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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-0372
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 9 3515-3520
Copyright © 2006 by The Endocrine Society

The A645D Mutation in the Hinge Region of the Human Androgen Receptor (AR) Gene Modulates AR Activity, Depending on the Context of the Polymorphic Glutamine and Glycine Repeats

Ralf Werner, Paul-Martin Holterhus, Gerhard Binder, Hans-Peter Schwarz, Michel Morlot, Dagmar Struve, Christine Marschke and Olaf Hiort

Departments of Pediatric and Adolescent Medicine (R.W., D.S., C.M., O.H.), University of Lübeck, 23538 Lübeck, Germany; Departments of Pediatric and Adolescent Medicine (P.-M.H.), Christian-Albrechts University, D-24105 Kiel, Germany; Departments of Pediatric and Adolescent Medicine (G.B.), Eberhard-Karls-University of Tübingen, D-72076 Tübingen, Germany; Departments of Pediatric and Adolescent Medicine (H.-P.S.), Ludwig-Maximilians University of Munich, D-80539 Munich, Germany; and Departments of Pediatric and Adolescent Medicine (M.M.), Endokrinologikum, D-30161 Hannover, Germany

Address all correspondence and requests for reprints to: Professor Dr. Olaf Hiort, Department of Pediatric and Adolescent Medicine, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. E-mail: hiort{at}paedia.ukl.mu-luebeck.de.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Background: Sufficient androgen receptor (AR) activity is crucial for normal male sexual differentiation. Here we report on two unrelated 46, XY patients suffering from undervirilization and genital malformations. Both patients had a short polyglycine (polyG) repeat of 10 residues and a relatively long polyglutamine (polyQ) repeat of 28 and 30 residues within the transactivation domain of the AR. In addition, they also harbor a rare A645D substitution.

Objective: We made a set of AR expression plasmid constructs with varying polyQ and polyG tract sizes in context with or without the A645D substitution and analyzed their in vitro transactivation capacity in transfected CHO cells.

Results: We found that a short polyG repeat downmodulated AR activity to approximately 60–65% of the wild-type receptor. This effect was aggravated by A645D in context of a long polyQ repeat to less than 50% activity. In contrast, in the context of a short polyQ and a short polyG repeat, the A645D mutation rescues AR activity to almost wild-type levels, demonstrating a contradictory effect of this mutation, depending on the size of the polymorphic repeats.

Conclusions: A combination of a short polyG repeat with a long polyQ repeat and an A645D substitution might contribute to the development of virilization disorders and explain the observed phenotypes of our patients as a form of androgen insensitivity. The whole recreation of AR sequence variations including individual polymorphic repeat sizes could unravel possible interference of mutations and variations on AR activity by in vitro transfection.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
ANDROGEN INSENSITIVITY (AIS) describes a clinical entity of variable virilization deficit in 46, XY individuals due to abnormalities in the molecular pathways of androgen action. It is usually caused by mutations in the androgen receptor (AR). The study of the molecular defects induced by mutations of the AR has greatly enhanced our knowledge of the biology of sex differentiation and functional aspects of the AR. However, it is difficult to find explanations for the phenotypic variability in patients with AIS, even if they bear the same mutation in the AR gene.

The AR belongs to the family of ligand-dependent nuclear steroid receptors and consists of three major domains: an N-terminal activation domain (NTD), a DNA-binding domain (DBD), and a ligand-binding domain (LBD). The DBD and LBD are connected by a hinge region (HR), which is defined by residues 628–669 (1). Although the DBD and LBD have been extensively studied, much less is known about the properties of the HR and the NTD.

The NTD is particular in that it harbors two polymorphic repeats, a polyglutamine encoded by a (CAG)nCAA trinucleotide repeat consisting of 9–36 amino acid residues and a polyglycine encoded by a (GGN)n stretch, consisting of 10–27 glycine residues within the normal population. The polymorphic repeats have been shown to play a prominent role in cofactor binding and N-C-terminal interaction of the receptor (2, 3, 4). For the HR, recent studies revealed evidence that it is a multifunctional domain that plays a role in nuclear trafficking (5, 6) but is also involved in the binding of AR cofactors and corepressors (7, 8, 9, 10) and the modulation of transactivation (1).

We here describe two unrelated 46, XY children with severe genital malformations suspective of AIS. We found a contraction of the AR-polyglycine (polyG) repeat to 10 residues in combination with a long polyglutamine (polyQ) repeat length of 28–30 residues in addition to a rare amino acid substitution A645D within the HR. Although short polyG repeats of 10 residues have been reported in normal populations (11, 12), these alleles are very rare. The A645D mutation was first detected by us (13) in one of our patients with partial AIS syndrome reported here. Later this mutation was described in a boy with a normal phenotype (14). Therefore, it must be presumed that genetic variations affecting the polymorphic repeats or the HR may be associated with a variable, even normal phenotype, calling for functional studies to explain their impact on AR function.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patients

All studies were approved by the Ethical Committee of the University of Lübeck, and the parents of the patients consented to molecular genetic studies.

