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- Case Report -
Novel mutation in the ligand-binding domain of the
androgen receptor gene (l790p) associated with complete
androgen insensitivity syndrome
Florina Raicu1,3,7, Rossella
Giuliani2, Valentina
Gatta2,3, Chiara Palka2, Paolo Guanciali
Franchi2, Pierluigi
Lelli-Chiesa4, Stefano
Tumini5, Liborio Stuppia2,3,6
Department of Clinical Sciences and Imaging,
2Department of Biomedical Sciences,
3Aging Research Center, 4Division of
Pediatric Surgery, 5Department of Pediatrics and Gynecology, G. d'Annunzio University Foundation, Chieti-Pescara
66013, Italy
6Institute of Molecular Genetics, National Research Council, IGM-CNR, Bologna 40100, Italy
7Carol Davila University of Medicine and Pharmacy, Bucharest 020027, Romania
Abstract
Mutations in the X-linked androgen receptor (AR) gene cause androgen insensitivity syndrome (AIS), resulting in
an impaired embryonic sex differentiation in 46,XY genetic men. Complete androgen insensitivity (CAIS) produces a
female external phenotype, whereas cases with partial androgen insensitivity (PAIS) have various ambiguities of the
genitalia. Mild androgen insensitivity (MAIS) is characterized by undermasculinization and gynecomastia. Here we
describe a 2-month-old 46,XY female patient, with all of the characteristics of CAIS. Defects in testosterone (T) and
dihydrotestosterone (DHT) synthesis were excluded. Sequencing of the
AR gene showed the presence in exon 6 of a T to C transition in the second base of codon 790, nucleotide position 2369, causing a novel missense Leu790Pro
mutation in the ligand-binding domain of the AR protein. The identification of a novel AR mutation in a girl with CAIS
provides significant information due to the importance of missense mutations in the ligand-binding domain of the AR,
which are able to induce functional abnormalities in the androgen binding capability, stabilization of active conformation,
or interaction with coactivators. (Asian J Androl 2008 Jul; 10: 687_691)
Keywords: androgen receptor; novel androgen receptor gene mutation; complete androgen insensitivity syndrome
Correspondence to: Dr Liborio Stuppia, G. D'Annunzio University Foundation, Via dei Vestini 35, Chieti 66013, Italy.
Tel: +39-0871-3554-137 Fax: +39-0871-3554-133
E-mail: stuppia@unich.it
Received 2007-06-20 Accepted 2007-11-10
DOI: 10.1111/j.1745-7262.2008.00376.x
1 Introduction
Androgens are the main steroid hormones that
determine the expression of the male phenotype. Their
activity is mediated by an androgen receptor (AR),
alternatively known as the dihydrotestosterone (DHT) receptor,
which, like all ligand-activated nuclear transcription
factors, translocates to the nucleus and binds to the
regulatory regions of specific chromosomal DNA sequences
(androgen response elements), to activate androgen
dependent genes [1]. The androgen_AR complex functions in conjunction with co-regulatory
proteins [2].
Disorders of AR function due to mutations in the
AR gene cause different forms of X-linked male
pseudo- hermaphroditism, known as androgen insensitivity
syndromes (AIS; OMIM ID: 300068), affecting XY female
individuals with normal androgen production and metabolisms.
AIS are estimated to be present in 1:20 000_64 000
male births. The presence of variable phenotypic
expression allows the classification of AIS into complete
androgen insensitivity (CAIS), partial androgen
insensitivity (PAIS) and mild androgen insensitivity
(MAIS).
In the most extreme form, CAIS-affected patients
have normal female external genitalia, absent pubic and
axillary hair ("hairless pseudofemale"), female breast
development, blind vagina, absent uterus and female
adnexa, and abdominal or inguinal bilateral testes
producing normal or high levels of testosterone, with
normal male karyotype [2, 3]. Cases affected by PAIS show
various degrees of ambiguous genitalia, ranging from
predominantly female external genitalia with signs of
external genital masculinization (clitotomegaly and
posterior labial fusion), to predominantly male genitalia
(Reifenstein syndrome) with impaired spermatogenesis,
hypospadias, micropenis and gynaecomastia [3_5]. MAIS is characterized by external male genitalia,
undermasculinization (including sparse facial and body
hair and micropenis) and gynecomastia at puberty
[6_8]. A subgroup of MAIS patients is represented by
phenotypically normal men with infertility as the sole clinical
symptom [2, 9].
