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Original Studies |
Department of Pediatrics, Medical University of Lübeck (P.-M.H., O.H.), 23538 Lübeck; and Department of Pediatric and Adolescent Medicine, Klinikum der Stadt Wolfsburg (G.H.G.S.), 38440 Wolfsburg, Germany
Address all correspondence and requests for reprints to: Paul-Martin Holterhus, M.D., Department of Pediatrics, Medical University of Lubeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. E-mail: holterhus{at}paedia.mu-luebeck.de
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The AR is a ligand-activated transcription factor of androgen-regulated
target genes (14, 15). Testosterone and dihydrotestosterone represent
the two most important physiological ligands. One of the major
determinants of AR function in vitro and in vivo
is the concentration of the ligand. Elevation of ligand concentration
is paralleled by increasing androgen binding to the receptor up to a
saturation state (16). Transcriptional activation of androgen-regulated
target genes due to the AR in vitro is clearly dependent on
hormone concentration (17). In vivo, inadequate ligand
concentration due to defects of testosterone biosynthesis or
5
-reduction leads to diminished androgen action, as evidenced by
defective masculinization of genetic male individuals (18, 19).
Mutations of the AR gene located within the ligand-binding receptor
domain can be associated with considerable loss of function due to
impaired hormone binding, thus also resulting in defective
masculinization. Transcriptional activity in vitro can be
severely reduced over the whole range of hormone concentrations
(20, 21, 22). However, in certain mutations, increasing ligand
concentration can compensate reduced activity. In some, but not all, of
these cases, one may also find a good clinical response to androgen
therapy (20, 21, 23, 24). Such observations have lead to speculations
about whether variations in androgen concentration during male sexual
differentiation could explain the phenotypic variability observed in
some AIS patients (6, 7, 8). In this context, we present a PAIS family
with unusually wide phenotypic diversity associated with the previously
uncharacterized L712F AR mutation. The association of our clinical and
experimental findings adds substantial evidence to the possible role of
variations in ligand concentration in particular cases of phenotypic
diversity in AIS patients.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Patients
Brother 1 (B1). B1 was 11 months old at initial
presentation. The child had ambiguous external genitalia, with a
scrotum bipartitum, a phallus 3 cm in length, and a well developed
glans penis 0.5–0.6 cm in diameter (Fig. 1
). A small introitus of a urogenital
sinus was located at the base of the phallus. Two gonads, 1
cm3 in volume (Prader orchidometer), were
palpable at the anulus inguinalis superficialis. The phenotype
corresponded to AIS type IIIb according to Sinnecker et al.
(4). Basal serum hormone concentrations were 0.17 nmol/L testosterone,
0.07 nmol/L dihydrotestosterone, and 0.2 IU/L LH (all values normal for
prepubertal age). After im injections of 1500 IU hCG every other day
over 14 days, testosterone increased to 25.3 nmol/L and
dihydrotestosterone to 2.19 nmol/L, thus excluding a defect of
testosterone biosynthesis or 5-reductase type II deficiency. The
basal sex hormone-binding globulin (SHBG) concentration in serum was
142.68 nmol/L (normal for age). After stanozolol administration
according to a previously published standardized protocol (4, 25), SHBG
decreased to 104.93 nmol/L (78.3% of the initial value; normal,
63.4%), indicating PAIS. Although hCG administration alone had no
effect on the external genitalia, a total of three injections of 100 mg
testosterone every 4 weeks increased the length of the phallus to 4 cm
and the diameter of the glans to 1.3 cm. Due to these data and to
clinical observations of the uncle of B1 (see below), it was decided to
raise the child in a male gender, and at the age of 15 months, surgical
correction of the external genitalia with a two-stage hypospadias
repair was performed.