Patient 1 was first presented at the age of 6 months with a micropenis, penile hypospadias, and severe chordae, corresponding to AIS grade 2b according to Sinnecker et al. (15). Mullerian structures were not visualized on ultrasound. At the age of 15 months, basal gonadotrophin concentrations as well as testosterone levels (LH < 1.5 IU/liter and FSH < 1.5 IU/liter, testosterone < 0.3 nmol/liter) were undetectable. After seven injections of 1.500 IU human chorionic gonadotropin (hCG) basal testosterone increased significantly to 7.3 nmol/liter, which made a defect of testosterone biosynthesis or testicular dysgenesis unlikely. The basal SHBG concentration in serum was 124.3 nmol/liter (normal range 76–137.4 nmol/liter). After stanozolol administration according to a published standardized protocol (15, 16), SHBG decreased to 56.9 nmol/liter (45% of the initial value), indicating a normal hepatic androgen response. AR ligand binding kinetics was estimated from genital skin fibroblasts obtained from genital surgery. Binding analysis was performed as reported previously (17). Molecular genetic investigation of both the 5{alpha}-reductase type II gene (SRD5A2) and AR gene was initiated. Within the coding region of SRD5A2, no genetic variations were demonstrated, making 5{alpha}-reductase type II deficiency unlikely. Analysis of the AR revealed an A645D substitution in exon 4 in conjunction with a long Q30 and a short G10 repeat in exon 1. The family history was uninformative, and further members were not available for testing.

Patient 2 was first presented at the age of 3.5 months. The child had ambiguous external genitalia with a micropenis of 1 cm stretched length, penile hypospadias, cryptorchidism on the right side, and a vaginal pouch with a long urogenital sinus. The phenotype corresponded to AIS grade 2b (15). LH was 0.9 mU/ml and FSH 3.8 mU/ml. Testosterone was determined after stimulation with hCG and was 16.4 nmol/liter. Furthermore, 5{alpha}-dihydrotestosterone (DHT) rose to 2.4 nmol/liter. These results ruled out an androgen biosynthesis defect as well as a 5{alpha}-reductase type II deficiency. The family history was negative; however, the mother gave consent for investigation of the carrier status of the mutations. After a combined treatment consisting of four doses of testosterone enanthate every 3 wk and afterward local treatment with 2.5% DHT cream two times daily, penile size increased significantly and strengthened the decision to raise the child as a boy. SRD5A2 and AR genes were investigated from blood DNA. Whereas a normal sequence of the SRD5A2 gene confirmed the results of hCG testing, the analysis of the AR gene revealed the A645D variant in addition to a long Q28 and a short G10 repeat. The mother was heterozygote for these variations.

Plasmids

We introduced the GGN-repeat sequence (GGT)3GGG(GGT)2(GGC)4 encoding 10 glycine residues into the AR expression vector pSVAR0 (a generous gift from Dr. Albert Brinkmann, Rotterdam, The Netherlands) (18). A 456-bp fragment was amplified from genomic DNA of patient 1 by Pfu-polymerase using the primers 5'-TCGCGACTACTACAACTTTCC-3' and 5'-GCCAGGGTACCACACATCAGGT-3'. Subsequently a BstEII-KpnI DNA fragment containing the shortened GGN-repeat was excised from the amplicon and ligated via the same restriction sites into pSVAR0, now termed pARQ20G10. The plasmid pSVAR0 itself has a polyQ tract length of 20 glutamine residues (CAG19CAA) and the GGN sequence (GGT)3GGG(GGT)2(GGC)10 and was included into the studies termed pARQ20G16. A different wild-type AR plasmid was obtained from Liao and colleagues (19) because it contains a longer (GGT)3GGG(GGT)3(GGC)18 fragment. It served for generation of pARQ20G25 via the same restriction sites. The Q20G25 construct reflects the upper range of the polyG repeat and is close to the most common polyG repeat length of 23 and 24 repeats, whereas the Q20G16 construct reflects the lower polyG repeat length of more than 95% of the European population. The plasmid pARQ20G0 was generated by a two-step PCR: the flanking regions of the GGN repeat of plasmid pARQ20G16 were amplified in two reactions using the primer pairs hAR-GGN-1s: 5'-GCA AGA GCA CTG AAG ATA CTG C-3' and hAR-GGN0–1: 5'-AGG GGG CTA CAG CTC CCG CCT CAC ACG GTC CAT ACA ACT GGC CT-3' for reaction 1 and hAR-GGN0–2: 5'-AGG CCA GTT GTA GGC GGG AGC TGT AGC CCC CT-3' and hAR-GGN-2A: 5'-CAT CAA AGA ATT TTT GAT TTT TCA GC-3' for reaction 2. The two amplification products were gel purified, denatured, annealed, and amplified in a third PCR using the primer pair hAR-GGN-1S and hAR-GGN2A. The resulting 1490-bp PCR fragment was digested with BstEII and KpnI and ligated into pARQ20G16. Thus, the four expression vectors were identical except for defined differences in GGN repeat length.

