This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mongan, N. P. Articles by Hughes, I. A. Search for Related Content PubMed PubMed Citation Articles by Mongan, N. P. Articles by Hughes, I. A.

Two de Novo Mutations in the AR Gene Cause the Complete Androgen Insensitivity Syndrome in a Pair of Monozygotic Twins

University Department of Pediatrics, Addenbrooke’s Hospital, University of Cambridge (N.P.M., J.J., K.G., N.S., I.A.H.), Cambridge, United Kingdom CB2 2QQ; Medical Research Council, Laboratory of Molecular Biology (J.W.S.), Cambridge, United Kingdom CB2 2QH; and Great Ormond Street Hospital for Children (M.D.), London, United Kingdom WC1N 3JH

Address all correspondence and requests for reprints to: Nigel P. Mongan, Ph.D., Department of Pharmacology, Room E-409A, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021. E-mail: . n.p.e.mongan.95{at}cantab.net

Abstract

The androgen insensitivity syndrome (AIS) is the most common cause of male undermasculinization and is typically caused by mutations in the AR gene. Affected individuals may exhibit either complete external feminization (complete AIS) or a partial phenotype (partial AIS). Here we describe monozygotic twins diagnosed with complete AIS who each possess two substitutions (CG at position 2930 and TC at position 2955, both in exon 7), leading to Phe⁸⁵⁶Leu and Ser⁸⁶⁵Pro mutations, respectively. Neither parent was found to be a carrier for these mutations, indicating that the double mutation arose de novo. Both mutations were recreated by site-directed mutagenesis and compared functionally with the wild-type receptor. The Phe⁸⁵⁶Leu mutation did not affect androgen binding when expressed in COS-1 cells, nor did this mutation decrease androgen-dependent trans-activation in transfected HeLa cells. However, the Ser⁸⁶⁵Pro mutation completely ablated androgen binding and trans-activation. In this study we demonstrate that the replacement of serine by proline at position 865 is sufficient in itself to cause complete AIS in these twins. Analyses of nuclear receptor structures suggest that this mutation is likely to perturb the conformation of helix 10/11, which plays a role in ligand binding, dimerization, and receptor activation. To our knowledge this is the first confirmed instance of AIS (complete or partial) due to an AR mutation occurring in twins. Furthermore, the phenotype was associated with two mutations that were both novel in nature.

THE AR IS a ligand-dependent transcription factor that is essential for normal male embryonic development and pubertal maturation (1). The AR gene is located on the X chromosome at Xq11–12 (2), and its eight exons encode a 110-kDa protein composed of a large N-terminal domain containing a strong constitutive activation function (AF-1) and a central DNA-binding domain. The C-terminal region of the AR includes the ligand-binding domain, the ligand-dependent activation function (AF-2), and residues that enable receptor dimerization. In the presence of androgens the AR binds as a homodimer to specific DNA response elements upstream of target genes (3). The ligand-AR complex has also been shown to recruit multiple transcriptional coactivators that enhance androgen-dependent trans-activation (4).

Androgens play a crucial role in normal male fetal development and pubertal maturation. The androgen insensitivity syndrome (AIS), often caused by AR gene mutations, causes a wide variety of phenotypic abnormalities in affected males. These vary from complete feminization of the external genitalia to a partial phenotype, ranging from hypospadias and micropenis to azoospermia in the absence of any external undermasculinization (1, 5). Many different mutations in the AR that interfere with receptor dimerization, hormone binding, or association of the receptor complex with DNA are associated with AIS (6, 7). These mutations have enabled detailed characterization of the functional domains of the receptor and have provided insight into the common mechanisms of gene regulation by nuclear hormone receptors.

In this study we present two mutations, Phe⁸⁵⁶Leu and Ser⁸⁶⁵Pro (both located in exon 7 in the ligand-binding domain of the AR), in a pair of twins presenting with complete AIS (CAIS). The functional effects of each mutation on AR trans-activation were investigated, and the structural consequences were considered with reference to known nuclear receptor structures.

