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Original Studies |
Department of Pediatrics, Medical University of Lübeck (P.M.H., O.H., K.K., G.H.G.S.), Lübeck, Germany; the Department of Endocrinology and Reproduction, Erasmus University (H.T.B., A.O.B.), Rotterdam, The Netherlands; and the Department of Gynecologic Endocrinology and Reproductive Medicine, University Hospital (A.K.H.), Hamburg-Eppendorf, Germany
Address all correspondence and requests for reprints to: Paul-Martin Holterhus, M.D., Department for Pediatrics, Medical University of Lübeck, Kahlhorststrasse 31–35, 23538 Lübeck, Germany.
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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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Single base mutations resulting in amino acid substitutions (missense mutations) represent the most common structural defects of the AR gene in either partial AIS (PAIS) or complete AIS (CAIS) (1, 2, 3, 8). More extensive structural alterations at the protein level can be due to nonsense mutations inducing the formation of a premature translation termination (stop) codon. This may be the result of either the direct conversion of amino acid codons into stop codons through point mutations (9, 10, 11) or the indirect formation after disarrangements of the translational reading frame by frameshift mutations (8, 11, 12, 13). Based on events at the molecular level [truncation of the AR, reduced AR messenger ribonucleic acid level (14, 15), and physiologically insufficient downstream initiation (9)], androgen action is seemingly completely abolished in vivo. As expectedly, in the current literature premature stop codon mutations have exclusively been associated with the CAIS phenotype (9–12; for review, see Ref. 1 and references therein).
To characterize causes for incongruent genotype-phenotype correlations in patients with androgen insensitivity, we investigated an AIS patient with partial virilization of the external genitalia despite the presence of a premature stop codon within the AR gene. Functional studies were performed at the DNA and protein levels, leading to elucidation of the underlying molecular mechanism. We discuss the clinical significance of an almost unrecognized and probably underestimated phenomenon for the clinical treatment and genetic counseling of patients with androgen resistance.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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A 23-yr-old woman of Polish origin contacted her physician
because of primary amenorrhea. Her external genitalia were
predominantly female. However, clitoral enlargement to 2.0 cm and pubic
hair Tanner stage 4 indicated partial virilization (Fig. 1
). Orificium urethrae externum and
introitus vaginae were separated. No fusion of the posterior labial
folds was present. In addition, bilateral axillary hair was observed,
and the voice was that of a normal female. Breast development
corresponded to Tanner stage 5. Two different karyotype analyses, on
blood lymphocytes and on genital skin fibroblasts, displayed a 46,XY
karyotype. Serum testosterone (31.2 nmol/L) was in the upper male
range, and LH (11.0 U/L) and the LH x testosterone product (343)
were elevated (normal LH x testosterone product, <170). As an
estimation of in vivo function of the AR, serum sex
hormone-binding globulin (SHBG) was measured before and after 3 days of
oral administration of 0.2 mg/kg BW·day of the anabolic steroid
stanozolol according to a standardized test protocol (4). Basal SHBG
was low but still normal for age (27.8 nmol/L). No decrease in SHBG
but, rather, an increase was observed in response to stanozolol. The
lowest value corresponded to 112.7% (31.3 nmol/L) of the initial
concentration [normal response, 51.4 ± 2.1% (±SE);
range, 35.6–62.1%] (4). On laparoscopy, bilateral ivory-colored
testis-like structures, each measuring 2–3 cm in length, were
demonstrated. No Mullerian remnants were detected. To prevent the
patient from further virilization, the gonads were removed.
Histological examination revealed testis-specific tissue
differentiation with atrophic germinal epithelium and Leydig cell
hyperplasia. After gonadectomy, hormone substitution therapy was
started with estradiol valerate.
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All cells were cultured at 37 C in 5% CO2. Genital skin fibroblasts of the patient, obtained during gonadectomy, and male control fibroblasts, derived from foreskin specimens, were maintained in MEM (Life Technologies, Grand Island, NY) supplemented with 10% (vol/vol) FCS, 1% (vol/vol) MEM nonessential amino acids (Life Technologie), and penicillin (200 IU/mL)-streptomycin (0.2 mg/mL). COS-1 and CHO (Chinese hamster ovary) cells were cultured in DMEM with the nutrient mix F-12 (Life Technologies), 5% (vol/vol) FCS, and antibiotics. For trans-activation studies, CHO cells were plated in medium containing 5% dextran-charcoal-treated FCS.