A polyQ tract of 30 residues was amplified from the DNA of patient 1 using the primer pair CAG-RW1s (5'-TGG TTC GTC CCG CAA GTT TC-3') and CAG-RW1a (5'-GCC TCG CTC AGG ATG TCT TTA-3') and PyroBest Taq-DNA-polymerase (Takara, Shiga, Japan). The amplicon was subcloned using the Zero Blunt TOPO PCR cloning kit (Invitrogen, Karlsruhe, Germany). The correct size of the polyQ repeat was verified by sequencing. The pARQ20G10 construct as well as the pCR4 vector containing the AR fragment with the Q30 repeat were propagated in the methylation-deficient Escherichia coli strain GM2163. Subsequently both vectors were digested with NheI and SexAI, and the Q30 fragment was ligated into pARQ20G10 resulting in a construct termed pARQ30G10.

The A645D variation was introduced into pSVAR0 using the QuikChange site-directed mutagenesis kit (Stratagene, Amsterdam, The Netherlands) and the primer pair AR645Mut_Sense (5'-GGA GGA AGG AGA GGA TTC CAG CAC CAC CAG C-3') and AR645Mut_Antisense (5'-GCT GGT GGT GCT GGA ATC CTC TCC TTC CTC C-3'). Finally, we also introduced the A645D mutation into pARQ20G0, pARQ20G10, pARQ20G25, and pARQ30G10 by cloning via the KpnI and BamHI sites. All introduced fragments as well as their near cloning borders were verified by plasmid sequencing.

Transient transfection assays

Transient transfections were preformed in Chinese hamster ovary (CHO) cells, which were maintained in 5% CO2 at 37 C in DMEM with the nutrient mix F-12 (Sigma, Taufkirchen, Germany) supplemented with 10% charcoal-stripped fetal calf serum (PAA Laboratories GmbH, Coelbe, Germany), penicillin (100 U/ ml), and streptomycin (100 µg/ ml; Sigma). Cells were transfected using the multicomponent lipid-based transfection reagent FuGENE 6 (Roche, Mannheim, Germany) according to instructions of the manufacturer. Activation of the androgen-responsive (ARE)2TATA luciferase reporter plasmid (a generous gift from Dr. G. Jenster, Rotterdam, The Netherlands) (20) due to our pAR expression constructs was investigated using 0–100 nmol/liter DHT or testosterone as indicated. All transfections were performed in triplicate, and at least three independent experiments were performed. Luciferase activity was determined using the Promega luciferase assay system (Promega Corp., Madison, WI). For means of comparison of experiments performed at different days, reporter gene induction due to pARQ20G16 and 10 nM DHT was defined to be 100%. All other induction values obtained in the same experiment were expressed relative to this value.

Western blotting

For protein analysis 3 x 105 CHO cells/well were grown overnight in six-well plates. Cells were transfected using 1 µg of (ARE)2TATA-Luc reporter plasmid, 150 ng of the respective AR expression plasmid, 25 ng of the renilla luciferase plasmid phRG-TK, and 2.35 µl Fugene 6. After 5 h cells were incubated with 10 nM DHT. Eighteen hours later cells were washed twice with PBS and harvested by adding 400 µl/well of M-Per protein extraction reagent (Pierce, Rockford, IL) containing 2 µg/ml aprotinin and 5 µg/ml leupeptin (Sigma). The cells were incubated on a shaker for a further 5 min, and cellular debris was pelleted by centrifugation for 10 min at 14.000 x g. Subsequently 25 µl of each lysate was analyzed in a dual-luciferase assay (Promega). Protein concentration was estimated according to Bradford using the Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA). Sample amounts were corrected for transfection efficiency using the renilla luciferase reporter expression and separated by SDS-PAGE. Western blots were detected with primary AR antibody F39.4.1 (Innogenex, San Ramon, CA) diluted 1:600 and a horseradish peroxidase-conjugated secondary antibody applying the Western Lightning Chemiluminescent Reagent Plus (PerkinElmer, Boston, MA). Relative AR amounts were estimated by densitometric scanning of signals on Hyperfilm ECL (Amersham Biosciences, Freiburg, Germany) using the quantification software package Quantity One (version 4.2.3; Bio-Rad).

Statistics

Statistical analysis was performed using the software package SigmaStat 3.0 (SPSS GmbH, Munich, Germany). Graphs were plotted using the software package Prism4 (GraphPad Software Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Effect of variation of polyG-length on AR transactivation

For the analysis of the impact of the polyG repeat length on AR transactivation capacity, four AR expression plasmids containing a deletion of the GGN repeat or 10, 16, or 25 GGN repeats were constructed under perpetuation of a CAG stretch coding for 20 glutamine residues (Fig. 1Go). The proper expression of the four plasmids was verified by Western immunoblotting of transient-transfected CHO cells using a monoclonal antibody directed against amino acids 301–320 of the human AR (SP61). All constructs showed a similar AR signal of approximately 110/112 kDa (Fig. 2AGo, inset).


