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Institute of Zoophysiology, Department of Endocrinology and Developmental Biology, University of Düsseldorf (W.W., B.P.), Düsseldorf; the Department of Internal Medicine, University of Bonn (G.R., H.-U.S.), Bonn; and the Department of General Zoology, University of Ulm (K.-D.S.), Ulm, Germany
Address all correspondence and requests for reprints to: Hans-Udo Schweikert, M.D., Endocrinology, Department of Internal Medicine, University of Bonn, Wilhelmstrasse 35–37, 53111 Bonn, Germany.
Abstract
Supplemental androgen therapy has enhanced virilization in only a few
patients with partial androgen insensitivity (PAIS). We herein report
on virilization in a patient with PAIS and a point mutation in the
DNA-binding domain of the androgen receptor. At the age of 19 yr, the
patient sought medical attention because of undervirilization.
Endocrine findings were typical for androgen insensitivity, but
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-reductase activity and androgen binding characteristics in
fibroblasts cultured from genital skin were normal. In an attempt to
improve virilization, high dose testosterone enanthate treatment (250
mg by im injection once a week) was begun. After 3.5 yr of this
treatment, marked promotion of virilization was achieved,
i.e. lowering of voice, male pattern secondary hair
distribution, marked growth of beard and coarse body hair, increase in
phallic size, increase in bone mineral density, and decrease in mammary
gland size. In addition, serum lipid levels were not affected. To our
knowledge this is the first documentation of successful treatment in a
patient with PAIS and a point mutation in the DNA-binding domain of the
androgen receptor.
SUBJECTS with androgen insensitivity syndromes (AIS) are characterized by a 46,XY karyotype, the presence of testes, normal or elevated androgen levels in blood, and impairment of the usual response to androgens associated with various aberrations of male differentiation and virilization. The spectrum of these disorders ranges from undervirilized or infertile men to individuals with ambiguous genitalia [partial AIS (PAIS)] to phenotypic females (complete AIS). Biochemical studies of androgen binding in cultured genital skin fibroblasts and molecular analysis of the androgen receptor gene of AIS patients have demonstrated that the usual cause of the disorder is a defective androgen receptor (1, 2).
Successful treatment with supplemental androgen therapy has been described in only a few patients with PAIS (see Ref. 2 for review), most of whom have mutations in the ligand-binding domain that causes defective androgen binding to the receptor.
Here we report the successful androgen therapy of a patient with PAIS and a mutation in the DNA-binding domain of the androgen receptor. The mutation, leading to the a change from arginine to glutamine in codon 607, has been reported previously (3).
Subjects and Methods
Subject 1
The index subject is a 23-yr-old man who displayed a phenotype
similar to that described by Reifenstein (4). He was noted to have
perineoscrotal hypospadias at birth. During childhood he underwent
several surgical procedures to correct this disorder, and at age 19 yr
he sought medical attention because of undervirilization (Fig. 1A
). Physical examination at this time
revealed an obese (height, 182 cm; weight, 91 kg) man with no acne or
temporal recession of the hairline and no beard growth. The voice was
high pitched, and mild gynecomastia (Tanner stage M2) was present.
Pubic hair distribution was female in character. The length of the
flaccid phallus was 5.5 cm; the circumference was 7.5 cm. Scars from
previous surgical procedures were present at the ventral surface of the
penis. Testicular size, as measured with the Prader orchidometer, was 6
mL on both sides. The patient reported a nonexisting libido (no
fantasies, no erection). Endocrine studies in pooled serum of the
patient at age 19 yr were as follow: testosterone, 4.6 µg/L (normal,
3–10); estradiol, 15 ng/L (normal, 10–35); LH, 21.2 IU/L (normal,
0.5–9.2); and FSH, 37.4 IU/L (normal, 1.6–11). Biochemical studies in
cultured genital skin fibroblasts showed a 5-reductase activity of
175 pmol/mg protein·h (normal, >10) and near-normal androgen binding
characteristics [binding capacity, 15 fmol/mg protein (normal, >18);
Kd, 0.22 nmol/L (normal, 0.22 ± 0.08);
thermostability, 100% (normal, >60)].
