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A
novel E153X point mutation in the androgen receptor gene in a patient
with complete androgen insensitivity syndrome
Silvia
B Copelli1,2, Serge Lumbroso1, Francoise Audran1,
Eliana H Pellizzari2, Juan J Heinrich2, Selva B
Cigorraga2, Charles Sultan1, Hctor E Chemes2
1L¶bor¶toire
d'Hormonologie, Hôpital Lapeyronie et INSERM U 439 Pathologie des
rcepteurs nuclaires.Unit d'Endocrinologie et Gyncologie Pdiatriques,
Hôpital A. de Villeneuve, Montpellier-France
2CEDIE-CONICET, Divisin de Endocrinologa, Hospital de
Niños Ricardo Gutierrez, Buenos Aires, Argentina
Asian J Androl 1999 Jun; 1: 73-77
Keywords: testicular feminization; androgen receptor; point mutation; Mullerian ducts
Abstract
Aim: To study a 46, XY newborn patient with a phenotype suggestive of an androgen insensitivity syndrome to confirm an anomaly in the AR gene. Methods: Genomic DNA from leukocytes was isolated in order to analyze SRY gene by PCR and sequencing of the eight exons of AR gene. Isolation of human Leydig cell mesenchymal precursors from the testis was performed in order to study testosterone production and response to hCG stimulation in culture. Results: Surgical exploration disclosed two testes, no Wolffian structures and important Mllerian derivatives. The SRY gene was present in peripheral blood leukocytes. Sequencing of the AR gene evidenced a previously unreported G to T transversion in exon 1 that changed the normal glutamine 153 codon to a stop codon. Interstitial cell cultures produced sizable amounts of testosterone and were responsive to hCG stimulation. Conclusion: This E153X nonsense point mutation has not been described previously in cases of AIS, and could lead to the synthesis of a short truncated (153 vs 919 residues) non functional AR probably responsible for the phenotype of complete androgen insensitivity syndrome (CAIS).1 Introduction
Androgen action in target cells requires classical and complex pathways of steroid receptors. Many alterations in the androgen receptor gene interfere with this action and lead to the androgen insensitivity syndrome. The androgen insensitivity syndrome (AIS) is the most frequent form of male pseudohermaphroditism. This disorder arises from a failure in the androgen receptor (AR) in target cells. The androgen action is essential for an appropriate virilization of the genital tract. The incomplete masculinisation found in patients with the AIS is most often the result of an alteration in the AR gene that leads to an abnormal response to testosterone and dihydrotestosterone. AIS is an X-linked recessive form of male pseudohermaphroditism with a phenotypic expression that varies from ambiguous external genitalia to normal female configuration[1]. The patients with complete androgen insensitivity syndrome (CAIS) are characterized by normal female external genitalia, shortened vagina, absence of uterus, intrabdominal or inguinal testis, normal breast development at puberty and absence of pubic/axillary hair. In the partial androgen insensitivity syndrome (PAIS) there is a wide spectrum of clinical disorders: from a mild clitoromegaly and/or slight labial fusion to an important ambiguity of external genitalia at birth. In other cases, there is a more extensive masculinization especially in Reifenstein Syndrome patients, with a male phenotype, perineal hypospadias and cryptorchidism[2]. The mildest form of expression is azoospermia in a normal male. The AR gene is located in the pericentromeric region of the X chromosome in Xq11-Xq12. It is a gene of 90 Kb and 8 exons that encodes a protein of 919 aminoacids[3,4]. This AR protein belongs to the nuclear receptor family with zinc finger motif and has three main functional domains. The extensive N-terminal domain encoded by exon 1, involved in transcriptional regulation is the least conserved region. The central domain encoded by exons 2 and 3, comprises two zinc finger structures and is responsible for binding to DNA at the androgen response element in the promoter sequences of target genes. The C-terminal region encoded by exons 4 to 8 is involved in high-affinity ligand binding[5]. The most frequent anomalies present in the AR gene in AIS patients are point mutations in the DNA-binding and ligand-binding domains contrasting with a low frequency of gene microdeletions[6]. We report here a novel E153X mutation in a CAIS patient with Mllerian remnants.2 Materials and methods
The patient was referred at birth as a normal female with bilateral inguinal hernia. The girl showed normal external genitalia but had a 46,XY karyotype. On surgical exploration at three months, two testes were found and bilateral orchidectomy was performed. Both gonads and the gonaducts were serially sectioned, embedded in Paraplast and the sections stained with Hematoxylin Eosin and PAS-HE. A fraction of both testes was used to isolate Leydig cell precursors according to techniques previously described[7]. Briefly, a piece of tissue was placed in a prechilled 1:1 mixture of Ham's F12 medium: Dulbecco's Modified Eagle Medium (DMEM: F12). The tissue was finely minced and was treated twice with a solution of 0.04% collagenase (type I, Sigma Chemical Co, 130 u/mg) and 0.01% trypsin inhibitor in the culture medium mentioned above, under constant agitation at 34 for 40 min. After this digestion procedure, cells obtained from supernatants were purified by use of a discontinuous Percoll density gradient and the interface between 34% and 60% was collected. Cells (2105/cm2) were plated in multiwell dishes in DMEM: F12. Culture was performed at 34 in a controlled humidified atmosphere of 5% CO2 in air. After an initial 72-h period a group of cells was stimulated with 50 ng/mL hCG (NIH-hCG-CR127) during the entire culture period. hCG was added with medium changes every 48-72 h. Basal cultures (no hCG added) were run in parallel.