Figure 1
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  		FIG. 1. Graphical display of the AR constructs. The lengths and positions of the different polyQ and polyG tracts within the N-terminal domain and the A645D mutation are shown.

 

Figure 2
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  		FIG. 2. Transcriptional activation of the AR depends on polyG repeat length. A, CHO cells were transiently cotransfected with the androgen-responsive firefly-luciferase reporter construct p(ARE)2TATA-Luc, the constitutively expressed renilla luciferase plasmid phRG-TK, and AR constructs containing a deletion of the polyG repeat (Q20G0) or a repeat of 10, 16, or 25 glycine residues. Five hours after transfection, cells were incubated for 18 h with vehicle alone or different concentrations (0.001–100 nM) DHT. Transfection efficiencies were normalized using the dual-luciferase assay kit (Promega). The activity of the ARQ20G16 construct in the presence of 10 nM DHT was set to 100%. Results are averages of at least seven separate experiments performed in triplicates (21 data points). Error bars, +1 SD. The AR activity between Q20G10 and Q20G25 constructs is significantly different (indicated by *, Student’s t test, P < 0.001). RLU, Relative luciferase units. Inset, The respective AR expression plasmids were also analyzed by immunoblot. The four plasmids are expressed to a similar level; a slight difference in AR size due to the different polyG tract length can be seen. Gel loadings were corrected for transfection efficiency by measurement of cotransfected constitutively expressed renilla luciferase. Relative levels of AR expression were estimated by densitometric analysis of the immunoblot. B, Dose-response curves of AR constructs with different polyG repeat length.

 
These four constructs were analyzed in transient expression assays using the androgen-responsive (ARE)2TATA-Luc reporter plasmid (20). Reporter gene activation of transfected cells was measured after treatment with vehicle alone or vehicle with 0.001–100 nM of DHT (Fig. 2Go) and testosterone (data not shown), respectively. All constructs showed a clear concentration-dependent induction of the androgen-responsive luciferase reporter, regardless which of the two steroid hormones had been used for induction. In both cases, shortening of the polyG stretch leads to a gradual reduction of AR transactivation capacity in transfected CHO cells with a higher transactivation at low concentrations with the more potent DHT. The transactivation capacity of the naturally occurring polyG10 variation in the Q20G10 construct was reduced by 34–41%, compared with Q20G25, over a range of four magnitudes of DHT (Fig. 2AGo) and testosterone concentration (data not shown). These differences were highly significant at all hormone values between 0.1 and 100 nM (Student’s t test, P < 0.001). The artificial Q20G0 mutant showed a further reduction to approximately half of the activity of the Q20G25 construct, demonstrating once more the increasing AR activity with increasing polyG-tract length. We also analyzed whether the polyG repeat length has an influence on the dose-response curves of our constructs (Fig. 2BGo). All four constructs showed the same dose-response curve for DHT with an EC50 of 1.06 x 10–10 M (95% confidence interval 9.27 x 10–11 to 1.21 x 10–10) or testosterone with an EC50 of 2.99 x 10–10 M (95% confidence interval 2.65 x 10–10 to 3.37 x 10–10, graph not shown).

Influence of the A645D mutation on AR transactivity

In a second set of experiments, we analyzed the impact of the A645D mutation on the transactivation capacity of AR constructs with various polyG tract length (Fig. 3Go). Therefore, we introduced the A645D mutation in our four polyG constructs with a constant polyQ tract length of 20 residues. Interestingly, the A645D mutation always improved the transactivation capacity of our constructs on the (ARE)2TATA promoter over a range of four magnitudes of DHT concentrations. This is most obvious and statistically significant for the Q20G0 and Q20G10 constructs (t test; P < 0.01). Similar results were received with the ligand testosterone (data not shown). In the case of the Q20G10 construct, the A645D mutation rescues AR transactivation capacity to almost the level of the Q20G16 construct.


Figure 3
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  		FIG. 3. Impact of the A645D mutation on AR activity of constructs with various polyG repeat length. CHO cells were transiently cotransfected with the androgen-responsive firefly-luciferase reporter construct p(ARE)2TATA-Luc, the constitutively expressed renilla luciferase plasmid phRG-TK, and AR constructs containing a polyQ repeat of 20 residues, a polyG repeat of 0, 10, 16, or 25 glycine residues, in combination with or without the A645D mutation. Five hours after transfection, cells were incubated for 18 h with vehicle alone or different concentrations (0.001–100 nM) of DHT. Transfection efficiencies were normalized using the dual-luciferase assay kit (Promega). The activity of the AR-Q20G16 construct in the presence of 10 nM DHT was set to 100%. Results are averages of at least three separate experiments performed in triplicates. RLU, Relative luciferase units. For statistical analysis, error bars indicate +1 SD (Student’s t test; *, P < 0.01.