Subjects and Methods

Subjects

One infant of a pair of monozygotic twins (confirmed with nine polymorphic markers on nine different chromosomes) with normal female external genitalia presented with a right inguinal hernia. On further examination both twins were found to have palpable inguinal testes and 46,XY karyotype and were therefore referred to the Cambridge Intersex Database research program for further investigation. Normal androgen responses to hCG stimulation and gonadotropin responses to GnRH stimulation (both tests performed at 5 months age) and the absence of female internal genitalia were consistent with the diagnosis of CAIS (Table 1). Gonadectomy or herniotomy have not yet been performed. Local ethics committee approval and informed consent were obtained for the use of patient samples as part of a sexual development disorders research program.

Genomic DNA was extracted from peripheral blood obtained from each affected twin and their parents. Mutations in the coding regions and flanking intronic sequences of exons 2–8 and non-polymorphic regions of exon 1 were screened by single strand conformation polymorphism analysis (SSCA) as previously described (8). The AR polyglutamine triplet repeat (AR [Gln]_n) encoding region of the AR gene was screened by direct sequencing. Only the amplification products of exon 7 of the patient’s AR gene showed an abnormal SSCA banding pattern and were therefore sequenced. This revealed two mutations, which resulted in amino acid substitutions Phe⁸⁵⁶Leu and Ser⁸⁶⁵Pro, according to the numbering system of Lubahn and colleagues (9). Exon 7 of each parent was also examined by direct sequencing and was found not to possess either mutation. The prevalence of the identified Phe⁸⁵⁶Leu substitution was examined in 70 normal and 71 undermasculinized males by electrophoresis of exon 7 PCR products (Table 2) digested with RsaI (which detects the Phe⁸⁵⁶Leu mutation), by SSCA, or by direct sequencing.

Mutations were introduced into the wild-type (WT) receptor cDNA vector pSVAR (3) using site-directed mutagenesis PCR. Briefly, 2 stages of PCR were used to amplify overlapping regions of the C-terminus of the AR cDNA. Primers P1 and P4 are upstream and downstream of the AR gene EcoRI sites, respectively. Primers P2 and P3 were designed to contain the mutations to be incorporated into the AR cDNA. The first stage used 15 cycles to independently amplify P1–P2 and P3–P4 regions. For the second stage, these products were size-separated on a 1% agarose gel and recovered using a Qiaquick gel extraction kit (QIAGEN, Crawley, UK). The purified fragments were then mixed in equal ratios and used as template for the fusion PCR stage (20 cycles), using primers P1 and P4. A thermostable, proof-reading polymerase, PfuTurbo (Stratagene, La Jolla, CA), and a limited number of amplification cycles were used to reduce the probability of PCR-induced error. The sequences of oligonucleotide primers used are given in Table 2. The PCR products were sequenced in both directions to confirm the presence of the point mutation and the products digested with EcoRI. The digestion products containing the mutation were then subcloned back into the EcoRI-digested WT vector to generate a full-length AR cDNA containing the appropriate mutation. The sequences of the final mutant constructs, pSVAR-Phe⁸⁵⁶Leu and pSVAR-Ser⁸⁶⁵Pro, were confirmed by direct sequencing.

Binding studies on transfected COS-1 cells

COS-1 cells (American Type Culture Collection, Manassas, VA) were maintained in DMEM (Life Technologies, Inc., Paisley, UK) containing penicillin (50 U/ml), streptomycin (50 µg/ml), glutamine (2 mM), MEM non-essential amino acids (1x), and 10% (vol/vol) FBS (Sigma, St. Louis, MO). Twenty-four hours before transfection, 2 x 10⁶ cells were transferred onto a 15-cm plate and cultured overnight in DMEM supplemented as above, but 10% dialyzed FBS (Life Technologies, Inc., Paisley, UK) was used instead of FBS. The cells were then transfected with 10 µg mutant or WT pSVAR and 30 µl Transfast reagent as directed by the manufacturer (Promega Corp., Madison, WI). After 48 h, the cells were transferred into serum-free DMEM, and 24 h later the binding assay was performed in duplicate as described previously (10).

Trans-activation assay

HeLa cells (American Type Culture Collection) which were transferred onto six-well plates and cultured with 10% dialyzed FBS and other supplements, as described above, were transfected at 80% confluence with 1.6 µg WT or mutated pSVAR, 5 µg pGRE-luciferase (androgen- and glucocorticoid-responsive firefly luciferase reporter vector) (11), 1 µg pRL-TK, a Renilla luciferase used as an internal control of transfection efficiency (Promega Corp.), and 22 µl Transfast/well as directed by the manufacturer. After 1 h, mibolerone (4-estren-7,17-dimethyl-17ß-ol-3-one) was added to a final concentration of 0.1–2 nM. After incubation for 48 h, the cells were lysed with passive lysis buffer (Promega Corp.) and assayed using a dual luciferase assay reagent system (Promega Corp.). The ratio of firefly to Renilla luciferase units was measured using a Turner TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). Three independent experiments were performed, each in triplicate. The values of each experiment were normalized to the maximal trans-activation ratio obtained for the WT pSVAR induced with 2 nM mibolerone.