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This 62-yr-old man is the maternal uncle of the propositus. Severe hypospadias was noted at birth, and breast enlargement occurred with the onset of puberty. He allowed blood to be drawn for assessment of androgen receptor gene status but refused further examination.
Subject 3
This 65-yr-old woman is the mother of the propositus.
Materials and Methods
Biochemical and molecular biological studies
Genital skin fibroblasts were established from biopsies as
described previously (5). Androgen receptor binding, determination of
5-reductase activity, and analysis of androgen receptor DNA were
performed as described previously (3).
Treatment protocol
Due to the severe undervirilization of this man, we began high dose testosterone treatment (250 mg testosterone enanthate, weekly im injections) because he and his family insisted that a therapeutic trial to improve his constitution should be instituted. Therapy was initiated at the age of 19 yr and has continued to the present; the clinical response has been documented during regular visits.
Results
Genetic and endocrine studies of the propositus revealed results
typical for androgen insensitivity (male karyotype, elevated
gonadotropin levels in the presence of normal testosterone blood
concentration). 5-Reductase activity and androgen binding
characteristics in cultured genital skin fibroblasts from subject 1
were essentially in the normal range, with a slightly decreased binding
capacity. Molecular analysis of the androgen receptor gene by
PCR-single strand conformation polymorphism analysis and DNA
sequencing, which was performed after beginning of androgen treatment,
revealed a mutation in the second zinc finger of the DNA-binding domain
(exon 3 of the androgen receptor), leading to the change of an arginine
to a glutamine in codon 607 (Fig. 2). No
other mutation in the androgen receptor gene was detected by single
strand conformation polymorphism analysis. The polymorphic Gln repeat
sequence in exon 1 of the patient’s androgen receptor gene contained
21 Gln residues, a value within the normal range (19–31 Gln residues).
The same mutation was detected in DNA from leukocytes of the patient’s
maternal uncle, and the mother was shown to be a heterozygous carrier
of the mutation.
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, B and C), and deepening of the
voice. Mammography performed before and after 3 yr of therapy
demonstrated a decrease in the mammary gland (right side from 3.5
x 2.0 to 3.0 x 1.5; left side from 3.0 x 2.5 to 2.5
x 2.0; height x depth in centimeters). Stretched penile length,
which was 5.5 cm before therapy, increased to 7.5 cm. The bone density
of the lumbar spine (L2 to L4) increased from
1.11 ± 0.01 to 1.21 ± 0.02 g/cm2 as well as red
cell blood parameters, which were subnormal before therapy
(erythrocytes, 4.6 to 4.9 x 106/µL; hematocrit,
39% to 44.6%; hemoglobin, 13.7 to 14.7 g/dL). Furthermore, the
patient reported a switch from a nonexisting to a normal libido with
regular intercourse.
To investigate whether this therapy has unwanted side-effects,
testosterone treatment was withdrawn for 6.5 weeks and then begun
again. As shown in Table 1 serum levels
of triglycerides, low and high density lipoprotein cholesterol, and
total cholesterol remained unchanged. Total and free testosterone as
well as estradiol and PRL increased as expected, whereas LH and FSH
decreased. No consistent effect was observed with regard to sex
hormone-binding globulin (Table 1).
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The benefits and risks of androgen therapy in a number of clinical disorders have been reviewed repeatedly (6, 7, 8), but successful treatment of AIS with supplemental androgen therapy has been achieved to date in only a few patients (9, 10, 11, 12, 13, 14, 15). Investigation of the androgen receptor gene, if performed, resulted exclusively in the detection of mutations in the ligand-binding domain impairing the binding of androgen to the receptor.
To our knowledge we here report for the first time a marked improvement of virilization in a patient with PAIS and a mutation in the DNA-binding domain of the androgen receptor. The mutation (Arg607Gln) is located in the tip of the second zinc finger, which is thought to play a role in receptor dimerization.
In a recent in vitro study, Poujol et al. (16) demonstrated that both an AR with the same mutation as that found in our patient and an AR with a mutation in an adjacent position (Arg608Lys) displayed weaker DNA binding to an isolated androgen response element than did the wild-type AR. Furthermore, cotransfection assays with an androgen-responsive reporter gene revealed a diminished trans-activation property of these mutated AR at an androgen concentration of 3 x 10-11 mol/L, which could be overcome by higher androgen levels (16).