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). Upon pulling up the tissue of the mons pubis,
penile length was 1.0 cm. The glans penis had a diameter of 0.9 cm. No
hypospadias was present. Both testes (2 cm3 each
in volume, by Prader orchidometer) were palpable in the scrotum. The
right testis previously underwent orchiopexy because of incarcerated
inguinal hernia. The basal serum testosterone concentration was 11.0
nmol/L; the LH concentration was 5.3 IU/L (both hormones were elevated
for age). At the age of 7 months, the basal serum SHBG concentration
was 146.96 nmol/L (normal for age). After stanozolol administration,
SHBG decreased to 108.9 nmol/L (74.5% of the initial value), again
indicating PAIS. Because of the good response to androgen therapy in B1
and his uncle and because of the same underlying AR gene mutation (see
below), B2 received three injections of 100 mg testosterone, im, every
4 weeks at the age of 1 yr. In addition, a thin layer of 2.5%
dihydrotestosterone gel had been administrated externally on the penile
skin twice daily during the same 3-month period. Penile length
increased from 1.5 cm before treatment to 3 cm after treatment. Because
of a penile chordae, a slight curvature of the penis appeared after
therapy, which was surgically corrected thereafter.
Brother 3 (B3). The external genitalia of B3 appeared
completely normal at birth. He was presented at the age of 7 weeks at
our department because of the previous diagnosis of AIS in his two
brothers and his uncle (Fig. 1). Penile length was 1.6 cm [slight
micropenis; normal for age, 2.7 ± 0.5 cm (26)], penile diameter
was 1.0 cm, the meatus urethrae was normal. A slight dorsal curvature
of the phallus due to a subtle chordae could be observed. Both testes
were palpable (1 cm3 in volume each, Prader
orchidometer) in a normally developed scrotum. Basal serum hormone
concentrations were 14.7 nmol/L testosterone and 7.5 IU/L LH (both
elevated for age). The serum SHBG concentration decreased to 85.5% of
the initial value in response to stanozolol at the age of 13 months,
thus indicating PAIS as in his two siblings. He was lost from
follow-up, and to our knowledge, androgen therapy was not initiated
until today.
Uncle. The uncle of the three brothers had a small penis
with penoscrotal hypospadias and chordee at birth, which was operated
upon at the age of 4 and 5 yr. Gynecomastia developed at the age of 12
yr. Pubertal development was lacking, no beard developed, and genitals
remained infantile, but erections were noted. When he was 14 yr old,
bilateral mastectomy was performed. Semen analysis revealed azoospermia
at the age of 23 yr. At the age of 37 yr he presented in good clinical
condition; height was 180.1 cm, weight was 88.5 kg, penis length was 4
cm, circumference was 7 cm, diameter of the glans was 2 cm, and
diameter of the corpora cavernosa was 0.5 cm (Fig. 1). The orificium
urethrae externum was located at the urethral ridge close to the glans.
Both testes had a volume of 10 cm3 (Prader
orchidometer). There were scars at the sites of mastectomy, no beard
but single dark hairs on the chin, and no recessus temporales. The
phenotype corresponded to AIS type IIb with otherwise normal clinical
findings.
Basal values for LH (between 8.1–10.4 IU/L), serum testosterone (between 57.4–59.8 nmol/L; normal, 10.3–34.2 nmol/L), and serum estradiol (between 173–401 pmol/L) were increased. The basal SHBG level was increased to 96.6 nmol/L (normal, 20.6–62.6 nmol/L). After stanozolol administration, SHBG decreased to 64.3 nmol/L or 66.6% of the initial value (normal, < 63.4%), which is slightly less than normal, indicating considerable remaining in vivo activity of the AR.
Treatment with high dose testosterone (500 mg testosterone enanthate, im, weekly) was initiated. All hormone measurements were performed 1 week after the last injection. LH decreased to 5.5–8.0 IU/L, serum testosterone increased to 75.2–102.5 nmol/L, estradiol increased to 269–346 pmol/L, and SHBG decreased to 78 nmol/L.
After 4 weeks of treatment, testosterone dose was increased to 1000 mg testosterone enanthate once a week. The LH level dropped further into the normal range (1.5–2.4 IU/L). Serum testosterone increased to levels between 102.5–191.4 nmol/L, estradiol increased to 140–508 pmol/L, and SHBG decreased to 50 nmol/L into the normal range (20.6–62.6 nmol/L).
Four months after initiation of the therapy a slight beard on the chin developed, the penis was 5–6 cm in length and 9 cm in circumference, the diameter of the glans was 2 cm, left testis was 8 mL, right testis was 8–10 mL, and pubic hair was Tanner stage 5 with slight hair at the linea alba. The patient reported being more active and stronger, and his libido was significantly increased. He shaved his chin every 3 days compared to once every 3 weeks before therapy. He reported having erections several times a day and being more sensitive to sexual arousal. His skin was more fatty and he sweated more. The volume of the ejaculate, however, remained very small. Semen analysis was not performed.