Results and Discussion

Two de novo mutations in the AR ligand-binding domain

Direct DNA sequencing revealed two novel mutations resulting in amino acid substitutions, Phe⁸⁵⁶Leu and Ser⁸⁶⁵Pro, in exon 7 of the affected individuals using the numbering system of Lubahn (9) (Fig. 1). The AR[Gln]_n of the affected individuals was 24, a value within the normal range (12). Neither parent was found to be a carrier for either mutation, indicating that these mutations arose de novo. None of the 141 controls screened was found to possess the Phe⁸⁵⁶Leu substitution. In excess of 200 AR mutations that cause the CAIS have been described (detailed information on these mutations are available at the McGill AR database (www.mcgill.ca/androgendb and references therein). Although there are rare reports of twins clinically diagnosed with testicular feminization (13, 14), this is the first confirmation of an AR gene mutation in twins resulting in CAIS.

View larger version (42K): [in this window] [in a new window]   Figure 1. DNA sequence of affected twin and unaffected mother. Partial DNA sequence chromatogram of exon 7 of the AR of one of the affected twins (A) and the sequence of the unaffected mother (B). The point mutations result in amino acid substitutions, Phe856Leu and Ser865Pro according to the codon numbering system of Lubahn et al. (9 ).

Binding assays in transfected COS-1 cells revealed that the Phe⁸⁵⁶Leu mutation did not disrupt androgen binding, and this mutated receptor exhibited a binding affinity (K_d = 1.1 nmol/liter) similar to that of the wild-type receptor (K_d = 0.9 nmol/liter). In marked contrast, the Ser⁸⁶⁵Pro mutant receptor displayed no specific androgen binding, indicating a key role for the serine residue in AR function. This was confirmed by the trans-activation assay carried out in transfected HeLa cells (Fig. 2), where the Phe⁸⁵⁶Leu substitution had no negative effect on androgen-dependent trans-activation. Indeed, at certain androgen concentrations, there was a mild enhancing effect on trans-activation compared with the WT receptor. However, the Ser⁸⁶⁵Pro mutation completely ablated androgen-dependent trans-activation, confirming a crucial role for this residue in AR action. Amino acid substitution Phe⁸⁵⁶Leu had no obvious deleterious effect on AR function, yet was not found in a control population. Thus, it is possible that this substitution has subtle functional effects in vivo that are not detected by the in vitro studies employed. It is interesting to note that single de novo mutations appear to occur at a high rate within the AR gene (15), and it is possible that this may underlie the rare accumulation of two de novo mutations found in these twins.

View larger version (28K): [in this window] [in a new window]   Figure 2. Mibolerone-induced trans-activation of the WT and two mutant AR expressed in HeLa cells. WT and mutant receptors (Phe856Leu and Ser865Pro) were transiently expressed in HeLa cells, and their trans-activation capacity was measured using the glucocorticoid response element-luciferase reporter system. The transfected cells were incubated for 48 h with 0–2 nM mibolerone (see Subjects and Methods). Each bar represents the mean ± SEM of nine assays (triplicate determinations in three independent experiments).

The structural consequences of each of these mutations were evaluated on the basis of the AR model (16) and crystal structure determined recently (17, 18). Phe⁸⁵⁶ is located near the N-terminus of helix 10/11 of the AR (Fig. 3) and is conserved in the related PR. Its side-chain is largely (70%) buried and is involved in hydrophobic packing interactions with helix 9. It is in close contact with Ile⁸³⁵, Leu⁸³⁸, and Ile⁸⁴². Substitution of this amino acid with a leucine would not be expected to be particularly deleterious, because the side-chain has a volume and character similar to those of phenylalanine. This is consistent with the empirical finding that this mutation does not perturb trans-activation by the AR.

