parental consent and patient assent were obtained prior to participation in these studies.
1
Supported in part by grants awarded by the National Institutes of Health to S.F.W. (HD34808) and to the General Clinical Research Center at the Children’s Hospital of Pittsburgh (M01RR00084). Also supported in part by grants from the Pennsylvania Chapter of the American Heart Association (S.F.W.), Research Advisory Committee of the Children’s Hospital of Pittsburgh (S.F.W.), Genentech Foundation (S.F.W.), and Serono Laboratories, Inc, Norwell, MA (S.F.W.). 137
1096-7192/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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TABLE 1 Clinical and Hormonal Characteristics (Mean 6 SD) in Patients with Premature Pubarche (PP) and Adolescent Hyperandrogenism (HA) PP Number Sex CA (years) BA (years) BA/CA BMI (kg/m 2) F-09 (mg/dl) F-309 (mg/dl)
25 21 F 7.1 6 1.8 8.6 6 2.5 1.2 6 0.2 19.3 6 4.6 16.8 6 8.2 31.8 6 4.2
HA 16 16 F 16.0 6 1.2 ND ND 29.4 6 8.1 14.0 6 6.3 32.4 6 11.0
1
2
3
4
F 15.6 ND ND 22.3 11.5 27.4
F 17.6 ND ND 22.9 5.7 23.5
M 9.5 13 1.37 29.1 11.0 29.5
F 15.9 ND ND 40.8 21.3 32.9
Note. Bone age assessments were not obtained in the HA patients (ND). Clinical and hormonal features of the four patients heterozygous for N363S are listed individually (F, female; M, male) in the four columns on the right side of the table.
Hormone Studies ACTH stimulation tests were performed as previously described (7). For one adolescent girl, an ACTH stimulation test had been performed elsewhere prior to referral and her cortisol values were excluded from statistical analysis. Genetic Studies Blood samples were obtained from all 41 subjects. Genomic DNA was extracted from peripheral blood lymphocytes. Using nucleotide sequence data generously provided by Dr. Sevilla Detera-Wadleigh and primer sequences kindly provided by Dr. Michael Karl (6), PCR primers were designed to amplify the coding and intron/exon boundary regions of the glucocorticoid receptor (GRL) gene. Exons 1 and 3– 8 were individually amplified. Exon 2 was amplified using four overlapping sets of primers. Exon 9 encoding both the a and the b isoforms of the glucocorticoid receptor was amplified using 15 overlapping sets of primer pairs. The PCR reactions consisted of 0.15 mg genomic DNA, 3 pmol of each primer, 1.25 ml 103 PCR buffer (Perkin–Elmer Cetus, Norwalk, CT), 200 mM dNTP, 0.09 ml [ 32P]dATP (10 mCi/ml, NEN, Boston, MA), 0.3 units of Taq polymerase (Perkin–Elmer Cetus), and 8.3 ml of sterile water in a total volume of 12.5 ml (PCR thermocycler conditions and primer sequences are available upon request). Single-strand conformational polymorphism (SSCP) analysis was performed using three distinct gel conditions as previously described (8). Following electrophoresis, gels were dried and autoradiography was performed. Unique conformers were excised from the gel and eluted into 100 ml 0.13 TE overnight. Using
these three gel conditions, we detect 85–90% of mutations (9,10). Eluted conformers were reamplified using the original primer pair and thermocycling conditions. The PCR product was sequenced in both sense and anti-sense orientations using manual sequencing (SequiTherm, Epicentre Technologies, Madison, WI) or an ABI automated sequencer 373A according to the manufacturer’s protocol (Applied BioSystems, Foster City, CA). The presence of sequence variants was confirmed by restriction fragment digestion of PCR-amplified genomic DNA. To determine the relative sizes of the PCR product obtained using primers 9D1F and 9D1R, a denaturing gel (8% Long Ranger, FMC BioProducts, Rockland, ME) was electrophoresed at 50 W room temperature for 2.5 h. Statistical Analysis Statistical analysis was performed using AbSTAT statistical software (Anderson-Bell, Arvada, CO). Data in the text are expressed as means 6 1 SD. RESULTS ACTH Stimulation Tests The 41 patients selected for molecular genotype analysis of the GRL showed stimulated cortisol responses greater than 21 mg/dl. Twenty-four (59%) had stimulated cortisol responses $30 mg/dl. The median stimulated cortisol response was 30.8 mg/dl. Mutation Screening For exons 1, 3, 4, 5, 6, 7, and 8, a single mobility pattern was observed for the 41 patients with all three gel conditions. SSCP analysis of the 39 portion
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of exon 2 showed 4 patients to have a distinct conformer. In addition, all 4 showed the wild-type conformer pattern suggesting that they were heterozygotes for the wild-type and a sequence variant. SSCP analysis of the PCR products amplified from the coding region of exon 9a showed identical conformers in all patients. The PCR product obtained with primers 9DF and 9DR, amplifed from the 39UTR of exon 9a, showed a unique pattern in one patient. SSCP analysis of 32 healthy controls using the same primers and electrophoretic conditions showed several distinct conformers in a pattern suggestive of a polymorphism. DNA Sequence Analysis of Exon 2 Wild-type and discrete conformers were eluted from the gels, reamplified, and sequenced. Nucleotide sequence analysis of the exon 2 conformer showed a point mutation, A 3 G at cDNA position 1220 causing a missense mutation, N363S. This mutation also causes the loss of a Tsp509I restriction site (Fig. 1). Restriction fragment analysis confirmed that all four patients were heterozygous for N363S. DNA Sequence Analysis of Exon 9 Nucleotide sequence analysis of the exon 9D conformer from 1 girl with premature pubarche showed a loss of 3 adenine nucleotides at positions 3218 – 3235 of the cDNA sequence. Sequence analysis of DNA samples from 3 healthy controls confirmed that the conformers were due to variation in the number of adenine nucleotides. To determine the relative size of this exon 9a 39-UTR region, a new primer pair, internal to the original primer pair, was used to amplify genomic DNA from the propositus, her family, and 32 healthy controls. Electrophoresis of the radiolabeled PCR product on a denaturing gel with a sequencing ladder verified that almost all control subjects were heterozygous for variants differing in the number of adenine nucleotides with the most common variants showing 15 or 18 adenine nucleotides. Hormone Concentrations vs Genotype Mean basal cortisol concentrations were 16.2 6 7.7 mg/dl among the subjects homozygous for N363 (wild type) and 12.4 6 6.5 mg/dl among those heterozygous for S363, P . 0.05. Mean stimulated cortisol concentrations were 32.4 6 7.6 mg/dl among those homozygous for N363 and 28.3 6 3.9 among