 
At last we combined the three mutations found in our patients and introduced the Q30 repeat of patient 1 as well as the A645D mutation into our G10 construct, resembling the situation in our patient (Fig. 4Go, white bar). This combination of all three variations found in our patient leads to over 50% reduction of activity, compared with our wild-type construct Q20G25 (Fig. 4Go, dark gray bar). Dissection of this combination revealed that truncation of the polyG repeat from 25 down to 10 residues under perpetuation of a Q20 repeat already diminished the hormone-induced expression of the reporter gene significantly (t test, P < 0.001) by approximately 34–41% (Q20G10, Fig. 4Go, lower right to upper left hatched bar). This effect was not further aggravated by introduction of a lengthened polyQ repeat with 30 glutamine residues, the repeat size found in our patient (Q30G10, Fig. 4Go, lower left to upper right hatched bar). Interestingly, the introduction of the A645D mutation into the Q30G20 construct (Q30G20A645D, Fig. 4Go, white bars) leads to a further reduction in transactivation of the reporter gene to less than 50% of the level of the Q20G25 construct (Fig. 4Go, dark gray bars). The difference between Q30-G10 and Q30G10-A645D at 1, 10, and 100 nM DHT was statistically significant (t test, P < 0.005). This was completely different from constructs with a polyQ repeat of 20 residues, in which the A645D mutation always improved the transactivation capacity to a greater (Fig. 3Go, Q20G0, Q20G10) or lesser (Fig. 3Go, Q20G16, Q20G25) extent. Therefore, in combination of a long Q30 repeat and a short G10 repeat, the A645D mutation can aggravate the diminishing effect on AR transactivation but in context of a short Q20 repeat the A645D mutation could rescue partly the negative effect of a G10 repeat (Fig. 4Go, Q30G10-A645D, white bars, and Q20G10-A645D, black bars). The difference between Q30-G10-A645D and Q20-G10-A645D at 1, 10, and 100 nM DHT is statistically significant (t test, P < 0.001).


Figure 4
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  		FIG. 4. The A645D modulates AR transactivation capacity, depending on the context of the polyQ and polyG repeat sizes. CHO cells were transiently cotransfected with the androgen-responsive firefly luciferase reporter construct p(ARE)2TATA-Luc, the constitutively expressed renilla luciferase plasmid phRG-TK, and AR constructs containing polyQ repeats of 20 or 30 residues, a polyG repeat of 10, 16, or 25 glycine residues, in combination with or without the A645D mutation. Five hours after transfection, cells were incubated for 18 h with vehicle alone or different concentrations (0.001–100 nM) of DHT. Transfection efficiencies were normalized using the dual-luciferase assay kit (Promega). The activity of the ARQ20G16 construct in the presence of 10 nM DHT was set to 100%. Results are averages of at least three separate experiments performed in triplicate. RLU, Relative luciferase units. For statistical analysis, error bars indicate +1 SD (Student’s t test; *, P < 0.001; X, P < 0.005.

 
Ligand binding characteristics

We analyzed the influence of the A645D mutation in context of a Q30 and G10 repeat on AR ligand binding kinetics of patient 1 using genital skin fibroblasts. Binding analysis performed with the synthetic androgen methyltrienolone as a ligand revealed a normal maximal binding capacity (Bmax) of 35.11 fmol/mg protein and dissociation constant (Kd) of 0.09 nM (Fig. 5Go) (normal range: Bmax, 13.35–115.98 fmol/mg protein; Kd, 0.03–0.13 nM).


Figure 5
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  		FIG. 5. Saturation binding curve and Scatchard plot of the AR of patient 1 using genital skin fibroblasts and methyltrienolone (R1881) as a ligand. Bmax = 35.11 fmol/mg protein; Kd = 0.09 nM.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
We describe two unrelated 46, XY patients with a severe virilization deficit who clinically fulfilled criteria for AIS. In patient 1, an SHBG AIS test had been performed, giving a normal decrease of SHBG after intake of stanozolol. However, patients with partial AIS may escape from this diagnostic strategy due to lacking sensitivity, and this procedure cannot entirely exclude an AR defect (15). Therefore, molecular genetic analysis of the AR gene as the most sensitive diagnostic tool for the diagnosis of AIS in childhood had been initiated in these patients.