View larger version (57K): [in this window] [in a new window]   Figure 3. Diagram showing the locations of the Phe856Leu and Ser865Pro mutations. The side-chains of residues at position 856 and 865 are indicated on the structure. The ligand R1881 (illustrated in pink), is a synthetic androgen and a close analog of mibolerone that was used in the binding and trans-activation studies described in this study. The AR structure (PDB accession = 1e3 g) (16 ) was manipulated using Swiss-PDB viewer and rendered using PovRay.

Helix 10/11 plays a key role in members of the nuclear receptor family. The C-terminal portion of the helix forms part of the walls of the ligand binding cavity. In the AR Leu⁸⁷³, Phe⁸⁷⁶, Ala⁸⁷⁷, and Leu⁸⁸⁰ make van der Waals contact with DHT. This region of helix 10/11 is also important for the positioning of helix 12, which is critical for coactivator binding. It seems likely, therefore, that any structural perturbation of helix 10/11 will render the receptor nonfunctional. This is consistent with the finding that this mutant is unable to activate transcription.

In nuclear receptors that homo- or heterodimerize, residues in the N-terminal portion of helix 10/11 of the ligand-binding domain (including Ser⁸⁶⁵) make the primary intermolecular contacts. It would seem, however, that the AR and PR cannot homodimerize in this fashion, as the interface is partly obscured by the C-terminal region beyond helix 12. This region adopts an extended conformation interacting in a groove between helix 10/11 and the loop between helexes 8 and 9. Consequently, the AR ligand-binding domain was found to be monomeric in the crystal lattice (17). The closely related PR ligand-binding domain was also found to be a monomer in solution, but crystallized as an apparent dimer. However, the PR dimerization interface does not involve the Ser⁸⁶⁵ position.

This study provides the first description of two novel AR mutations occurring in twins with AIS. The functional analysis of these mutations illustrates that the Ser⁸⁶⁵Pro mutation alone was sufficient to result in the complete AIS phenotype. Structural analysis revealed that alteration of this residue located in the AR ligand-binding domain may disrupt receptor dimerization and the precise positioning of helix 12, both of which are required for AR function. These findings confirm the cause of complete feminization in the twins and represent a further step toward defining the structural and functional roles of key residues in the AR ligand-binding domain.

Acknowledgments

We are grateful to Dr. A. Brinkmann (Erasmus University, Rotterdam, The Netherlands) for generously providing the WT AR vector, and to Dr. A. Allera (Otto von Guericke University, Magoleburg, Germany) for providing the GRE-luciferase vector.

Footnotes

This work was supported by grants from the Birth Defects Foundation (to N.P.M.), an ESPE Research Fellowship, sponsored by Novo Nordisk A/S (to J.J.), Endocrine Society Summer Fellowship (to K.G.), and the European Union BioMed Program.

Abbreviations: AIS, Androgen insensitivity syndrome; CAIS, complete androgen insensitivity syndrome; SSCA, single strand conformation polymorphism analysis; WT, wild-type.

Received September 25, 2001.

Accepted December 5, 2001.

