A Novel Mutation in the D-Box of the Androgen

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Jan 22, 2016 - Phenotype and Severe PAIS. Yvonne L. Giwercmana Sten A. Ivarssonb Jonas Richthoffa. Kristina B. Lundina Aleksander Giwercmana.

Original Paper Received: April 30, 2003 Accepted after revision: September 10, 2003 Published online: December 1, 2003

Horm Res 2004;61:58–62 DOI: 10.1159/000075240

A Novel Mutation in the D-Box of the Androgen Receptor Gene (S597R) in Two Unrelated Individuals Is Associated with both Normal Phenotype and Severe PAIS Yvonne L. Giwercman a Sten A. Ivarsson b Jonas Richthoff a Kristina B. Lundin a Aleksander Giwercman a Departments of a Urology and b Paediatrics, Malmö University Hospital, Lund University, Malmö, Sweden

Abstract Background: An absent or dysfunctional androgen receptor (AR) in 46,XY individuals is the most common cause of various degrees of undermasculinization. Therefore, we routinely perform sequencing of the AR gene in all cases with suspected androgen insensitivity. Methods: In a newborn 46,XY male diagnosed with partial androgen insensitivity syndrome and a phenotypically normal man, who in childhood had bilateral cryptorchidism, the AR was directly sequenced. Seven additional men with cryptorchidism in infancy were chosen as controls. Results: An AR variant (S597R) was identified in both males. Treatment of the newborn with 1% dihydrotestosterone ointment locally, resulted in normal penile size for age. Sequencing of the region in 7 other men with cryptorchidism in infancy did not reveal any additional deviation from the normal reference sequence. Conclusion: The same mutation at this codon can cause


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significantly different phenotypes as shown by the variation in masculinization of these individuals, with 1 severely affected child and 1 normally developed man. However, the S597R mutation does not seem to be a common cause of undescended testes in boys. Despite the S597R mutation and severe undermasculinization, as seen in the baby, normal male phenotype for age could be achieved with treatment. Copyright © 2004 S. Karger AG, Basel


The androgen receptor (AR) belongs to the family of steroid hormone receptors and as the other members of this family – glucocorticoid receptor (GR), mineralocorticoid receptor (MR) and progesterone receptor (PR) – it is divided into three functional domains. The N-terminal domain, which is involved in transcriptional activation of androgen-responsive genes, the central part, which harbors a DNA-binding domain (DBD), and the C-terminal part, which consists of a hormone-binding domain [1]. The DBD of the AR, encoded by exons 2 and 3, common-

Yvonne L. Giwercman Department of Urology, Wallenberg Laboratory, Entrance 46 Malmö University Hospital, SE–205 02 Malmö (Sweden) Tel. +46 40 337904, Fax +46 40 337043 E-Mail [email protected]

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Key Words Androgen receptor W Androgen insensitivity syndrome W Mutation, S597R W D-box

Androgen Receptor Mutation Causing Different Phenotypes

Fig. 1. a Subject 1, a newborn 46,XY male, diagnosed with PAIS. He had one palpable testis, a small vaginal cavity, and perineoscrotal hypospadias. b Treatment with 1% DHT ointment was initiated during the first month of life and resulted in normalied penile size within 2 months.

Subjects and Methods Subjects Subject 1 was a newborn 46,XY individual, diagnosed with PAIS. At birth the baby was considered to be a girl with a suspected hypertrophic clitoris. Subsequently, the child was found to have a bifid scrotum and both testes were localized to the pelvic cavity by ultrasound and magnetic resonance tomography. Furthermore, ureterography disclosed a short but male-type urethra and the gender assignment was changed to a male with micropenis and hypospadias (fig. 1a). An hCG test (1,500 IU i.m.) resulted in a rise in serum testosterone (T) from 2.2 to 8.8 nM and dihydrotestosterone (DHT) from 0.08 to 0.34 nM and a swelling of the genitals was noted at inspection. Treatment with 1% DHT ointment was initiated during

