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Original Studies |
University Department of Growth and Reproduction (A.G., T.K., H.L., N.E.S.), Rigshospitalet, DK 2100 Copenhagen, Denmark; University Department of Clinical Genetics (M.S.), Rigshospitalet, DK 2100 Copenhagen, Denmark; Department of Molecular Medicine (Y.L.G., H.Z., A.W.), Karolinska Hospital, SE 17176 Stockholm, Sweden; and Department of Urology, Malmö University Hospital (A.G.), SE 20502 Malmö, Sweden
Address correspondence and requests for reprints to: Aleksander Giwercman, University Department of Urology, Malmö University Hospital, SE 20502 Malmö, Sweden. E-mail: aleksander.giwercman{at}kir.mas.lu.se
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Abstract |
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Two adult brothers, referred because of gynecomastia and hormonal
levels in serum indicating androgen insensitivity (high sex
hormone-binding globulin, and LH levels, despite extremely high
testosterone concentration), turned out to be
relatives to a third young man, referred independently of the two
others and exhibiting identical clinical and hormonal stigmata. In all
three men, we found a C
A substitution at position 2470 (exon 7) in
the androgen receptor gene, leading to a Gln824Lys mutation in the
ligand-binding domain of the receptor. Exploring the family history
revealed that their grandfathers, on their mothers’ side, were
brothers; and the Gln824Lys mutation was also found in the one of them
who was still alive.
Binding studies with the mutant receptor in transfected COS-7 cells, with mibolerone as ligand, exhibited equal Kd (0.7 vs. 1.0 nmol/L), IC50 (0.8 vs. 1.1 nmol/L), and maximum binding (7.1 vs. 8.9 fmol/106 cells), as compared with the wild-type (WT) receptor. In a chloramphenicol acetyl transferase trans-activation assay, the activity of the mutant receptor was identical to that of the WT, when the synthetic androgen R1881 was used as a ligand; but with dihydrotestosterone, in concentrations up to 10 nmol/L, the activity of Gln824Lys mutated receptor was 10–62% of the WT variant.
Thus, Gln824Lys mutation was found, both in vivo and in vitro, to cause slight impairment of receptor function but was compatible with preservation of male fertility. The patients inherited the mutation from their grandfathers through their mothers, and one of the young men possessing the mutation has fathered a daughter.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Testicular specimens from AIS individuals do not only exhibit a high risk of carcinoma in situ (6) but also other qualitative and quantitative disturbances of spermatogenesis, and patients with even mild forms of PAIS are considered infertile. It has been suggested that AR mutations may be the cause of male infertility without other phenotypical signs of androgen insensitivity (7). However, the assumption of androgen resistance was mainly based on hormonal profiles of the infertile men and not on molecular studies of the AR. Recently, a case of male infertility with verified single amino acid substitution in the AR receptor gene was reported (8).
Thus, mutations of AR causing reduced sensitivity to androgens are considered as incompatible with preserved fertility. We report a mutation in the ligand-binding domain (LBD) of the AR, inherited from a grandfather and with preserved fertility potential, despite a hormonal profile showing reduced androgen sensitivity. Functional in vitro studies confirmed slightly decreased receptor function.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Patient A1 was referred, at the age of 21 yr, because of gynecomastia and high androgen levels. Endocrine evaluation revealed hormonal levels typical for AIS, with high LH, despite increased testosterone and estrogen level and relatively low FSH. The patient reported that he entered the puberty somewhat later than school friends of the same age (approximately 14 yr old), and the enlargement of breasts began contemporary with the start of puberty and progressed during a period of 1 yr. Afterwards, the gynecomastia had been stationary.
At the time of clinical examination, the patient presented with normal body proportions. The development of both breasts was estimated to be Tanner stage IV, with a diameter of approximately 15 cm. The hair distribution was normal on arms, legs, and in the axillary region. Some hairs around the areolae were also present.
Genital examination revealed pubic hair at Tanner stage VI and genital development at Tanner stage V, including normal penile size and without genital abnormalities. Both testes (each 20 cm2) were located in the scrotum.
Patient A2 was a brother of A1 (2 yr younger) and presented with similar medical history, physical signs, and hormone levels.