Androgen binding studies
Androgen binding studies were performed as previously described
(16). In brief, confluent cultures of genital skin fibroblasts were
incubated 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) in either the presence or
absence of a 200-fold molar excess of unlabeled ligand. All incubations
were performed in duplicate. After 1 h at 37 C, 50 µL culture
medium were taken from each dish for determination of total counts.
Cell monolayers were washed with 2 mL Tris-saline (20 mmol/L Tris and
0.15 mol/L NaCl, pH 7.4) and scraped in 1 mL TEG buffer [20 mmol/L
Tris, 1.5 mmol/L ethylenediamine tetraacetate, 10% (vol/v) glycerol,
600 µmol/L phenylmethylsulfonylfluoride, and 500 µmol/L bacitracin,
pH 7.4]. After centrifugation of the cell suspensions (10 min,
800 x g), pellets were washed in TEG without protein
inhibitors and dissolved in 1 mL 0.5 N NaOH (30 min, 56 C).
Five hundred microliters were taken for liquid scintillation counting;
100 µL were used in duplicate for protein determination. Scatchard
calculations were performed on a Microsoft Excel Personal Computer
software (Microsoft Corp., Richmond, WA).
Immunoblot analysis
Western immunoblot analysis was performed as described previously (17) with only minor changes. Briefly, confluent cultures of genital skin fibroblasts (175 cm2) or transfected COS-1 cells (75 cm2) were lysed in a buffer containing 40 mmol/L Tris-HCl (pH 7.4), 1 mmol/L ethylenediamine tetraacetate, 10% glycerol, 10 mmol/L dithiothreitol, 1% Triton X-100, 0.08% SDS, 0.5% sodium deoxycholate, 600 µmol/L phenylmethylsulfonylfluoride, and 500 µmol/L bacitracin. AR was immunoprecipitated from whole cell lysates by the monoclonal antibody F39.4 (18) coupled to goat antimouse agarose beads (Sigma Chemical Co., St. Louis, MO). Samples were separated by SDS-PAGE (4% stacking gel and 7% separating gel) for 60 min at 200 V. Electroblotting on cellulose nitrate membranes was performed for 1 h at 100 V. After drying and rinsing several times with PBS-0.1% Tween-20 (Bio-Rad, Richmond, CA), the membranes were blocked with PBS, 0.1% Tween-20, and 5% nonfat dry milk (blocking buffer) and subsequently incubated for 1 h in a moist chamber with primary antibody SP197 or SP061 (each diluted 1:1000 in blocking buffer), directed against amino acids 1–20 or 301–320 of the AR, respectively (17, 18). As second antibody, an antirabbit peroxidase conjugate (Sigma) was used in a 1:4000 dilution. Protein detection was performed by chemiluminescence using the Renaissance Western Blot Chemiluminescence Reagent (DuPont-New England Nuclear, Boston, MA).
DNA studies
Genomic DNA was extracted from peripheral blood leukocytes
according to standard procedures and served as template for the PCR.
Exons 1–8 of the AR gene were individually amplified, followed by
mutation screening using nonisotopic single strand conformation
polymorphism (SSCP) analysis as reported previously (11, 19, 20).
Resulting from an aberrant migration pattern on SSCP, a 414-bp PCR
fragment representing segment 2 of exon 1 was purified using the
Qiaquick extraction kit (Qiagen, Hilden, Germany) and directly
sequenced using [
-32P]ATP end-labeled primers with the
Sequenase sequencing kit (Amersham Buchler, Braunschweig, Germany)
(19). AflII (New England Biolabs, Beverley, MA) restriction
site analysis of the 414-bp exon 1 PCR fragment was performed on
genomic DNA derived from either blood leukocytes or genital skin
fibroblasts. Several controls for wild-type AR DNA contamination of the
PCR reactions were performed by repeating separate experiments in
different laboratories and using different genomic DNA preparations.
Template-free conditions were always included. The samples were
analyzed on a 5% glycerol polyacrylamide gel followed by silver
staining.