Interestingly, both patients had a similar combination of polymorphic repeat variations within the NTD, namely a shortening of the polyG repeat to 10 residues and a relative long polyQ repeat of 28 or 30 residues and a rare A645D mutation in exon 4. The amino acid substitution A645D has been described in individuals with highly variable phenotypes ranging from complete AIS to normal genital appearance. In the one case of an A645D substitution described recently in a patient with complete AIS, a R752Q mutation (21) also was demonstrated. Because there are several reports of complete AIS syndrome patients carrying a R752Q mutation (22, 23, 24), it can be concluded that the severe malfunction of the AR is due to R752Q rather than the double-mutant R752Q/A645D, and the impact of A645D on AR functionality may in fact be low; however, functional studies have been lacking. This opinion is further enhanced by the description of a phenotypically normal boy bearing the A645D variation (14). For the polymorphic repeats within the NTD, several studies (11, 12, 25, 26) determined the normal ranges. Although a polyQ repeat length of 28 or 30 residues is reported to be at the upper end of the normal range within human populations (25, 26), the impact of the polyQ repeat length on AR transcriptional activity has been established well in in vitro studies (27, 28, 29, 30). Much less is known about the polyG repeat; however, reports on short repeats of 10 residues are rare (11, 12). In our own studies of normal and infertile males, neither a G10 nor an A645D substitution has been found, making it a seemingly rare variation in the normal German population (31). Therefore, we aimed at investigating all three genetic variations, the polymorphic Q and G repeats in conjunction with the A645D substitution, in their impact on AR function in comparison with the wild-type allele Q20G25A645.

Our transfection results also demonstrate that the length of the polyG repeat can modulate AR transactivation capacity in vitro in CHO cells. Whereas longer polyQ repeats are correlated with a decreased AR transactivation capacity (28, 29, 30), longer polyG tracts strengthen it (Fig. 2Go). Thus, influence on AR functionality is inversely related comparing polyQ and polyG repeat lengths. Moreover, in our transfection experiments, the negative effect of a short G10 tract on AR function is similar to that of a long polyQ tract, which is reported to be associated with undermasculinization of male genitalia (32) and male infertility (33, 34, 35, 36) or teratozoospermia (26).

Our transfection studies regarding the influence of A645D mutation on AR constructs with various polyG tract lengths revealed that it had only a slightly positive effect on AR activity of our wild-type constructs (Q20G16, Q20G25) but could rescue AR activity of the Q20G10 construct up to the wild-type level of ARQ20G16 and also improve the activity of the artificial Q20G0 construct remarkably. The rescue of AR activity of the Q20G10 construct by the A645D mutation might lead to a normal phenotype in some males with a short polyG tract of 10 residues. On the other hand, we saw a significant reduction of AR activity by this mutation in context of a long Q30 repeat. The combination of a short G10 repeat with a long Q30 repeat and the A645D mutation diminished AR activity to less than 50%, compared with the wild-type allele Q20G25 construct, and this effect may significantly affect AR function in our patients. The hormone binding data from patient fibroblasts (Fig. 5Go) as well as the dose-response curves of our constructs demonstrated that neither the short polyG repeat nor the A645D mutation influenced hormone binding of the AR. Instead, a short polyG repeat might have a negative impact on AR coactivator binding. This might be rescued partially by conformational changes due to a A645D substitution. In turn, this positive effect might be reversed by a long polyQ repeat due to the N/C-terminal interaction. This speculative explanation needs to be proved by further experiments.

Because the polymorphic repeats modulate AR activity, one would expect this combination of repeats also in apparently normal men, but these repeats might predispose for virilization disorders or infertility, depending on the androgen levels of the individual. This opinion is enhanced by our observation that patient 2 of our study showed a good response to hormone therapy. In addition, we conclude that both of the variable repeat regions influence AR functionality in sex development. They may predispose to a well-characterized virilization disorder and enhance defective virilization in otherwise normal children. Furthermore, complete in vitro recreation of the patients’ AR reading frame sequence for transfection analysis might be useful for the estimation of the pathogenetic impact of AR gene mutations. Apart from the genetic origin of the mutations (hereditary vs. somatic) and differences in individual androgen levels, the particular context of the two polymorphic regions of the AR may be a third factor strongly influencing AR function in patients with partial AIS. This should be kept in mind in future studies of genotype-phenotype correlation in AIS.


   Acknowledgments
 
The authors are grateful to Drs. S. T. Liao (Chicago, IL), A. Brinkmann (Rotterdam, The Netherlands), and G. Jenster (Rotterdam, The Netherlands) for providing plasmids.


   Footnotes
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft for the Clinical Research Group "Intersex—from gene to gender" (KFO 111/1-2).

Disclosure statement: the authors have nothing to disclose.

First Published Online June 27, 2006

Abbreviations: AIS, Androgen insensitivity; AR, androgen receptor; Bmax, maximal binding capacity; CHO, Chinese hamster ovary; DBD, DNA-binding domain; DHT, 5{alpha}-dihydrotestosterone; hCG, human chorionic gonadotropin; HR, hinge region; Kd, dissociation constant; LBD, ligand-binding domain; NTD, N-terminal activation domain; polyG, polyglycine; polyQ, polyglutamine; SRD5A2, 5{alpha}-reductase type II gene.

Received February 17, 2006.

