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Departments of Endocrinology and Diabetology, University Children’s Hospital (S.R., A.B.-L., F.N., E.J.S.), 8032 Zurich, Switzerland; and Department of Pediatrics, Addenbrooke’s Hospital, University of Cambridge (N.P.M.), Cambridge, United Kingdom CB2 2QQ
Abstract
Mutations in the X-linked androgen receptor (AR) gene cause the androgen insensitivity syndrome by impairing androgen-dependent male sexual differentiation to varying degrees. Complete androgen insensitivity (CAIS) yields an external female phenotype, whereas affected cases of partial androgen insensitivity have various ambiguities of the genitalia. Here we describe a 46,XY phenotypically female patient with all of the characteristics of CAIS, i.e. primary amenorrhea, no axillary or pubic hair, female external genitalia, no uterus, and undescended testes. Defects in testosterone and dihydrotestosterone synthesis were excluded. The molecular basis of the disease was clarified by means of direct sequencing of PCR-amplified exonic fragments of the AR gene. An A to C transition in exon 4 of the AR gene led to a novel missense His689Pro mutation in the ligand-binding domain of the AR protein. Functional studies demonstrated that the mutated AR is unable to efficiently bind its natural ligand dihydrotestosterone and to trans-activate known androgen response elements. Analysis of the structural consequences of the His689Pro substitution suggests that this mutation is likely to perturb the conformation of the second helix of the AR ligand-binding domain, which contains residues critical for androgen binding.
THE ANDROGEN receptor (AR) is a ligand-activated nuclear transcription factor that mediates the cellular effects of androgens, including differentiation, homeostasis, morphogenesis, and growth. The AR is encoded by the AR gene located on the X-chromosome at Xq11–12. The gene consists of eight exons, of which exons 2–3 code for the DNA-binding domain, and exons 4–8 code for the ligand-binding domain (1). More than 200 mutations (www. mcgill.ca/androgendb and references therein) have been reported, to date linked to prostatic carcinoma and breast cancer in males, but the classical phenotypical expression of AR mutation is the androgen insensitivity syndrome (AIS) (2, 3, 4). In the most extreme form, the complete AIS (CAIS), the 46,XY individual presents at birth as a phenotypically normal girl. However, the patients have undescended testes and no Mullerian duct-derived structures. Molecular structures have now been determined for the ligand-binding domain (LBD) of several members of the nuclear hormone receptor superfamily, including the thyroid receptor (5), estrogen receptor 
(6), progesterone receptor (7), and most recently the AR (8, 9). All share a similar LBD structural organization based on an arrangement of 10–12 helixes. With such detailed understanding of the androgen LBD now available, it is possible to evaluate the structural consequence of AR mutations. Here we describe a case of CAIS in which a novel mutation in the AR gene is the cause of the disorder. The mutation found in exon 4 leads to the missense H689P rearrangement in the LBD of the AR, significantly impairing ligand binding and the trans-activation potential of the mutant receptor.