AR expression vectors and transfections
The human AR expression vector pSVAR0 (22) served as a starting point to create a mutant complementary DNA construct containing the stop codon TGA at codon position 172. After AflII digestion of pSVAR0, the overhanging 5'-ends were blunted by a 15-min incubation with 0.06 U/µL S1 nuclease (Pharmacia). This was followed by BamHI digestion, thus removing a 2318-bp AflII/S1-BamHI fragment from the vector. Two oligonucleotides (5'-TGA-ACT-AGT-CGA-TG-3' and 5'-GAT-CCA-TCG-ACT-AGT-TCA-3') were hybridized to construct a linker containing the TGA stop codon, an additional SpeI site, and a BamHI 5'-overhanging end. Subsequently, the linker fragment was cloned into the prepared vector. In-frame ligation was verified by plasmid sequencing. In a second cloning step, the above-mentioned 2318-bp fragment was recloned into the first step product. The latter was pretreated before ligation by SpeI digestion followed by blunting of the 5'-overhanging SpeI end (S1 nuclease) and an additional BamHI digestion. The final construct pSVAR172-stop was verified for the correct sequence by plasmid sequencing. Construction of the expression vector pSVAR121 representing an N-terminal-deleted AR complementary DNA encompassing codons 189–910 was previously reported by Jenster et al. (23).
CHO and COS-1 cells were maintained as described above and transiently transfected by the calcium phosphate precipitation method (24). For trans-activation studies, CHO cells were cultured in 10-cm2 six-well multidishes, using 12.5–2,500 ng AR expression vector/dish (50–10,000 ng/mL precipitate) and 0.5 µg (2 µg/mL) of the reporter plasmid mouse mammary tumor virus (MMTV)-Luc (Organon, West Orange, NJ) (25), adjusting to a final amount of 5 µg (20 µg/mL) plasmid DNA with the pTZ19 carrier plasmid. Transfections were performed in triplicate in three independent experiments (100–10,000 ng/mL precipitate: two experiments, each performed in triplicate). Twenty-four hours before cell lysis, cells were incubated with medium containing either no hormone or 1 nmol/L R1881 (DuPont-New England Nuclear). Luciferase activity was determined as previously described (25). For studying expression of the AR, 75-cm2 subconfluent cultures of COS-1 cells were transfected with 1.5 µg (1 µg/mL) expression plasmid (pSVAR0, pSVAR121, or pSVAR172-stop, respectively), adjusted to a final amount of 30 µg (20 µg/mL) with the carrier plasmid pTZ19. Twenty-four hours after transfection, cells were glycerol shocked by a 1.5-min incubation with 15% glycerol-MEM without FCS. Whole cell lysates were generated 24 h later, preceding immunoprecipitation of the AR.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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All eight exons of the AR gene of the patient’s genomic DNA were
successfully amplified by PCR and resulted in PCR fragments of the
predicted length (11). Exons 2–8 were individually amplified in a
single step, exon 1 was divided into seven overlapping segments.
Because of an abnormal migration pattern on SSCP analysis, the 414-bp
segment of exon 1 of the gene was sequenced. At codon position 172, a
point mutation, TTA to TGA, was detected that replaced the original
leucine residue and created a premature stop codon (opal; Fig. 2), thus eliminating a restriction
recognition site for the enzyme AflII. In addition to the
mutant DNA sequence, the wild-type DNA sequence was observed (Fig. 2).
To confirm these data, an AflII restriction recognition site
analysis of the respective 414-bp PCR fragment was performed.
Restriction of PCR products from normal controls lead to the formation
of two fragments, 205 and 209 bp in length, respectively. In separate
DNA preparations from peripheral blood leukocytes and genital skin
fibroblasts of the patient, however, the PCR products were partially
digested (Fig. 3, lanes 2 and 4,
respectively). Hence, sequencing data and restriction site analysis
both demonstrate the presence of mutant and wild-type AR DNA sequences
in the patient’s blood leukocytes and genital skin fibroblasts.
Because karyotype analyses excluded chromosomal mosaicism, and
sequencing of the CAG repeat in exon 1 supplemented the presence of
only one X-chromosome, mosaicism due to a somatic mutation of the AR
gene was considered.