Patient B was referred, at the age of 16, because of gynecomastia and slightly diminished penile size. The patient has entered puberty at the age of 13, and enlargement of the breast was one of the first signs recognized by him. At the time of referral, the gynecomastia had been stationary for 2 yr. Initial investigation at the local department of pediatrics revealed normal 46,XY karyotype and high testosterone, LH, and estradiol levels. The patient was referred to the pediatric endocrinology and andrology center for further investigation and treatment.
His weight was 69.4 kg, and his height was 175.9 cm at the time of referral, increasing to a final height of 178.7 cm at the age of 20. The body proportions were normal, with a sitting height/total height ratio of 0.52. The development of breasts was estimated to be Tanner stage III-IV. The body hair was sparse with no hair on the thorax.
Genital examination revealed pubic hair of stage V; genital development, stage IV-V; with diminished penile size. Both testes were located in the scrotum, and their volume was 20 cm2 for the right and 23 cm2 for the left testis. No scrotal abnormalities were found.
From the age of 18 to the age of 20, he was treated with im testosterone enanthate injections (250 mg every 2nd week). During this treatment, pubic hair growth progressed from stage V to stage VI. As compared with the off-treatment period, the serum levels of testosterone, LH, estradiol, and FSH were unaffected by testosterone injections. However, the level of sex hormone-binding globulin (SHBG; 61 nmol/L) was significantly lower during the androgen replacement than when the patient did not receive androgen supplementation (6 measurements; mean and 95% confidence interval, 77 nmol/L; 62–90 nmol/L).
At the age of 23 yr, he fathered a healthy girl. The patient’s wife conceived in a natural way.
The clinical and laboratory patient data are summarized in Tables 1
and 2.
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Because patients A1, A2, and B had the same middle name and an identical mutation of the AR gene, they were interviewed about their family relations, to disclose possible common relatives.
Sequencing of the AR gene
Molecular analyses. DNA was isolated from EDTA-stabilized blood samples by routine methods. All exons and part of the flanking introns were PCR-amplified using the primers described by Batch et al. (9). One of the primers in each set was 5'-end labeled with biotin. The PCR products were scanned for mutations by single-strand conformation analysis (SSCA). Products with an aberrant SSCA pattern were sequenced and subsequently sequenced using the Sequenase kit version 2.0 (USB).
SSCA. PCR products were denatured by mixing 3 µl of the PCR product with 3 µl of loading mixture (95% formamide, 20 mmol/L EDTA, 0.05% bromophenolblue, and 0.05% xylene cyanol), followed by boiling for 5 min and cooling on ice. Two microliters of each denatured sample was loaded on a 20% homogeneous PHAST-gel with native buffer strips (Pharmacia, Uppsala, Sweden). The separation condition consisted of a prerun for 10 avh (average volt-hours) at 5 mA, 1W, and 400V; a sample application for 2 avh at 5 mA, 1W, and 25 V; and a separation for 450 avh at 5 mA, 1W, and 400V. Single-stranded products were visualized by silver staining.
Construction of Gly824Lys expression plasmid and transfection into COS-7 cells
To create the CA base change at position 2470 in the AR,
site-directed mutagenesis technique was used (10, 11). The procedure
consists of two main steps, a primary PCR and a secondary PCR.
For the primary PCR, two different sets of primers were used. Each set of primers consists of one flanking primer that hybridizes at one end of the template DNA and one internal primer (mutagenesis primer) that hybridizes at the site of the nucleotide alteration.
The mutagenesis sense and antisense primers were designed as follows (the overlapping sequences in italics and the mutated nucleotide underlined): sense primer, 5'-GATGGTCTCAAAAATAAGAAATTCTTTGATGAA-3'; antisense primer, 3'-GTCACCTACCAGAGTTTTTAT-TCTTT-5'. The 5' upstream flanking sense primer was designed to include the wild-type (WT) sequence from the 5' ATG methionine codon no. 1 extending 18 nucleotides downstream. A Kozak sequence was added at the 5' end. The Kozak messenger RNA sequence 5'-GCCGCCA/GCCAUGG has been reported as a consensus sequence for initiation of translation of highly expressed genes in higher eukaryotes (12). Upstream, to this Kozak sequence a Bg1II- and a BamHI cleavage sites were added, together with four additional nucleotides, the function of which was to facilitate the subsequent digestion. The 3' downstream flanking antisense primer was designed to overlap the stop codon extending 21 nucleotides further upstream. A BamHI cleavage site plus four additional nucleotides were included in the 5' end of this primer.