Androgen binding analyses
Only from subject B2 could genital skin fibroblasts be obtained
during surgical correction of the external genitalia. Control
fibroblasts were derived from a foreskin specimen of a normal
prepubertal male subject. Binding analyses were performed as previously
reported (12). In brief, the cells were cultured at 37 C with 5%
CO2 in MEM (Life Technologies, Inc.,
Grand Island, NY), supplemented with 10% FCS, 1% (vol/vol) MEM
nonessential amino acids (Life Technologies, Inc.), and
penicillin (200 IU/ml)/streptomycin (0.2 mg/ml). For androgen binding
studies, confluent cultures of genital skin fibroblasts were incubated
in duplicate with medium containing increasing concentrations of
17ß-hydroxy-17-[3H]-methyl-4,9,11-estrotrien-3-one
([3H]R1881; 0.02–3.0 nmol/L) from
DuPont-NEN Life Science Products (Boston, MA) in either
the presence or the absence of a 200-fold molar excess of unlabeled
hormone. Two independent binding assays were performed on the
fibroblasts of the patient. For Scatchard calculations and statistics,
Excel personal computer software (Microsoft Corp.,
Richmond, WA) was used.
Mutation detection analyses
Detection of a CTT (L)
TTT (F) point mutation at codon
position 712 in subject B1 was reported in a previous overview of AR
gene mutations (27). In detail, from this subject (B1) and a male
control, genomic DNA had been extracted from peripheral blood
leukocytes according to standard procedures. The DNA served as a
template for the PCR. All eight exons of the AR gene had been amplified
individually by PCR, followed by screening for mutations using
nonradioactive single strand conformation analysis as described
previously (2, 28). Resulting from a band shift on single strand
conformation analysis, the exon 4 PCR product of subject B1 was
purified using the Qiaquick extraction kit (QIAGEN,
Hilden, Germany) and subsequently directly sequenced with the Sequenase
sequencing kit (Amersham Pharmacia Biotech Buchler,
Braunschweig, Germany) using [
-32P]ATP
end-labeled primers (2).
In the present study, exon 4 genomic DNA PCR products of the AR gene of subjects B2 and B3, who had not been born at the time of molecular diagnosis in B1, were directly sequenced. The same was performed on genomic DNA of their mother and their uncle.
Plasmids, transient transfections, and expression studies
Construction of a 712F point mutated AR expression plasmid (pSVAR712F) was based on the wild-type human AR expression plasmid pSVAR0 (29), which was a gift from Dr. A. O. Brinkmann, Erasmus University (Rotterdam, The Netherlands). In the first step, the mutation 712 TTT (F) was introduced into pSVAR0 using the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA) according to recommendations of the manufacturer. Using pSVAR0 as template, 14 PCR cycles with Pfu DNA polymerase and the mutagenic primers 5'-G-GGA-GAG-AGA-CAG-TTT-GTA-CAC-GTG-GTC-AAG-TGG-3' and 5'-CCA-CTT-GAC-CAC-GTG-TAC-AAA-CTG-TCT-CTC-TCC-C-3' were run. Subsequently, the methylated, but not mutated, pSVAR0 template DNA was removed by DpnI digestion. Remaining mutant plasmids were transformed into Escherichia coli bacteria (Epicurian coli XL1-Blue supercompetent cells, Stratagene). Accuracy of mutagenesis was verified in plasmid preparations by plasmid sequencing covering also the restriction recognition sites KpnI and BamHI located upstream and downstream of the mutation. In the second step, the mutant 1350-bp KpnI-BamHI fragment was excised from the first step product and recloned into a new pSVAR0 plasmid, leading to the final mutant plasmid pSVAR712F. The latter was verified again by plasmid sequencing for in-frame ligation and correctness of mutant DNA sequence.