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Androgen-binding properties were studied in a whole cell assay of
genital skin fibroblasts using the synthetic androgen methyltrienolone
(R1881). In Fig. 4, a Scatchard plot is
presented that demonstrates specific R1881 binding of genital skin
fibroblasts of the patient. The calculation of maximal binding
(Bmax) revealed a value of 22.56 fmol/mg protein, which is
below the normal male range (39.0–169.0 fmol/mg protein). A normal
dissociation constant (Kd) of 0.063 nmol/L was found
(normal male range, 0.03–0.13 nmol/L). This result indicates the
expression of an AR containing a functionally intact androgen-binding
domain.
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Investigation of AR expression in the patient’s tissue was
performed by immunoprecipitation of the AR from whole cell lysates of
equally grown confluent cultures of genital skin fibroblasts preceding
Western immunoblot analysis. Using the monoclonal antibody F39.4 for
immunoprecipitation and the polyclonal antibody SP061 for
immunodetection on cellulose nitrate membranes, a normal 110/112-kDa AR
doublet was found in the genital skin fibroblasts of the patient (Fig. 5, lane 3), indicating expression of the
wild-type AR. The signal intensity of the 110/112-kDa band in the
patient was always reduced compared to that of the male control genital
skin fibroblast strain (Fig. 5, lane 2 vs. lane 3). This is
in accordance with the low Bmax observed in the Scatchard
analysis and could be an indication of a reduced amount of wild-type AR
expressed in a given population of genital skin fibroblasts of the
patient. However, any quantitative interpretation should be restrained
because the ratio of fibroblasts in tissue culture containing either
the mutant or the wild-type AR allele does not necessarily reflect
in vivo conditions.
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AR expression using pSVAR172-stop, pSVAR121, or pSVAR0 expression
plasmids has been investigated in transiently transfected COS1 cells.
Antibody F39.4 has been used for immunoprecipitation, whereas either
SP197 or SP061 served for immunodetection on Western blots. Figure 5
, lane 1, illustrates the formation of a normal 110/112-kDa AR after
transfection of the wild-type AR construct pSVAR0. Construct pSVAR121,
encoding amino acids 189–910 (23), demonstrated the expression of the
predicted N-terminal-deleted 87-kDa AR fragment using SP061 in
immunodetection (Fig. 5, lane 5). In accordance, no signal could be
obtained using antibody SP197 (Fig. 5, lane 7). Remarkably, a weaker
band following the same pattern in migration (87 kDa) and
immunodetection (SP061, signal; SP197, no signal) as pSVAR121 was found
for pSVAR172-stop (Fig. 5, lanes 4 and 6, respectively), indicating the
expression of an 87-kDa AR fragment most likely caused by downstream
initiation of translation at Met189. The 87-kDa band that
is visible in lane 3 representing the patient’s genital skin
fibroblasts is also consistent with downstream initiation, because no
87-kDa signal could be obtained in a different blot using antibody
SP197 (data not shown). Whether the smaller bands in lane 2
representing the control fibroblast strain are also caused by the use
of alternative start sites or by proteolysis has not been
investigated.
Trans-activation studies
To exclude the possibility that partial virilization of the
patient could have been significantly influenced by the N-terminal
truncated AR caused by the mutant AR allele, the
trans-activation properties of pSVAR172-stop compared with
those of pSVAR121 and pSVAR0 were investigated. Induction of the
MMTV-Luc reporter plasmid using transiently transfected CHO cells was
measured in either the presence or absence of 1 nmol/L R1881. Maximum
reporter gene induction by the wild-type AR plasmid pSVAR0 was found
using 12.5 ng expression plasmid in transfections of 10-cm2
cultures of CHO cells; a 46.0- fold induction (range, 45.2–46.8)
relative to basal activity in the absence of R1881 was observed (Fig. 6). Under these conditions, no
significant trans-activation of the reporter gene by
pSVAR172-stop was present (1.4-fold; range, 0.4–2.6; Fig. 6). Only at
highly elevated concentrations of pSVAR172-stop could a partial
trans-activation of MMTV-Luc be observed, with a maximum
induction of 13.0-fold (range, 10.3–15.7) using 1250 ng expression
plasmid (5 µg/mL precipitate; Fig. 6). With respect to the AR
expression studies (Fig. 5), this activity is most likely due to the
N-terminal-truncated 87-kDa AR caused by downstream initiation. The
pSVAR121 expression plasmid represents the same N-terminal truncated AR
as the downstream initiation product caused by pSVAR172-stop. Partial
trans-activation of the reporter plasmid was observed using
pSVAR121 (6.8-fold; range, 5–8.4), corresponding to previously
reported data (23) (Fig. 6).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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DNA sequencing and restriction site analysis demonstrated the
presence of a somatic mosaic of mutant and wild-type AR alleles in the
patient. This was confirmed by the presence of normal androgen-binding
properties in cultured genital skin fibroblasts and the detection of a
normal 110/112-kDa AR doublet in Western immunoblots, both indicating
expression of the wild-type AR. A physiological significance of
downstream initiation of translation at Met189 resulting in
an 87-kDa AR, as demonstrated in Fig. 5, seems unlikely, because only
relatively high plasmid concentrations of the pSVAR172-stop plasmid
lead to relevant reporter gene induction in cotransfection assays.