Accepted June 19, 2006.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

  1. Wang Q, Lu J, Yong EL 2001 Ligand- and coactivator-mediated transactivation function (AF2) of the androgen receptor ligand-binding domain is inhibited by the cognate hinge region. J Biol Chem 276:7493–7499[Abstract/Free Full Text]
  2. Irvine RA, Ma H, Yu MC, Ross RK, Stallcup MR, Coetzee GA 2000 Inhibition of p160-mediated coactivation with increasing androgen receptor polyglutamine length. Hum Mol Genet 9:267–274[Abstract/Free Full Text]
  3. Callewaert L, Christiaens V, Haelens A, Verrijdt G, Verhoeven G, Claessens F 2003 Implications of polyglutamine tract in function of the human androgen receptor. Biochem Biophys Res Commun 306:46–52[CrossRef][Medline]
  4. Buchanan G, Yang M, Cheong A, Harris JM, Irvine RA, Lambert PF, Moore NL, Raynor M, Neufing PJ, Coetzee GA, Tilley WD 2004 Structural and functional consequences of glutamine tract variation in the androgen receptor. Hum Mol Genet 13:1677–1692[Abstract/Free Full Text]
  5. Zhou ZX, Sar M, Simental JA, Lane MV, Wilson EM 1994 A ligand-dependent bipartite nuclear targeting signal in the human androgen receptor. Requirement for the DNA-binding domain and modulation by NH2-terminal and carboxyl-terminal sequences. J Biol Chem 269:13115–13123[Abstract/Free Full Text]
  6. Poukka H, Karvonen U, Yoshikawa N, Tanaka H, Palvimo JJ, Janne OA 2000 The RING finger protein SNURF modulates nuclear trafficking of the androgen receptor. J Cell Sci 113:2991–3001[Abstract]
  7. Gao N, Zhang J, Rao MA, Case TC, Mirosevich J, Wang Y, Jin R, Gupta A, Rennie PS, Matusik RJ 2003 The role of hepatocyte nuclear factor-3{alpha} (forkhead box A1) and androgen receptor in transcriptional regulation of prostatic genes. Mol Endocrinol 17:1484–1507[Abstract/Free Full Text]
  8. Poukka H, Aarnisalo P, Santti H, Janne OA, Palvimo JJ 2000 Coregulator small nuclear RING finger protein (SNURF) enhances Sp1- and steroid receptor-mediated transcription by different mechanisms. J Biol Chem 275:571–579[Abstract/Free Full Text]
  9. Hong CY, Gong E-Y, Kim K, Suh JH, Ko H-M, Lee HJ, Choi H-S, Lee K 2005 Modulation of the expression and transactivation of androgen receptor by basic helix-loop-helix transcription factor Pod-1 through recruitment of histone deacetylase 1. Mol Endocrinol 19:2245–2257[Abstract/Free Full Text]
  10. Liao G, Chen L-Y, Zhang A, Godavarthy A, Xia F, Ghosh JC, Li H, Chen JD 2003 Regulation of androgen receptor activity by the nuclear receptor corepressor SMRT. J Biol Chem 278:5052–5061[Abstract/Free Full Text]
  11. Kittles RA, Young D, Weinrich S, Hudson J, Argyropoulos G, Ukoli F, Adams-Campell L, Dunston GM 2001 Extent of linkage disequilibrium between the androgen receptor gene CAG and GGC repeats in human populations: implications for prostate cancer risk. Hum Genet 109:253–261[CrossRef][Medline]
  12. Lundin KB, Giwercman A, Richthoff J, Abrahamsson PA, Giwercman YL 2003 No association between mutations in the human androgen receptor GGN repeat and inter-sex conditions. Mol Hum Reprod 9:375–379[Abstract/Free Full Text]
  13. Hiort O, Sinnecker GHG, Holterhus PM, Nitsche EM, Kruse K 1996 The clinical and molecular spectrum of androgen insensitivity syndromes. Am J Med Genet 63:218–222[CrossRef][Medline]
  14. Nordenskjöld A, Söderhall S 1998 An androgen receptor gene mutation (A645D) in a boy with a normal phenotype. Hum Mutat 11:339
  15. Sinnecker GHG, Hiort O, Nitsche EM, Holterhus PM, Kruse K 1997 Functional assessment and clinical classification of androgen sensitivity in patients with mutations of the androgen receptor gene. Eur J Pediatr 156:7–14[Medline]
  16. Sinnecker GHG, Köhler S 1989 Sex hormone-binding globulin response to the anabolic steroid stanozolol: evidence for its suitability as a biological androgen sensitivity test. J Clin Endocrinol Metab 68:1195–1200[Abstract]
  17. Holterhus PM, Brüggenwirth HT, Hiort O, Kleinkauf-Houcken A, Kruse K, Sinnecker GHG, Brinckmann AO 1997 Mosaicism due to a somatic mutation of the androgen receptor gene determines phenotype in androgen insensitivity syndrome. J Clin Endocrinol Metab 82:3584–3589[Abstract/Free Full Text]
  18. Brinkmann AO, Faber PW, van Rooij HC, Kuiper GG, Ris C, Klaassen P, van der Korput JA, Voorhorst MM, van Laar JH, Mulder E 1989 The human androgen receptor: domain structure, genomic organization and regulation of expression. J Steroid Biochem 34:307–310[CrossRef][Medline]