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ly referred to as the zinc fingers, is the most highly conserved region among members of the steroid hormone receptor family, with approximately 80% amino acid identity [2]. Moreover, all members GR, MR, PR, and AR recognize the same hormone response elements (HREs) in the promoters of target genes [3]. The ability to determine the specificity of AR interaction with DNA resides in three amino acids at the base of the first zinc finger (glycine 577, serine 578 and valine 581), located in the so-called P-box [2, 4]. These three amino acids are identical in the GR, MR and PR. The second zinc finger stabilizes DNA receptor interaction by contacting the DNA phosphate backbone [5]. In this part of the AR, a five amino acid residue long distal box (D-box), which is participating in forming a dimerizing interface for receptor monomers and in specifying the half-site spacing requisite at HREs, has been identified [6]. However, other domains of the AR might also be involved in hormonedependent dimerization [5, 7]. Deficient function of the AR is the cause of the androgen insensitivity syndrome (AIS), characterized by varying degrees of incompletely virilized genital phenotypes in karyotypic males (46,XY) [8]. In its most severe form, complete AIS (CAIS), affected individuals present with female external genitalia apart from a lack of pubic and axillary hair [9]. Individuals with partial forms (PAIS) present with undermasculinization ranging from almost female appearance to primarily male with undervirilization such as hypospadias or micropenis and even men without genital malformations, but with gynecomastia and/or hampered virilization at the other end of the spectrum [10, 11]. Mutations in the P-box have exclusively been associated with CAIS, whereas mutations in the D-box seem to cause milder forms of androgen resistance, seen as PAIS (AR mutation database: http://www. mcgill.ca/androgendb/). Successful supplementation, in attempts to improve virilization, has been described in only a few cases with mutations in the DBD [9]. We have found a mutation in the D-box of the AR (S597R) in 2 unrelated males – in a normal man, with cryptorchidism in childhood as the only manifestation of reduced androgen sensitivity, and in a newborn baby with PAIS. The newborn boy showed significant response to hCG stimulation and subsequent androgen treatment resulted in normalized penile size.

Big Dye Primer Cycle Sequencing Ready Reaction Kit and the ABI Prism310 DNA sequencer (PE Corp., Foster City, Calif., USA). Semen Analysis Subject 2 delivered one semen sample. The ejaculate was obtained by masturbation after 72 h of sexual abstinence. The assessment of volume, concentration, motility and morphology was performed as recommended by the World Health Organization (WHO). The study was approved by the local ethical committee.


In the first zinc cluster, the P-box, which is involved in HRE recognition, is indicated. In the second zinc cluster, the D-box, which is involved in dimerization and HRE half site recognition, is indicated. Serine 597 is marked with an arrow.

his first month of life and resulted in penile growth to 20 mm within 2 months (fig. 1b). The pregnancy was normal and the mother of the boy was healthy during the whole period. Both parents were of Swedish origin and there was no consanguinity between them. The father has a normal boy from a previous marriage. Blood samples were available from both the mother and grandmother of subject 1. Subject 2 was an 18-year-old (46,XY) military conscript, who initially participated as a voluntary in a study of reproductive function in young Swedish males. He was selected as a normal sample for establishment of sequencing conditions of the AR gene. The man presented with normal body proportions and normal hair distribution. Genital examination revealed pubic hair at Tanner stage VI and genital development at Tanner stage V, including normal penile size and without genital abnormalities. At the time of birth he had suffered from bilateral cryptorchidism, but both testes had descended spontaneously before 1 year of age and were at the time of clinical examination located in the scrotum. The testis volume was 20 cm3 for the left and 25 cm3 for the right testis. His sperm output was normal (208 ! 106/ml) and endocrine evaluation revealed FSH, LH, sex hormone-binding globulin (SHBG), and estradiol values within normal range in the circulation. Inhibin B was slightly elevated. In addition, 7 military conscripts with self-reported cryptorchidism at birth were selected to study whether this particular mutation (S597R) is a common cause of testicular undescent. Sequencing of the AR Gene Genomic DNA was prepared from peripheral leukocytes. For sequencing of the AR gene, PCR amplification of the whole gene was performed, using sets of flanking intronic primers at concentrations of 0.5 ÌM. Each 50-Ìl reaction was done using 50 ng DNA, 1.0 mM MgCl2, 0.2 mM dNTP, 50 mM KCl, 10 mM Tris-HCl (pH 8.4 at 70 ° C), and 0.1% Tween 20. Amplification was performed for 30 cycles; each cycle included denaturation for 1 min at 96 ° C, primer annealing at 54–58 ° C for 30 s, and primer extension for 3–5 min at 72 ° C. One microliter of each PCR product was used for subsequent nested PCR. The DNA was analyzed by cyclic sequencing using the