Different reports by others on premature stop codons of the AR gene in
the human (9) as well as in the mouse (27) being associated with
partially active N-terminal truncated AR fragments caused by downstream
initiation support our findings. The respective phenotypes have always
been CAIS despite the occurrence of downstream initiation (9, 27).
Thus, we conclude that the partial virilization of our patient is most
likely due to the expression of the wild-type AR based on somatic
mosaicism. The absent SHBG decrease in response to stanozolol may be
due to variations in the tissue distribution of mutant and wild-type AR
alleles. One would expect, with respect to this consideration, that
liver parenchymatous cells predominantly, if not exclusively, contain
the mutant form of AR alleles.
The occurrence of somatic mosaicism in genetic diseases is not a
rare event. For example, the McCune-Albright syndrome is due to somatic
mutations of the gene coding for the -subunit of the G protein
(Gs) (28, 29). The variability in the severity of
clinical manifestations in this disease is consistent with the presence
of different ratios of mutated and wild-type alleles in tissues of
individual patients (28, 29). Concerning AIS, the possibility of
somatic mosaicism hardly received attention in the current literature.
Publications dealing with somatic mutations of the AR gene have mostly
been restricted to malignant disease (30, 31, 32). To date, only one case
of a somatic mutation of the AR gene in AIS has been published (19).
Partial virilization of that patient has been suggested to be most
likely due to the expression of the wild-type AR based on the somatic
mosaic. A comparable animal model has been studied with the
XXTfm-Sxr mouse in detail (33, 34). Different ratios of
androgen-responsive and -unresponsive cells in the somatic mosaics of
these mice are responsible for phenotypes ranging from predominantly
male with hypospadias to severely impaired masculinization with
predominantly female appearance (33).
Somatic mosaicism of the AR gene represents the first clearly defined mechanism significantly influencing the genotype-phenotype correlation in patients with AIS. Expression of the wild-type AR plays a crucial role in this molecular genetic constellation by shifting the AIS subtype to a higher degree of virilization than expected from the mutant allele alone. For clinical purposes, knowledge about somatic mosaicism of the AR gene in a particular patient with AIS provides important information for further management. First, somatic mosaicism can elucidate possible discrepancies occurring among phenotype, genotype, and the SHBG androgen sensitivity test and therefore be of help in interpretation of the data obtained. Second, it provides the basis for genetic counseling of these families, as the risk for another child with AIS is low if somatic mosaicism is present in the index patient. Third, early gonadectomy is prudent in all patients rendered female to prevent undesired virilization during puberty (clitoromegaly and deepening of the voice) because of presumed partial androgen action.
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Acknowledgments |
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Footnotes |
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2 Recipient of a visiting scholarship from the European Society for
Pediatric Endocrinology.
Received April 2, 1997.
Revised July 30, 1997.
Accepted August 1, 1997.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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mutation is present in fibrous
dysplasia of bone in the McCune Albright syndrome. J Clin
Endocrinol Metab. 79:750–755.[Abstract]
18) in the androgen
receptor gene in human prostate cancer. Biochem Biophys Res Commun. 198:74–80.[CrossRef][Medline]
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