FIG. 1. Restriction digest analysis of N363S. The top portion of the figure shows the results of Tsp509I digestion on a 4% agarose gel. The A 3 G mutation at cDNA position 1220 causes the loss of a Tsp509I site. When the wild-type allele is digested with Tsp509I, four DNA fragments, 136, 135, 56, and 18 bp, are observed. For the mutant allele, three fragments, 154, 135, and 56 bp, are generated. The anticipated results of digestion are illustrated in the cartoon on the lower portion of the figure for homozygous N363 (WT), heterozygous (HET), and homozygous S363 (MUT). Lanes 1, 2, 3, 4, and 8 are heterozygous for the N363S variant, whereas lanes 5, 6, 7, 9, and 10 are homozygous for the wild-type, N363, allele. Lanes 1 and 2 contain samples from hyperandrogenic patients and lanes 3–10 contain samples from healthy controls. The molecular weight marker is in the lane on the right. The 18-bp fragment is difficult to visualize and not shown on the gel image. SSCP analyses confirmed heterozygosity for all individuals.
those heterozygous for S363, P . 0.05. Basal and stimulated cortisol concentrations are listed individually in Table 1 for the four heterozygotic carriers. Frequency of N363S Variant Genomic DNA samples from 65 healthy controls were assayed by SSCP and restriction digest analyses for the N363S variant. Five of 65 healthy controls (7.7%) were found to be heterozygous carriers of the N363S variant. Frequency of the N363S allele was comparable for the hyperandrogenic patients and the healthy adult controls, 4.9 vs 3.8%.
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DISCUSSION Phenotypes described for glucocorticoid resistance associated with mutations in the glucocorticoid receptor include premature pubarche, hirsutism, oligo/amenorrhea, and elevated cortisol concentrations (1– 6). None of 41 hyperandrogenic patients in whom mutation screening of the GRL was performed were found to have GRL mutations leading to glucocorticoid resistance. We speculate that the 9a 39-UTR variant involving a variable number of adenine nucleotides among patients and control subjects is a common polymorphism. Thus, glucocorticoid resistance due to GRL mutations appears to be an infrequent cause of premature pubarche and adolescent hyperandrogenism. However, we cannot exclude the possibility that some of our patients have glucocorticoid resistance due to other factors affecting glucocorticoid signal transduction such as intracellular hormone availability (11), ligand-binding affinity (12), intracellular trafficking, and glucocorticoid receptor phosphorylation (13). False-negative mutation screening analysis cannot be entirely excluded. Nonetheless, since our protocol ascertains 85–90% of sequence variants (9,10), the expenditure of limited resources, i.e., time and money, to sequence the GRL from all 41 patients is difficult to justify. As part of the Rotterdam population-based study on glucocorticoid sensitivity, SSCP analysis identified six sequence variants. Only two mutations led to amino acid changes: R23K and N363S (14). Among our patient population, we detected the N363S variant. Transient transfection assays showed that the N363S variant exhibits transcriptional activation and repression comparable to the wild type (6,15,16). Interestingly, individuals carrying this variant showed increased glucocorticoid and decreased insulin sensitivity (17). Among our patients, there was a trend for the heterozygotic carriers to have lower basal and stimulated cortisol concentrations as would be anticipated with increased glucocorticoid sensitivity. The frequencies of the N363S allele in our patients and our healthy control subjects, 4.9 and 3.8%, respectively, are comparable to that of the Rotterdam study, 3.0%. Insulin resistance/hyperinsulinemia and hyperandrogenism are characteristic features of polycystic ovary syndrome (PCOS). Premature pubarche appears to be an early manifestation of PCOS (18 – 22). As glucocorticoids induce insulin resistance through a poorly defined mechanism (23,24), it is
interesting that we found 9.8% of our patients to carry a genetic marker which seems to be associated with decreased glucocorticoid and insulin sensitivities in an elderly Dutch population. Thus, glucocorticoid resistance due to GRL mutations is an uncommon cause of mild hyperandrogenism. Additional studies are essential to determine if this variant influences insulin sensitivity in children, adolescents, and women with PCOS. ACKNOWLEDGMENTS We appreciate Peter A. Lee, M.D., Ph.D., for providing DNA samples from his patients. We are indebted to Eric P. Hoffman, Ph.D., and Donald B. DeFranco, Ph.D., for helpful discussions. We thank Amy Jones-Gilliland and Tamara Johnston for nursing assistance. We gratefully acknowledge Debbie Cleary for technical assistance with hormone assays.
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Selma F. Witchel Rhonda R. Smith Division of Pediatric Endocrinology Children’s Hospital of Pittsburgh University of Pittsburgh 3705 Fifth Avenue Pittsburgh, Pennsylvania 15213 Received December 3, 1998, and in revised form December 28, 1998