Transient transfections were performed on CHO (Chinese hamster ovary) cells. They were maintained in 5% CO2 at 37 C in DMEM with the nutrient mix F-12 (DMEM/F-12, Life Technologies, Inc.), 10% (vol/vol) FCS, and antibiotics as described above for genital skin fibroblasts. For trans-activation studies 10% dextran/charcoal-treated FCS was used. The cells were transfected by the Ca2+ phosphate precipitation method (30) with only minor changes as previously described (12, 13). Activation of the androgen-responsive mouse mammary tumor virus-luciferase reporter plasmid (Organon, West Orange, NJ) due to pSVAR712F or pSVAR0 was investigated using 0.01, 0.1, 1.0, 10.0, and 100.0 nmol/L testosterone as well as 0.01, 0.1, 1.0, and 10.0 nmol/L dihydrotestosterone, respectively. All transfections were performed in triplicate. Four independent experiments were performed at different times. Mutant and wild-type AR constructs were always investigated at the same time. Transcriptional activity was expressed as fold induction of luciferase activity related to basal activity in the absence of hormone. Transfection efficiency was controlled by cotransfection of the constitutively active pRL-SV40 Renilla luciferase expression plasmid (Promega Corp., Madison, WI). Firefly and Renilla luciferase activity were determined using the dual luciferase reporter gene assay (Promega Corp.).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Exon 4 genomic DNA PCR fragments of B2, B3, their mother, and
their uncle were investigated by DNA sequencing because of the previous
detection of a new CTT (L)TTT (F) point mutation at codon 712 of the
AR gene in subject B1 (27). In subjects B2 and B3 and the uncle, the
same mutation as that in B1 was found, while the mother was
heterozygous.
Androgen binding analyses
Only genital skin fibroblasts of patient B2 could be obtained for
binding studies. Scatchard analysis of R1881 binding on these cells
revealed a slightly elevated Kd (Exp 1, 0.21
nmol/L; Exp 2, 0.22 nmol/L) and a normal maximal binding capacity (Exp
1, 38.2 fmol/mg protein; Exp 2, 53.4 fmol/mg protein), indicating
moderate malfunction of the ligand-binding AR domain. Expectedly, the
normal control strain revealed normal binding parameters
(Kd, 0.08 nmol/L; binding capacity, 26.4 fmol/mg
protein; Fig. 2).
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The pSVAR712F construct demonstrated partial
transactivation deficiency in the presence of
dihydrotestosterone compared with the wild-type AR. Maximum activity
was about three fourths of wild-type activity at 0.1 nmol/L
dihydrotestosterone and higher. Using testosterone as ligand, again a
partial trans-activational deficit of the mutant AR was
observed. This was mainly observed at 0.01 and 0.1 nmol/L testosterone,
respectively. However, testosterone concentrations of 1 nmol/L and
higher showed increasing compensation of the functional defect of the
mutant AR (Fig. 3). Differences in
reporter gene induction between the two constructs were not due to
different AR protein expression levels, as evidenced by Western
immunoblot analyses on cell lysates of transiently transfected CHO
cells (data not shown).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Interestingly, trans-activation studies revealed a very particular pattern of functional impairment of the mutant 712F-AR. Defective transcriptional activation was restricted to low hormone concentrations, whereas increasing androgen supply was accompanied by a considerable gain of function. Testosterone at concentrations higher than 1.0 nmol/L could even completely neutralize the defect of the mutant AR. Hence, the 712F-AR is able to change its functional constitution in vitro from subnormal to (almost) normal within the physiological concentration range of testosterone (dihydrotestosterone) in vitro.
Fetal testosterone formation starts at about the 9th week of gestation (31, 32) with peak concentrations appearing at about the 16th week of gestation. They are comparable to those of the normal adult male range (33, 34). The urethral folds have usually fused completely in the midline to form the cavernous urethra and corpus spongiosum by 12th to 14th weeks (Ref. 35 and references therein). Remarkably, testosterone concentrations in these early stages of fetal development show considerable individual variation (32, 34, 36). It is interesting in this respect that there are many reports on partially active mutations within the ligand-binding domain of the AR that are characterized by substantial changes of transcriptional activity in the presence of different ligand concentrations. Androgen levels in the upper physiological concentration range or above can sometimes fully compensate a receptor defect evident at low concentrations in trans-activation assays. As in our family, such mutations have often been associated with a PAIS phenotype, and some patients have been reported with a good response to high dose androgen therapy (20, 23, 24). Based on such clinical and experimental observations, it has been proposed by different researchers that variations of ligand concentration during male sexual differentiation could be one relevant factor for the phenotypic variability in AIS (6, 7, 8). It is conceivable that variations in androgen levels in fetal target tissues within a critical concentration range that would be able to elicit either subnormal or normal trans-activation function of the 712F-AR may have had relevant consequences for the phenotype of the presented subjects. Although in certain patients, the androgen concentration could just have been high enough to induce (almost) full biological AR activity, as suggested for B2 and B3, this will not have been the case in the more severely affected patients, B1 and the uncle.