Subjects and Methods
A 16-yr-old girl was brought to our attention because of primary amenorrhea. Physical examination revealed the absence of axillary and pubic hair, normal female external genitalia, and normal breast development. Pelvic ultrasound showed the presence of abdominal gonads and the absence of uterus and tubes. The karyotype was normal male 46,XY. Testosterone (T) and dihydrotestosterone (DHT) synthesis defects were excluded by the normal rise of T and DHT after hCG stimulation. The gonads were removed at the age of 17 yr, and histopathological examination demonstrated testicular tissue. Genomic DNA was extracted from peripheral blood leukocytes of the patient and normal controls. PCR amplification of exonic fragments (including the intron-exon boundaries) was carried out using the primers depicted in Table 1
and Fig. 1. Direct sequencing of these PCR products was performed using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit and was analyzed by electrophoresis on the ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems, Rotkreuz, Switzerland). Site-directed mutagenesis was carried out using PCR strategy based on overlapping primers bearing the desired mutation and flanking primers carrying a BamHI (5') and EcoRI (3') sites, respectively, to facilitate ligation. As template we used the human wild-type (WT) AR cDNA (pSVAR0, Dr. Brinkmann, Rotterdam, The Netherlands). The two PCR products obtained were then mixed and used as templates for the synthesis of a mutated full-length cDNA that was subcloned into pCMV-Script (Stratagene, Basel, Switzerland) and used for functional studies. Confluent (80%) monkey kidney COS-1 cells were transfected with 10 µg DNA/60-mm plate using the TransFast transfection system reagent (Promega Corp., Wallisellen, Switzerland) as directed by the supplier. The influence of the rearrangement on protein translation or stability under DHT treatment was assayed via Western blot analysis on protein extracts from transfected COS-1 cells using a 1:50 dilution of a mouse antihuman antibody (Santa Cruz Biotechnology, Inc., Nunningen, Switzerland). Androgen binding was investigated by transiently expressing WT and mutant receptors in COS-1 cells. The cells were exposed to increasing concentrations of [3H]DHT (1–12.5 nM; NEN Life Science Products, Zaventem, Belgium) in the presence or absence of a 100-fold excess of unlabeled DHT. Specific androgen binding was assayed at 37 C on whole cells as previously described (10), and the results were analyzed using Scatchard analysis (PRISM, GraphPad Software, Inc., San Diego, CA). To check the thermostability of AR complexes, the highest DHT concentration point (12.5 nM) was also duplicated at 40 C in the presence of 100 µM cycloheximide (11). To further investigate the stability of the AR-ligand complexes, we determined the intracellular dissociation rate for WT and mutant AR by plating 106 COS-1 cells/well in six-well tissue culture plates. Twenty-four hours after the initial plating, the cells were transfected with 5 µg pSVAR0 (WT) or mutant AR DNA per well as described above. The dissociation rates of 5 nM [3H]DHT from WT and H689P AR were determined as previously described (12). The analysis of the dissociation experiments was carried out using PRISM (GraphPad Software, Inc.).
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Results
Direct sequencing analysis of PCR products revealed the presence of an A to C transition in exon 4 resulting in the previously unreported histidine 689 proline mutation. Histidine 689 is located in the LBD of the AR protein (Fig. 1
, A and B). The mutation does not affect protein translation or stability, as demonstrated by Western blot analysis (Fig. 1C). However, as expected from the location of the mutation in the LBD, the H689P mutant receptor is significantly defective with regard to ligand binding. In particular, the binding capacity, which measures the concentrations of binding sites, is highly reduced in COS-1 cells expressing the mutant receptor compared with that in the WT construct (3.43 vs. 32.4 fmol/mg protein; Fig. 2), whereas the affinity of the mutant H689P AR for DHT appears to be comparable to that of the WT (1.55 vs. 1.96 nM; Fig. 2). The mutant H689P receptor differs significantly (P < 0.05) in the percent decrease in DHT binding at 40 C compared with the binding at 37 C (61 ± 5.6%), indicating a lower heat stability of the mutant AR compared with WT (32 ± 7.3%). To further characterize the nature of the binding defect, we measured the rate of dissociation of bound receptor (off-rate). As shown in Fig. 3, the mutation increases the dissociation rate of the AR about 6-fold compared with WT, strengthening the idea that the mutated receptor will not be able to stably bind its natural ligand in vivo.
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). This is consistent with the severe clinical androgen insensitivity.
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), but does not interact directly with the ligand. However, this residue is located in a tripeptide domain (Gly-His-Asp) conserved in all AR identified to date and in the structurally related PR (Fig. 6), suggesting a crucial role in receptor function. Although histidine and proline are similar in volume, substitution of this residue by proline would be expected to result in dramatic perturbation of the structure due to the imino character of the proline residue side-chain replacing the positively charged, hydrophilic histidine residue.