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Sequencing of the AR Gene In both subjects 1 and 2, who are unrelated, an AGC→AGG substitution was found at codon 597, causing an amino acid change from serine to arginine (S597R). The mutation is localized in the D-box, in exon 3, of the AR gene (fig. 2). DNA was also available from the mother and grandmother of subject 1. Sequencing of the region revealed that both women were carriers of the S597R mutation. In subject 1 a CAG repeat length of 23 and a GGN repeat length of 23 were found. Subject 2 had 21 CAG and 24 GGN. No deviations from the normal reference sequence of exon 3 were found in any of the other 7 conscripts with cryptorchidism. Semen Analysis For subject 2 the mean values of seminal volume, sperm concentration, and percentage progressively motile spermatozoa were as follows: 1.6 ml, 208.0 ! 106/ml, and 24%. For comparison the reference values, as recommended by WHO, are: volume 62.0 ml, concentration 620 ! 106/ml, and progressively motile 650%. Hormone Analyses In subject 1, a rise in T from 2.2 to 8.8 nM and DHT from 0.08 to 0.34 nM was seen after stimulation with hCG (1,500 IU i.m.), indicating normal testicular function. In subject 2, all hormone concentrations were within normal levels, except for inhibin B which was elevated: T 23.0 nM (ref interval 8.7–33.0 nM ), FSH 0.49 Ìg/l (ref interval 0.2–2.1 Ìg/l), LH 0.55 Ìg/l (ref interval 0.2– 1.6 Ìg/l), SHBG 25 nM (ref interval 13–65 nM), estradiol 85 pM (ref interval 60–150 pM), and inhibin B 279 ng/l (100–240 ng/l). Prostate-specific antigen was measured in ejaculate, with a value of 627 mg/l. Normal for this hormone is 230–1,485 mg/l in ejaculate.

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Fig. 2. The AR DNA-binding domain, arranged as two zinc fingers.

In order to establish sequencing conditions for the AR gene, 3 military conscripts, among 302 others, were randomly chosen as normal controls. They initially participated as voluntaries in a study of reproductive function in young Swedish males. Unexpectedly, a missense mutation was found in the D-box of the AR gene in 1 of the young men, changing serine to arginine at codon 597 (S597R). In this man, cryptorchidism in childhood was the only manifestation of possible androgen resistance. Notably, he also had normal sperm output, although slightly reduced volume and motility. Exactly the same amino acid substitution was later found in a newborn boy with severe hypospadias and the testes located in the pelvic cavity. Currently, 59 missense mutations have been reported in the DBD, of which 5 were found in the D-box, 3 affecting codon 596 and 2 codon 597. All cases with D-box mutations were diagnosed with PAIS (AR mutation database: http://www.mcgill.ca/androgendb/). Some individuals, affected with PAIS, show a significant response to androgen treatment, in particular if the mutations are located in the LBD of the AR [12–15]. In vitro studies of receptors with mutated LBD have supported the in vivo situations, as supraphysiological concentrations of hormones have been shown to improve, or in some cases even overcome, the mutation [12–14]. However, this phenomenon has not frequently been reported among cases with mutations in the DBD and treatment of this category of patients with high doses of hormones has only occasionally been tried [16]. Encouraged by the fact that despite the genetic defect, as seen in the military conscript, normal male development could occur, treatment with DHT ointment locally was tried in the newborn boy. Within 2 months the penile size was normal for age. Serine 597 has earlier been described as mutated to threonine (S597T) and glycine (S597G) respectively. The S597T mutation was found in 7 cases with hypospadias and cryptorchidism [17], whereas the S597G mutation was present in a patient diagnosed with PAIS [18]. This individual had two separate mutations in exon 3, S597G and in addition R617P. The R617P mutation has earlier been described in a patient with CAIS [19]. The mutant, recreated in vitro, was able to bind ligand in a normal fashion but not to activate the transcription of the androgen-responsive mouse mammary tumor virus promoter in co-transfection studies [18]. Interestingly, in accordance with the phenotypes seen in the patients, the presence of the S597G substitution appeared to compensate for the