It is an important observation concerning the biological interpretation of the molecular data in this case that all three androgen-treated patients showed very good clinical response to either testosterone alone (B1 and uncle) or in combination with external dihydrotestosterone (B2). This is additionally reflected by the successive down-modulation of LH and SHBG in response to increasing serum levels of testosterone during therapy in the adult uncle. Thus, in this family, the in vivo effects of androgen therapy principally support the trans-activation data and vice versa. However, trans-activation data alone should usually be considered carefully while discussing a direct physiological relevance (37, 38). It is important to note that partial activity in reporter gene assays is not necessarily associated with sufficient androgen action in vivo (Ref. 20 and references therein; 39). Also intriguingly in this respect, the high serum testosterone levels between 57.4–59.8 nmol/L in the uncle before therapy did not lead to sufficient in vivo androgen action, although full transcriptional activity of the mutant 712F-AR was demonstrated in vitro at this concentration. This indicates that even the interpretation and comparability of distinct hormone concentrations as used in cultured transfected cells with respect to possible physiological actions during embryonic development or in later life is very difficult. In the presented family, the well documented good clinical and biochemical response to androgens observed in the uncle as well as the considerable bioactivity of the mutant receptor in vivo as deduced from the SHBG androgen sensitivity test encouraged us to assign B1 a male gender and treat him accordingly. Whether the quantitative difference between testosterone and dihydrotestosterone concerning activation of the 712F-AR in transfected CHO cells may be significant with respect to physiological target tissues exceeds the capability of the used overexpression model and has not been investigated in more detail.
It may be presumed that different molecular mechanisms independent from AR sequence alterations and ligand concentration may also influence the genotype phenotype correlation in AIS (8). In particular, it cannot be excluded that such mechanisms could also have influenced the variability of the phenotypic appearance in the presented kindred. This, however, has not been investigated in detail in the present study. Remarkably, only a few factors have actually been demonstrated in individual AIS patients (13). One such mechanism that could play a role in phenotypic diversity in AIS is an individually different AR messenger ribonucleic acid level (8). Rodien et al. investigated this hypothesis in an AIS family with striking phenotypic diversity and a M780I point mutation of the AR gene. However, the researchers did not find significant differences (6). Unfortunately, we could not examine this possibility in the present study, because only fibroblasts of one subject (B2) were available. In an earlier study, however, we did not find decreased AR messenger ribonucleic acid levels in fibroblast strains of five patients with missense mutations of the AR gene compared with four control cell lines (40). Increasing knowledge of cross-modulation of AR mediated gene transcription (9, 10, 11) and coactivators and corepressors of nuclear receptor function (41) will presumably uncover further insights into the understanding of androgen action and phenotypic diversity in AIS.
In summary, the presented clinical data in combination with the particular pattern of transcription activation due to the mutant 712F-AR clearly delineate the importance of ligand concentration in phenotypic diversity in AIS. It is very likely that in certain cases, different phenotypes could be based on variation of receptor function caused by variations of androgen availability in the critical time of genital differentiation during early embryonic development.
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Acknowledgments |
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Footnotes |
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Received February 21, 2000.
Revised May 24, 2000.
Accepted June 5, 2000.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-dihydrotestosterone interact differently with the
androgen receptor to enhance transcription of the MMTV-CAT reporter
gene. Mol Cell Endocrinol. 88:15–22.[CrossRef][Medline]
-reductase deficiency. Eur J Pediatr. 155:445–451.[CrossRef][Medline]
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) and a control strain
(•).