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Characterization of mutations in the AR gene serves as a reliable tool for the diagnosis and molecular subclassification of AIS. Knowledge of the mutation in the AR and its functional consequences will improve the management of such cases of male pseudohermaphroditism with regard to gender assignment, genital surgery, and gonadectomy. Our patient presented with the classical characteristics of the complete form of the disease, being a 46,XY individual completely feminized at birth with breast development and complete absence of axillary and pubic hair. Molecular analysis of her AR gene revealed the presence of an A to C substitution, leading to the predicted missense His to Pro mutation in the AR LBD. The His689Pro mutation has never been described, and the histidine residue is conserved among different species (mouse, rat, pig, and human). The functional characterization of this novel mutation showed that the mutant receptor has normal affinity for its physiological ligand DHT, but significantly decreased binding capacity despite comparable amounts of AR protein. This impaired binding capacity is probably due to increased instability of the hormone- receptor complex, as demonstrated by the higher thermolability of the H689P mutant receptor/DHT complex compared with the WT receptor and by the faster dissociation rate of the radioligand from the mutant receptor. Because AR is rapidly degraded in its unbound form, it may be hypothesized that at physiological peripheral DHT concentrations, the H689P mutant receptor will be unstable. Thus, although we could not perform a genital skin biopsy in our patient, it is likely that no efficient DHT binding will take place in vivo.
The trans-activation assay showed that the mutant receptor retains only 3% activation ability with DHT as ligand and close to zero with T as ligand compared with the WT counterpart. That is consistent with the severe clinical phenotype.
The recent determination of the crystal structure of the human (8) and rat (9) AR LBD in addition to detailed molecular modeling studies (13) have provided unparalleled insight into the roles of specific residues in androgen recognition and receptor dimerization. Furthermore, recent functional studies have demonstrated the importance of the AR LBD in coactivator recruitment (10). Therefore, mutations that disrupt AR LBD structure inevitably have a dramatic effect on AR function.
Mutations in the AR LBD have long been known to be a cause of CAIS. Although several substitutions resulting in CAIS have been reported in the N-terminal region of the AR LBD (described in the comprehensive McGill AR database at www.mcgill.ca/androgendb and references therein), none was previously reported at position 689. Given the highly conserved nature of these residues, they are likely to play a critical role in creating the correct structural architecture of the AR LBD. Proline residues are frequently found within helical bends of folded proteins, and Pro694 serves to introduce a bend in the loop domain linking the first and second helixes. It is likely that accommodating a second proline residue at position 689 would cause significant perturbation of helix 3, which contains residues essential for androgen binding (13), and would reduce AR function. The N-terminal region of the AR LBD composed of helixes 1 and 2 shows a high degree of conservation within AR from divergent species (Fig. 7) and with the related PR (Fig. 6). Although residues in this region of the AR do not interact directly with the hormone (8, 9, 14), each residue is likely to play an important role in ordering the structural domains containing the residues that contribute to the ligand-binding pocket. This is consistent with the observation that substitution mutations in this region (Table 2; Refs. 15, 16, 17, 18, 19, 20, 21) result most frequently in the complete form of androgen insensitivity. Our receptor binding and dissociation data suggest that residue 689 plays a crucial role in the kinetics of androgen binding, rather than in the affinity of the receptor for its ligand.
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Acknowledgments
We are grateful to Dr. Brinkmann (Rotterdam, The Netherlands) for the generous gift of the pSVAR0 construct, and to Prof. C. W. Heizmann for his continuous support.
Footnotes
Address all correspondence and requests for reprints to: Dr. Anna Biason-Lauber, Steinwiesstrasse 75, Departments of Endocrinology and Diabetology, University Children’s Hospital, 8032 Zurich, Switzerland. E-mail: .
This work was supported by the Swiss National Science Foundation (Grant 32-063629.0).
S.R. and A.B.-L. contributed equally to this work.
Abbreviations: AR, Androgen receptor; ARE, androgen responsive element; CAIS, complete androgen insensitivity; DHT, dihydrotestosterone; LBD, ligand-binding domain; PR, progesterone receptor; T, testosterone; WT, wild type.
Received January 31, 2002.
Accepted June 10, 2002.
References
This article has been cited by other articles:
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  A. Biason-Lauber, D. Konrad, F. Navratil, and E. J. Schoenle A WNT4 Mutation Associated with Mullerian-Duct Regression and Virilization in a 46,XX Woman N. Engl. J. Med., August 19, 2004; 351(8): 792 - 798. [Abstract] [Full Text] [PDF]
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) and mutant H689P (
) AR. The H689P substitution accelerates 6-fold the dissociation of DHT from the mutant receptor compared with WT. The data shown are the mean ± SD of three independent experiments.