Androgen Receptor Mutation Causing Different Phenotypes

loss of DNA binding caused by mutation R617P, since the receptor containing both aberrations was able to partially restore DNA-binding activity. Furthermore, although there was no mutation present in the LBD, an accelerated rate of dissociation of DHT from the double mutated AR was observed with both the endogenous and recreated mutant ARs in vitro. These findings propose that mutations in amino acid residues crucial to the binding of the AR to target DNA sequences are a common cause of receptor-binding positive androgen resistance and that variable impairment of DNA binding can lead to distinctive phenotypes. Moreover, the results indicate that despite the mutations location in the DBD, androgen-binding kinetics to the LBD can be changed and that high doses of androgens could stabilize the AR-hormone complex sufficiently to enable proper DNA recognition, in spite of suboptimal D-box function. An intriguing question is, how distinct biological effects are exerted by different steroids when steroid hormone receptors share very conserved DNA-binding domains, bind to similar DNA sequences, and although more than one SHR is present in a particular cell. Specificity might be introduced by tissue-specific metabolism of hormones, expression levels of receptors, the presence of specific co-activators, interaction with other nuclear receptors or through HREs that are organized differently than the generally accepted palindromic array [20–22]. Interestingly, serine 597 is conserved between mouse, monkey, rabbit and human, but not among the members of the steroid hormone receptor family, as the other members (GR, PR, and MR) all have glycine (G) at codon 597. This could propose that this particular amino acid contributes to the specificity of the DBD of the AR, but is not an absolute prerequisite for its function. Furthermore, since the mutation S597R was not present in any of the other 7 military conscripts with self-reported cryptorchidism in childhood, we therefore do not believe that it is a common cause of undescended testes in boys. Rather this mutation, as many others in the AR gene, can give rise to different effects even within families, implying that other factors are co-acting to determine the final phenotype. Hence, it could be suggested that different phenotypes, as seen in our study, could be the results of variations in genes encoding for those co-factors. Furthermore, mutations in the AR gene, as the one reported by us, might make the fetus more sensitive to the intrauterine endocrine milieu, the variations in the latter a possible explanation for the diverging phenotype in individuals presenting with identical AR genotype.

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In conclusion, the same mutation at codon 597 of the AR can cause different phenotypes as shown by the variation in masculinization of 2 individuals, 1 normally developed man with cryptorchidism in childhood as the only sign of androgen resistance and 1 severely affected child with micropenis and hypospadias. Furthermore, the newborn boy responded well to local application of DHT ointment, proving that there indeed is a marked sensitivity to androgens. A long-term follow-up is needed, but it is important for the life quality of affected baby boys with PAIS, in some cases to consider treatment.

Acknowledgments This study was supported by grants from the Swedish Governmental Funding for Clinical Research, the Crafoordska Fund, Ove Tulefjords Fund, the Foundation for Urological Research, the Faculty of Medicine, University of Lund, and the Health Services Administration, Malmö University Hospital.



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