The AR is encoded by the AR gene (Xq11_12). This
gene spans more than 90 kb and codes for a protein
with three major functional domains. The N-terminal
domain, which serves a modulatory function, is encoded
by exon 1 (1586 bp); the DNA-binding domain is
encoded by exons 2 and 3 (152 bp and 117 bp, respectively); and the androgen-binding domain (LBD)
is encoded by exons 4_8, which vary from 131 bp to
288 bp in size [10, 11]. The C-terminus of the LBD
mediates the hormone dependent transcription
activation function. Functional studies demonstrate that the
mutations in the AR ligand-binding domain perturb the
conformation of the helix, which is unable to efficiently
bind the ligand DHT and to transactivate known
androgen response elements [12].
The present version of the AR database contains
more than 600 AIS specific mutations (www.mcgill.ca/androgendb), with the total number of reported
mutations rising from 374 to 605 in only 4 years [13].
Approximately 90% of molecular defects in the AR gene
are single base mutations, mostly missense mutations.
In addition to the point mutations, the AR gene contains
regions of repetitive DNA sequences, trinucleotide
repeat CAG and GGN, that have been associated with a
number of disorders, such as androgen insensitivity,
male infertility and prostate cancer [14].
Here we describe a novel mutation in the AR
gene (L790P) detected in a patient affected by CAIS.
2 Patients and methods
A 2-month-old girl was referred to us because of
the presence of a bilateral mass in the inguinal canal
associated with inguinal herniae. Physical examination
revealed a short and blind-ending vagina with female
external genitalia. Testosterone (T) and DHT synthesis
defects were excluded given the normal rise of T and
DHT after HCG stimulation (basal T: 1.3 nmol/L; basal
DHT: 0.7 nmol/L, T/DHT ratio after human chorionic
gonadotropin (HCG) stimulation: 7:9). gonadotropin
levels were normal (follicle stimulating hormone: 1.1
UI/L; luteinizing hormone: 1.2 UI/L). Chromosomal analysis
showed diploid 46, XY male karyotype. During the
intervention the presence of testes was confirmed and
gonadal histology was consistent with AIS. After clinical,
hormonal and cytogenetic examinations the girl was
diagnosed as affected by CAIS.
peripheral blood samples were obtained from the girl
and her mother for molecular analysis. The subjects
gave informed consent for molecular analysis of their
blood samples and the study was approved by a local
ethical committee. Genomic DNA was extracted with
the use of the High Pure PCR Template Preparation kit
(Roche Applied Science, Penzberg, Germany). PCR amplification of AR exonic fragments 1_8 (including the
intron exon boundaries) was carried out using the
primers described by Ishii et al. [15], except for exons 1 and 5,
which were amplified using the primers reported in
Table 1.
Direct sequencing of the PCR products was performed using the ABI PRISM BigDye Terminator Cycle
Sequencing Ready Reaction kit and analyzed using an
ABI PRISM 3100 Genetic Analyzer (PE Applied Biosystems, Monza, Italy).
3 Results and discussion
Direct sequencing analysis of PCR products revealed
in the patient the presence of a T to C transition in exon
6 resulting in the previously unreported leucine 790
proline substitution (Leu790Pro) (Figure 1). The same
mutation was detected in heterozygous form in the mother of the patient. The mutation was confirmed in
both the patient and her mother in two independent
experiments. Leucine 790 is located in the LBD of the
AR protein (Figure 2). Mutations of a single amino acid
in the LBD of the AR can induce functional abnormalities
in androgen binding, stabilization of active conformation,
or interaction with coactivators. In the AR C-terminal
LBD, there is a clear predominance of missense
mutations, with a significantly greater number of CAIS and
PAIS. Out of 193 mutations, 103 causing CAIS are
located in the LBD, 51 in the N-terminal and 24 in the DNA
binding domain [13]. Substitutions resulting in CAIS
have been reported also in the N-terminal region of the
AR LBD, while only one mutation at position 790 has
been previously reported (Leu790Phe), but was
associated with MAIS [8].
Given the highly conserved nature of these residues,
they likely play a critical role in creating the correct
structural architecture of the AR LBD. Although residues in
this region of the AR do not interact directly with the
hormone, each residue probably has an important role in
ordering the structural domains containing the residues
that contribute to the ligand-binding pocket. This is
consistent with the observation that substitution mutations
in this region result most frequently in the complete form
of androgen insensitivity. Therefore, the different
phenotypes showed by our patient and the one described by
Tsukada et al. [8], both carriers of a mutation involving
codon 790, can be explained by the different amino acid
substitution. In fact, the insertion of a proline instead of
a leucin in the amino acidic chain could act as a
structural disruptor in the middle of the regular secondary
structural elements, because of the exceptional
conformational rigidity of proline cyclic structure compared to
other amino acids. In contrast, the substitution of the
leucin with a phenylalanine, as in the case reported by
Tsukada et al. [8], would lead to a less severe structural
disruption in the conformational status of the AR protein,
causing a milder phenotype.