Our patient responded well to androgen therapy. This response was marked not only with respect to muscle bulk and sexual hair development, but external genital growth was also favorably affected. Stretched penile length increased by 40% from 5.5 to 7.5 cm, a value somewhat below (17), respectively within the lower limit of normal (18). Libido, which had been nonexistent before therapy, developed normally, and the patient now reports regular intercourse. In addition, lipid levels remained unaffected.
Thus, androgen therapy not only may be useful in PAIS individuals with androgen receptor gene mutations in the ligand-binding domain associated with defective androgen binding, but in some instances may also be successful in patients with mutations in the DNA-binding domain of the androgen receptor, presumably not affecting androgen binding to the receptor.
At this point it is of interest that recently the effect of long term (5-yr) high dose androgen treatment in another patient with a mutation in the DNA-binding domain of the androgen receptor at position 608 (Arg to Lys) directly beside the mutation found in our patient has been reported (15). However, in contrast to our patient, long term treatment with testosterone and its analogs over many years had no effect. He was reported to have a limited clinical response to extremely high doses of testosterone (500 mg, three times a week), which resulted in an increase in libido, more frequent erections, and the production of penile discharge during sexual arousal. The reason for the discrepancy between these two patients remains unclear.
The same mutation (Arg607Gln) found in our patient has also been detected in the androgen receptor of two brothers with AIS, who displayed clinical symptoms of Reifenstein’s syndrome, i.e. penoscrotal hypospadias and microphallus at birth (19). At the age of 55 and 75 yr, respectively, both patients developed breast cancer, a rare disease in men (20). In addition, a third patient with PAIS (hypospadias, microphallus, and gynecomastia) due to a mutation in the neighboring codon (Arg608Lys) developed breast cancer (21, 22). Two main hypotheses have been proposed to explain breast cancer development associated with the androgen receptor mutations found in these men. First, prolonged abnormal sex steroid exposure of the epithelium of the mammary gland, i.e. loss of the protective effect of androgens due to a defective androgen receptor in favor of estrogens, could eventually trigger malignancy. Second, the mutated androgen receptor itself could activate estrogen-regulated genes through binding to estrogen-responsive elements and in this way cause cancer.
As mentioned, our patient developed gynecomastia at puberty. Thus, we have monitored the patient’s mammary gland size, performing a mammography before and after 2 and 3 yr of treatment. Interestingly, a marked decrease in mammary gland size was achieved with therapy. A possible explanation for this outcome is that the deficient action of androgen (in the presence of a normal response to estrogen) on the epithelium of the mammary gland was partially reversed by high dose androgen treatment and thus might be protective against prolonged abnormal estrogen exposure. However, at the moment we do not know the long term effect of this therapy with respect to the development of mammary cancer in men with mutations of the androgen receptor. Consequently, regular examinations of the breasts during high dose androgen therapy for AIS are necessary.
In conclusion, to our knowledge no clear and general applicable parameter or functional test is available to predict the outcome of androgen treatment in PAIS patients. Our results indicate that even in the case of a mutation in the DNA-binding domain of the androgen receptor, high dose testosterone therapy might be successfully instituted and thus appears to be warranted for a limited period of months to decide whether long term treatment should be continued. It is clear that thorough clinical monitoring is mandatory.
Acknowledgments
The authors are grateful to Dr. Jean D. Wilson, Division of Endocrinology, Department of Internal Medicine, University of Texas Health Science Center (Dallas, TX), for helpful suggestions.
Footnotes
1 This work was supported by the Deutsche Forschungsgemeinschaft (SFB
351, A1). Part of this work has been presented at the 10th
International Congress of Endocrinology, San Francisco, CA, June
12–15, 1996 (Abstract P1–165). 
Received October 22, 1997.
Revised December 17, 1997.
Accepted December 23, 1997.
References
-reductase deficiency and disorders of the androgen receptor. J Clin Invest. 74:1496–1508.
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