Afterwards, the BamHI digested PCR fragments (full-length AR), and WT as the Gly824Lys mutant, were inserted into the pTEJ-8 expression vector (13) by cohesive end ligation. Sequence confirmation of the natural-occurring and site-directed mutation, compared with WT androgen receptor (AR), was performed. After transformation, followed by purification, the human AR-genes were inserted into COS-7 cells using the calcium phosphate precipitation technique (14).
Binding studies on transfected COS-7 cells
The ligand-binding properties of mutant and WT AR were studied in transfected COS-7 cells by means of a competition assays. In this assay, to the transfected cells were added 0.05 nmol/L [3H]-mibolerone and nonradioactive mibolerone in concentrations between 10-3 and 103 nmol/L. Mock transfected COS-7 cells and nontransfected COS-7 cells were used as negative controls. Bound [3H]-mibolerone was plotted as a function of the logarithmic competitor ligand concentration. The following parameters were computed: the radioligand affinity for the receptor (Kd)l; the concentration of the nonradioactive competitor that is able to displace half of the radioligand from the receptor (IC50); and the maximum binding of [3H]-mibolerone to the receptor. Each single experiment was performed in triplicate at 37 C. Five independent experiments were performed.
In addition, AR protein expression was verified by Western immunoblot
analysis (Fig. 1).
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Approximately 1 x 106 COS-1 cells were transfected by electroporation [Bio-Rad Laboratories, Inc. (Hercules, CA) Gene Pulser, 1250 V, 25 µF] with 5 µg of each of the pTEJ-8-AR constructs together with 1 µg of the ß-galactosidase vector pCH110 (Pharmacia, Sweden) and 2 µg of the reporter construct pGM-CAT. They were subsequently seeded in 2.5-cm Petri dishes, and incubated for 24 h in DMEM containing 10% FBS and 0.002% gentamycin.
pGM-CAT contains the chloramphenicol acetyl transferase (CAT) gene under the control of mouse mammary tumor virus promoter, which contains two androgen response elements. Twenty-four hours after transfection, the medium was changed to contain 10% charcoal-stripped serum, and the cells were stimulated with increasing concentrations of the synthetic androgen methyltrienolone (R1881) or the native ligand dihydrotestosterone (DHT) (0.01–10 nmol/L ). As controls, cells were transfected in parallel with pTEJ-8 without the AR, pCH110, and pGM-CAT. Twenty-four hours after adding the ligand, the cells were washed twice in PBS, harvested, and lysed for measurements of CAT protein levels using an enzyme-linked immunosorbent assay (Roche Molecular Biochemicals GmBH, Mannheim, Germany). The results were corrected for differences in protein concentrations using the Bradford assay (15) and the efficacy of transfection, as expressed by ß-galactosidase activity. The numbers of independent experiments performed with the two ligands were four for R1881 and two for DHT stimulation, respectively.
Hormone analyses
Serum levels of FSH and LH were measured by time-resolved
immunoflurometric assay (DELFIA; Wallac, Inc.,
Turku, Finland); whereas testosterone, estradiol, and SHBG were
analyzed by an RIA (Coat-a-count; DPC Biermann GmbH, Bad
Nauheim, Germany). For Inhibin B analysis, an immunometric
assay, as described by Illingworth et al. (16), was used.
This assay uses a monoclonal capture antibody against the
ßB and a secondary monoclonal enzyme-conjugated
antibody raised against the sequence of the 
subunit. The assay
is specific for bioactive inhibin dimer
-ßB.
Semen analysis
Two of the patients (A1 and B) delivered four semen samples each. The ejaculates were obtained by masturbation after at least 2 days of sexual abstinence. The assessment of volume, concentration, motility, and morphology was performed as recommended by the World Health Organization (WHO) (17).