In conclusion, the characterization of mutations in
the AR gene serves as a reliable tool for the diagnosis and
molecular subclassification of AIS. The present case
demonstrates that, in addition to the localization of the
mutation within the gene sequence, the kind of amino
acid substitution in a case of missense mutation strongly
affects the resulting phenotype. Knowledge of the
mutation in the AR and its functional consequences can
provide useful information about the genotype-phenotype
correlation, improving the management of cases of male
pseudohermaphroditism with regard to gender assignment,
genital surgery and gonadectomy.
References
1 Li BY, Liao XB, Fujito A, Thrasher JB, Shen FY, Xu PY.
Dual androgen-response elements mediate androgen
regulation of MMP-2 expression in prostate cancer cells. Asian J
Androl 2007; 9: 41_50.
2 Quigley CA, De Bellis A, Marschke KB, el-Awady MK,
Wilson EM, French FS. Androgen receptor defects: historical
clinical, and molecular perspectives. Endocr Rev 1995; 16:
271_321.
3 Boehmer AL, Brinkmann O, Bruggenwirth H, van Assendelft
C, Otten BJ, Verleun-Mooijman MC,
et al. Genotype versus phenotype in families with androgen insensitivity syndrome.
J Clin Endocrinol Metab 2001; 86: 4151_60.
4 Rodien P, Mebarki F, Mowszowicz I, Chaussain JL, Young J,
Morel Y, et al. Different phenotypes in a family with
androgen insensitivity caused by the same M780I point mutation in
the androgen receptor gene. J Clin Endocrinol Metab 1996;
81: 2994_8.
5 Evans BA, Hughes IA, Bevan CL, Patterson MN, Gregory
JW. Phenotypic diversity in siblings with partial androgen
insensitivity syndrome. Arch Dis Child 1997; 76: 529_31.
6 Grino PB, Griffin JE, Cushard WG Jr, Wilson JD. A mutation
of the androgen receptor associated with partial androgen
resistance, familial gynecomastia, and fertility. J Clin
Endocrinol Metab 1988; 66: 754_61.
7 Pinsky L, Kaufman M, Killinger DW. Impaired
spermatogenesis is not an obligate expression of receptor-defective
androgen resistance. Am J Med Genet 1989; 32: 100_4.
8 Tsukada T, Inoue M, Tachibana S, Nakai Y, Takebe H. An
androgen receptor mutation causing androgen resistance in
undervirilised male syndrome. J Clin Endocrinol Metab 1994;
79: 1202_7.
9 Ferlin A, Vinanzi C, Garolla A, Selice R, Zuccarello D,
Cazzadore C, et al. Male infertility and androgen receptor
gene mutations: clinical features and identification of seven
novel mutations. Clin Endocrinol (Oxf) 2006; 65: 606_10.
10 Migeon BR, Brown TR, Axelman J, Migeon CJ. Studies of
the locus for androgen receptor: localization on the human X
and evidence for homology with the Tfm locus in the mouse.
Proc Natl Acad Sci U S A 1981; 78: 6339_43.
11 Lubahn DB, Joseph DR, Sullivan PM, Willard HF, French FS,
Wilson EM. Cloning of human androgen receptor
complementary DNA and localization to the X chromosome. Science
1988; 240: 327_30.
12 Adachi M, Takayanagi R, Tomura A, Imasaki K, Kato S, Goto
K, et al. Androgen-insensitivity syndrome as a possible
coactivator disease. N Engl J Med 2000; 343: 856_62.
13 Gottlieb B, Beitel LK, Wu HJ, Trifiro M. The androgen
receptor gene mutation database ARDB. Update Hum Mutat
2004; 23: 527_33.
14 Rajender S, Singh L, Thangaraj K. Phenotypic heterogeneity
of mutations in androgen receptor gene. Asian J Androl 2007;
9: 147_79.
15 Ishii T, Sato S, Kosaki K, Sasaki G, Muroya K, Ogata T,
et al. Micropenis and the AR Gene: mutation and CAG
repeat-length analysis. J Clin Endocrinol
Metab 2001; 86: 5372_8.
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