References

1. Quigley CA, De Bellis A, Marschke KB, el-Awady MK, Wilson EM, French FS 1995 Androgen receptor defects: historical, clinical and molecular perspectives. Endocr Rev 16:271–321[CrossRef][Medline]
2. Brown CJ, Goss SJ, Lubahn DB, Joseph DR, Wilson EM, French FS, Willard HF 1989 Androgen receptor locus on the human X chromosome: regional localization to Xq11–12 and description of a DNA polymorphism. Am J Hum Genet 44:264–269[Medline]
3. Brinkmann AO, Faber PW, van Rooji HC, Kuiper GG, Ris C, Klaassen P, van der Korput JA, Voorhorst MM, van Laar JH, Mulder E, Trapman J 1989 The human androgen receptor: domain structure, genomic organisation and regulation of expression. J Steroid Biochem 34:307–310[CrossRef][Medline]
4. McKenna NJ, Lanz RB, O’ Malley BW 1999 Nuclear receptor coregulators: cellular and molecular biology. Endocr Rev 20:321–344[Abstract/Free Full Text]
5. Ahmed SF, Cheng A, Dovey L, Hawkins JR, Martin H, Rowland J, Shimura N, Tait AD, Hughes IA 2000 Phenotypic features, androgen receptor binding and mutation analysis in 278 clinical cases reported as androgen insensitivity syndrome. J Clin Endocrinol Metab 85:658–665[Abstract/Free Full Text]
6. Gottlieb B, Lehvaslaiho H, Beitel LK, Lumbroso R, Pinsky L, Trifiro M 1998 The androgen gene mutations database. Nucleic Acids Res 26:234–238[Abstract/Free Full Text]
7. Gottlieb B, Beitel LK, Lumbroso R, Pinsky L, Trifiro M 1999 Update of the androgen receptor gene mutations database. Hum Mutat 14:103–114[CrossRef][Medline]
8. Batch JA, WIlliams DM, Davies HR, Brown BD, Evans BA, Hughes IA, Patterson MN 1992 Androgen receptor gene mutations identified by SSCP in fourteen subjects with androgen insensitivity syndrome. Hum Mol Genet 1:497–503[Abstract/Free Full Text]
9. Lubahn DB, Brown TR, Simental JA, Higgs HN, Migeon CJ, Wilson EM, French FS 1989 Sequence of the intron-exon junctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity. Proc Natl Acad Sci USA 86:9534–9538[Abstract/Free Full Text]
10. Bevan CL, Brown BB, Davies HR, Evans BAJ, Hughes IA, Patterson MN 1996 Functional analysis of six androgen receptor mutations in patients with partial androgen insensitivity syndrome. Hum Mol Genet 5:265–273[Abstract/Free Full Text]
11. Knoke I, Allera A, Wieacker P 1999 Significance of the CAG repeat length in the androgen receptor gene (AR) for the transactivation function of an M780I mutant AR. Hum Genet 104:257–261[CrossRef][Medline]
12. Edwards A, Hammond HA, Jin L, Caskey CT, Chakraborty R 1992 Genetic variation in five trimeric and tetrameric tandem repeat loci in four human population groups. Genomics 12:241–253[CrossRef][Medline]
13. Marshall HK, Harder HI 1958 Testicular feminizing syndrome in male pseudohermaphrodite: report of two cases in identical twins. Obstet Gynecol 12:284–293[Free Full Text]
14. Chen FP 1996 Testicular feminization with incomplete Müllerian regression in twin patients: laparoscopic diagnosis and treatment. Acta Obstet Gynecol Scand 75:304–307[Medline]
15. Hiort O, Sinnecker GHG, Holterhus PM, Nitsche EM, Kruse K 1998 Inherited and de novo androgen receptor gene mutations: Investigation of single-case families. J Pediatr 131:939–943
16. Poujol N, Wurtz JM, Tahiri B, Lumbroso S, Nicolas JC, Moras D, Sultan C 2000 Specific recognition of androgen by their nuclear receptor. J Biol Chem 275:24022–24031[Abstract/Free Full Text]
17. Matias PM, Donner P, Coelho R, Thomaz M, Piexoto C, Macedo S, Otto N, Joschko S, Scholz P, Wegg A, Basler S, Schafer M, Egner U, Carrondo MA 2000 Structural evidence for ligand specificity in the binding domain of the human androgen receptor. J Biol Chem 275:26164–26171[Abstract/Free Full Text]
18. Sack JS, Kish KF, Wang C, Attar RM, Kiefer SE, An Y, Wu GY, Scheffler JE, Salvati ME, Krystek SR, Weinmann R, Einspahr HM 2001 Crystallographic structures of the ligand binding domains of the androgen receptor and its T877A mutant complexed with the natural agonist dihydrotestosterone. Proc Natl Acad Sci USA 98:4904–4909[Abstract/Free Full Text]

This article has been cited by other articles:

				S. E. Hannema, I. S. Scott, J. Hodapp, H. Martin, N. Coleman, J. W. Schwabe, and I. A. Hughes Residual Activity of Mutant Androgen Receptors Explains Wolffian Duct Development in the Complete Androgen Insensitivity Syndrome J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5815 - 5822. [Abstract] [Full Text] [PDF]

				N. Poujol, S. Lumbroso, B. Terouanne, J.-M. A. Lobaccaro, W. Bourguet, and C. Sultan Pathophysiology of Androgen Insensitivity Syndromes: Molecular and Structural Approaches of Natural and Engineered Androgen Receptor Mutations at Amino Acid 743 J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5793 - 5800. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mongan, N. P. Articles by Hughes, I. A. Search for Related Content PubMed PubMed Citation Articles by Mongan, N. P. Articles by Hughes, I. A.