Testicular biopsy
Bilateral testicular biopsy was performed in patient A2 as an open surgical procedure (18). Tissue specimens (4 mm in diameter) were fixed in Stieve’s fixative and embedded in paraffin. Sections of 4-µm thickness were stained with hematoxylin-eosin, and light-microscopical assessment with semiquantitative evaluation of spermatogenesis and Leydig cells was performed.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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An interview with the three patients disclosed that they were
closely related to each other (Fig. 2). The grandfathers of A1, A2, and of
B, on their mothers’ side, were brothers. DNA from the grandfather of
patient B and from his mother was obtained and tested for the mutation
in the AR gene.
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We found a CA substitution at position 2470 (exon 7) in the AR
gene, leading to a Gln824Lys in the proband A1. DNA analysis revealed
the same mutation in the other two patients and in B’s mother
(heterozygote carrier) and in B’s grandfather.
Hormone analyses
The results of hormonal evaluation are summarized in Tables 1
and 2. The hormone levels were characteristic for patients with AIS. In all
three men, the levels of LH and SHBG were high, despite significantly
increased serum testosterone. Serum inhibin B levels were 355 pg/mL,
287 pg/mL, and 141 pg/mL for A1, A2, and B, respectively. The normal
range for this hormone is 35–312 pg/mL.
Semen analysis
The mean values of seminal volume, sperm concentration, percent
immotile, and morphologically normal spermatozoa were as follows: A1:
1.2 mL, 4.0 x 106/mL, 81%, and 23%; B:
1.9 mL , 4.7 x 106/mL, 57%, and 36%. For
comparison, the reference values, as recommended by WHO (17), are: vol
2.0 mL; concentration 20 x 106/mL;
immotile 
50%; and morphologically normal 30%.
Testicular biopsy
Bilateral testicular biopsy, performed in patient A2, disclosed an
ongoing spermatogenesis, including a spermatid stage in the majority of
tubules (right, 99%; left, 70%). However, the number of late
spermatids was reduced. On both sides, in approximately 1% of tubules,
the Sertoli cells were undifferentiated; and, on the left side, the
spermatogenesis was arrested at the primary spermatocyte stage in 29%
of tubules (Fig. 3). The number of Leydig
cells was apparently normal.
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There was no significant difference between the mutant and the WT
receptor for any of the binding characteristics that were tested. The
results are summarized in Table 3.
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In a trans-activation assay, the Gln824Lys mutated AR
exhibited CAT activity equal to that of WT when the synthetic androgen
R1881 was used as a ligand. However, in experiments
performed with the naturally occurring androgen, DHT, the
property of the mutated receptor was between 10% (0.1 nmol/L) and 62%
(10 nmol/L) of the WT. These results are illustrated in Fig. 4.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The semiquantitative evaluation of a testicular biopsy from one of the men revealed some disturbance in spermatogenesis, including presence of immature Sertoli cells and some tubules, with a maturation arrest at the stage of primary spermatocyte. However, in the majority of tubules, there was ongoing spermatogenesis, with some reduction of the spermatid number. This histological picture was in agreement with the normal levels of inhibin B, indicating that the early stages of spermatogenesis were relatively unaffected by the AR mutation (21).
Both clinical and in vitro studies have shown that the Gln824Lys mutation found by us is of significance for the function of the AR. All three men presented clinically with gynecomastia, although they appeared as otherwise-normal males. Furthermore, high levels of testosterone, estradiol, and SHBG, in combination with increased LH and low FSH, is a typical endocrine profile for patients with AIS (22). The Gln824Lys mutation affects the LBD of the AR. However, the affinity of the mutant receptor, as expressed by Kd, as well as IC50, and the maximum binding of [3H]-mibolerone were equal to the values for the WT, indicating that the binding of androgen to the receptor is not significantly affected. Similar to the binding studies, experiments confirmed the relatively mild form of the Gln824Lys mutation. Thus, with the synthetic androgen R1881, the activities in COS-1 cells transfected with the mutant and the WT receptor were equal. However, in experiments with DHT, a naturally occurring androgen, the activity of the Gln824Lys mutated receptor was significantly lower than that of the WT. CAT activity was found to be 10% of the normal activity, at a DHT concentration of 0.01 nmol/L, and it increased to 62% at 10 nmol/L DHT.