  19. Chang CS, Kokontis J, Liao ST 1988 Structural analysis of complementary DNA and amino acid sequences of human and rat androgen receptors. Proc Natl Acad Sci USA 85:7211–7215[Abstract/Free Full Text]
  20. Jenster G, Spencer TE, Burcin MM, Tsai SY, Tsai MJ, O’Malley BW 1997 Steroid receptor induction of gene transcription: a two step model. Proc Natl Acad Sci USA 94:7879–7884[Abstract/Free Full Text]
  21. McLean HE, Ball EMA, Rekaris G, Warne GL, Zajac JD 2004 Novel androgen receptor gene mutations in Australian patients with complete androgen insensitivity syndrome. Hum Mutat Online 10.1002/humu. 9221
  22. Cabral DF, Marciel-Guerra AT, Hackel C 1998 Mutations of androgen receptor gene in Brazilian patients with male pseudohermaphroditism. Braz J Med Biol Res 31:775–778[Medline]
  23. Komori S, Kasumi H, Sakata K, Tanaka H, Hamada K, Koyama K 1998 Molecular analysis of the androgen receptor gene in 4 patients with complete androgen insensitivity. Arch Gynecol Obstet 261:95–100[CrossRef][Medline]
  24. Sakai N, Yamada T, Asao T, Baba M, Yoshida M, Murayama T 2000 Bilateral testicular tumors in androgen insensitivity syndrome. Int J Urol 7:390–392[CrossRef][Medline]
  25. Lund A, Tapanainen JS. Lahdetie J, Savontaus ML, Aittomaki K 2003 Long CAG repeats in the AR gene are not associated with infertility in Finnish males. Acta Obstet Gynecol Scand 82:162–166[CrossRef][Medline]
  26. Milatiner D, Halle D, Huerta M, Margalioth EJ, Cohen Y, Ben-Chetrit A, Gal M, Mimoni T, Eldar-Geva T 2004 Associations between androgen receptor CAG repeat length and sperm morphology. Hum Reprod 19:1426–1430[Abstract/Free Full Text]
  27. Mhatre AN, Trifiro MA, Kaufman M, Kazemi-Esfarjani P, Figlewicz D, Rouleau G, Pinsky L 1993 Reduced transcriptional regulatory competence of the androgen receptor in X-linked spinal and bulbar muscular atrophy. Nat Genet 5:184–188[CrossRef][Medline]
  28. Chamberlain NL, Driver ED, Miesfeld RL 1994 The length and location of CAG trinucleotide repeats in the androgen receptor N-terminal domain affect transactivation function. Nucleic Acids Res 22: 3181–3186
  29. Kazemi-Esfarjani P, Trifiro MA, Pinsky L 1995 Evidence for a repressive function of the long polyglutamine tract in the human androgen receptor: possible pathogenic relevance for the (CAG)n-expanded neuropathies. Hum Mol Genet 4:523–527[Abstract/Free Full Text]
  30. Wang Q, Udaykumar TS, Vasaitis TS, Brodie AM, Fondell JD 2004 Mechanistic relationship between androgen receptor polyglutamine tract truncation and androgen-dependent transcriptional hyperactivity in prostate cancer cells. J Biol Chem 279:17319–17328[Abstract/Free Full Text]
  31. Hiort O, Holterhus PM, Horter T, Schulze W, Kremke B, Bals-Pratsch M, Sinnecker GHG, Kruse K 2000 Significance of mutations in the androgen receptor gene in males with idiopathic infertility. J Clin Endocrinol Metab 85:2810–2815[Abstract/Free Full Text]
  32. Lim HN, Chen H, McBride S, Dunning AM, Nixon RM, Hughes IA, Hawkins JR 2000 Longer polyglutamine tracts in the androgen receptor are associated with moderate to severe undermasculinized genitalia in XY males. Hum Mol Genet 9:829–834[Abstract/Free Full Text]
  33. Tut TG, Ghadessy FJ, Trifiro MA, Pinsky L, Yong EL 1997 Long polyglutamine tracts in the androgen receptor are associated with reduced trans-activation, impaired sperm production and male infertility. J Clin Endocrinol Metab 82:3777–3782[Abstract/Free Full Text]
  34. Dowsing AT, Yong EL, Clark M, McLachlan, RI, de Kretser DM, Trounson AO 1999 Linkage between male infertility and trinucleotide repeat expansion in the androgen receptor gene. Lancet 354:640–643[CrossRef][Medline]
  35. Yoshida KI, Yano M, Chiba K, Honda M, Kitahara S 1999 CAG repeat length in the androgen receptor gene is enhanced in patients with idiopathic azoospermia. Urology 54:1078–1081[CrossRef][Medline]
  36. Mifsud A, Sim CK, Boettger-Tong H, Moreira S, Lamb DJ, Lipshultz LI, Yong EL 2001 Trinucleotide (CAG) repeat polymorphisms in the androgen receptor gene: molecular markers of risk for male infertility. Fertil Steril 75:275–281[CrossRef][Medline]



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