Genomic
DNA (0.5-1 g) extracted from peripheral blood leucocytes was
used as template for polymerase chain reaction (PCR). In addition to the
patient, both parents and a sister were studied.
3 Results
3.1
Molecular biology of the SRY and AR genes
As
expected, the SRY (The testis determining factor) was present in peripheral
blood leucocytes from the patient. Exons 2-8 of AR gene were amplified
by PCR with a primer set each. For the analysis of exon 1, five sets of
overlapping primers were used to cover its complete length. PCR analysis
of all AR exons in the patient, her parents, and a normal 46,XX sister
showed no microdeletions. Sequencing of each exon in the patient detected
a G to T transversion in exon 1, resulting in a change from the normal
153 glutamine codon (GAG) to a stop codon (TAG). Her mother was heterozygous
for the mutation, while father and sister were normal (Figure 1).
Figure 1. Partial sequence of exon 1 of the androgen receptor
gene in the normal sister, patient, carrier mother and father. The mutation
consists in a G to T transversion in the 153 codon. The column to the
left represents the normal sense sequence with the G to T transversion(*)
in codon 153 resulting in the change from the normal Glu codon (GAG) to
a stop codon (TAG).
3.2
Pathologic examination
Both gonads were ovoid structures 9 mm6 mm3 mm with the typical appearance of testes. The tunica albuginea was very well developed. The seminiferous tubules were 45-50 m in diameter with a thin tubular wall, immature Sertoli cells and very abundant gonocyte-like immature spermatogonia. In the intertubular spaces, there were some vacuolated cells with Leydig cell differentiation. There were no sings of testicular dysgenesis (Figure 2). A tubular 3 cm long structure was observed on the left side, with macro and microscopic features of a Fallopian tube (Figure 2). There were also proliferations of smooth muscle cells along the course of the tube. No uterine remnants or Wolffian derivatives (epididymis or vas deferens) were found.
Figure 2. A: Gonadal histology showing normal testicular architecture with seminiferous cords populated by immature Sertoli cells and abundant infantile spermatogonia. B: The left side gonaduct evidences Fallopian tube differentiation. Bars A and B: 25 m.
3.3
In vitro testosterone production by interstitial cell cultures
Immature
interstitial cells isolated from both testes developed in culture as a
monolayer of fibroblast-like cells. The cells secreted testosterone to
the culture medium in a time-dependent fashion, with a peak at d 9 (118.52.5)
pg T/g DNAh. At this time, addi-tion of hCG elicited a
significant increase in T production (155.36.1) pg T/g DNAh
(Figure 3).
4 Discussion
The
AR gene is the nuclear receptor gene in which the most numerous and heterogeneous
variety of mutations have been described, usually localized in exons 2
to 8 that encode the DNA- and ligand-binding domains. These mutations
induce changes in the AR protein that are responsible for the complete
or partial phenotypes of AIS. There have been more than 200 point mutations
described as a cause of AIS, but deletions or microdeletions of the AR
gene have been only occasionally found[6]. The E153X point
mutation here described has not been reported previously in cases of AIS. This
is a nonsense mutation responsible for the synthesis of a truncated androgen
receptor (153 vs 919 aminoacids) devoid of most of the functional domains.
Although the mutation has not been reproduced in vitro, the mutant AR
is highly suspected to be completely inactive and thus explains the complete
phenotype of CAIS in our patient. Exon 1 is a large exon, infrequently
hit by mutations, but in many cases, the reported mutations are stop codons
that lead to CAIS[6]. Furthermore, the complete phenotype of
our patient seems to exclude the theoretical possibility of a downstream
initiation (from methionine 189), leading to the partial production of
functional AR.
The
in vitro T production by interstitial cell cultures and its response
to hCG is similar to that reported by Berensztein et al[10]
for normal prepubertal testes obtained at necropsy. This indicates that
the interstitial cell function in our patient is normal, as has been previously
reported for prepubertal
testes from CAIS patients[7].
An
unusual feature in this patient is a presence of important Mllerian
derivatives. Under normal circumstances the Mllerian ducts suffer involution
under the influence of AntiMllerian Hormone (AMH) a protein produced by
Sertoli cells of the fetal testis. On the other hand, Wolffian derivatives
(the epidydimis and vas deferens) are stimulated to grow and differentiate
by fetal testosterone secretion[11]. In patients with CAIS
AMH secretion is not affected leading to complete Mllerian regression,
while Wolffian ducts fail to grow due to androgen insensitivity. The asymmetrical
presence of a Fallopian tube in our patient suggests that her left testis
was unable to suppress the development of the homolateral Mllerian duct.
Other mechanisms that have been also proposed to explain the rare Mllerian
persistence in androgen insensitivity are the peripheral
failure of AMH receptors or early testicular descent[12], but
there has not been any conclusive evidence to support any of these interpretations.
Moreover, the report of Rutgers & Scully[13] that up to
35 % of patients with CAIS have persistent Mllerian derivatives, indicates
that this phenomenon is not such a rare event as it was previously thought.
5
Acknowledgements
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Supported
by grants PMT-PICT 0090 from CONICET, and PICT 0450 from ANPCyT, Argentina,
and Institut National de le Sant et de la Recherche Mdicale, INSERM,
France.
Correspondence to Dr Silvia B Copelli.
Centro de Investigaciones Endocrinolgicas, Hospital de Niños
Ricardo Gutierrez, Gallo 1330, Buenos Aires, 1425 ARGENTINA.
Tel: +54-11-4962 4035 Fax: +54-11-4963 5930
E-mail: copelli@fend.guti.sld.ar
Received 1999-03-25 Accepted 1999-05-10