Because the mutation is located in a highly conserved region of the LBD, with 100% amino acid identity with the progesterone receptor, mineralocorticoid receptor, and glucocorticoid receptor (23), it is supposed to have a critical role in receptor structure and function. Furthermore, codon 824 is conserved among several species, including human, chimpanzee, dog, rat, rabbit, mouse, rainbow trout, and xenopus. Therefore, it may be a little surprising that the Gln824Lys mutation causes relatively discrete clinical symptoms and only slight reduction of AR function shown in vitro.
Recently, the crystal structure of unliganded human retinoic acid
receptor LBD (24), the liganded human estrogen receptor LBD (25), and
rat thyroid hormone receptor were solved (26). The overall structure of
the different LBDs seemed to be quite similar; and, therefore, the
structure of the AR-LBD could be predicted as containing 12 -helices
and 2 ß-strands connected by linker regions (27). Our data might
indicate that the part of helix 9, in which the Gln824Lys mutation is
located, may play a lesser role for hormone binding but, rather, be
involved (directly or via coactivators) in function. Similar findings
were recently presented by Wang et al. (8), who found
another mutation in the LBD of the AR causing decreased
transactivation, despite unaffected in vitro binding to the
receptor.
The Gln824Lys mutation of AR has not previously been reported. As most of the AR mutations found until now, it is a single-base mutation in the C-terminal end of the AR, resulting in an amino acid substitution in the receptor protein. However, in virtually all cases where mutations of the AR have been reported, the patient presented with severe signs of intersex, either as CAIS or PAIS (3). In the present case, apparently, the sensitivity to testosterone and DHT in fetal life was sufficiently high to ensure a normal development of internal and almost normal (micropenis in patient B) external genitalia. Also, the postnatal receptivity to androgens was adequate to obtain a normal development of secondary male sex characteristics, spermatogenesis with sperm production, and even fertility. However, the patients developed gynecomastia attributable to high estrogen levels resulting from conversion of testosterone to estradiol.
Mutations in the AR are usually considered as being incompatible with preservation of normal male fertility (7). Wang et al. (8) recently reported on an azoospermic man possessing a mutation in exon 6 of the AR. This patient was, except for somewhat sparse body hair, normally virilized, but a testicular biopsy revealed Sertoli-cell-only pattern as the cause of azoospermia. One case of pregnancy, after androgen treatment of a patient with mutation in the AR gene, was reported by Yong et al. (28). However, this patient did not exhibit the classical biochemical signs of androgen insensitivity: high LH levels despite increased testosterone concentration. In this man, both testosterone and LH were within the normal range, whereas the FSH level was high. One can, therefore, not exclude that androgen sensitivity was rather unaffected in this man despite the mutation in the AR. In the cases presented by us, the levels of reproductive hormones and the presence of gynecomastia clearly indicated that the sensitivity to androgens was reduced.
In an animal model for AIS, the Tfm rat, a substitution in the LBD (exon 5) of the rat AR causes a decrease in hormone binding capacity and PAIS (29). Interestingly, the PAIS could be overcome by administration of high doses of testosterone. Similarly, the results of our in vitro experiments may indicate some therapeutic possibilities in management of adult men with poor semen quality associated with androgen insensitivity caused by mutations in the AR. Because the intratesticular androgen concentration is approximately 100 higher than the serum levels, it is hard to imagine that replacement therapy with testosterone might improve the spermatogenetic function. The effect would be rather opposite because testosterone becomes converted to estradiol, which suppresses the pituitary FSH production. On the other hand, as indicated by the experiment, the effect of the mutation in the LBD of the AR may, at least in some cases, be overcome by use of synthetic androgens. R1881 may be a potential candidate, because it apparently activates the mutant receptor similarly to the WT variant. It remains to be seen whether these in vitro results can be reproduced in an in vivo situation.
In conclusion, the phenotype of individuals with mutated AR gene may vary from CAIS to apparent normal phenotype, and our example of a Gln824Lys mutation in the LBD of the AR is an additional part of this broad spectrum. This mutation affected somewhat the in vitro and in vivo function of the receptor but was compatible with grossly normal male phenotype, except gynecomastia, and preserved spermatogenesis, sperm production, and even fertility.
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Footnotes |
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Received October 13, 1999.
Revised February 17, 2000.
Accepted March 3, 2000.
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References |
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, heterozygote.
