1
ORIGINAL ARTICLE
Three novel mutations in the androgen receptor gene
associated with partial androgen insensitivity syndrome:
H570R, G589E and S759T
Três mutações novas no gene receptor de andrógeno associado à síndrome de insensibilidade
parcial do andrógeno: H570R, G589E e S759T*
Patrícia Renovato Tobo1, Angela Silva Barbosa 2, Lene Garcia Barbosa3, Angela Maria Spínola-Castro4,
Carlos Alberto Moreira-Filho5
ABSTRACT
Three novel mutations in the androgen receptor gene were detected
by PCR-SSCP and characterized by DNA sequencing of genomic
DNA samples from 3 unrelated patients with male pseudo
hermaphroditism caused by partial androgen insensitivity. The
patients’ phenotypes suggest that these mutations impaired but
did not abolish the AR function.
Keywords: Mutation; Androgens; Androgen-insensitivity syndrome;
Polymerase chain reaction; Polymorphism, Single stranded
conformational; Pseudohermaphroditism; Male
RESUMO
Três mutações novas causando troca de aminoácidos no gene do
receptor de andrógeno foram detectadas por PCR-SSCP e caracterizadas
por seqüenciamento de DNA em 3 pacientes não aparentados com
pseudo-hermafroditismo masculino por insensibilidade periférica a
andrógenos. O fenótipo dos pacientes sugere que essas mutações
alteraram mas não aboliram a função do receptor.
Descritores: Mutação; Androgênios; Síndrome de resistência a
andrógenos; Reação em cadeia da polimerase; Polimorfismo
conformacional de simples fita; Pseudo-hermafroditismo; Masculino
INTRODUCTION
Mutations in the androgen receptor (AR) gene are associated with
the androgen insensitivity syndrome (AIS) in 46, XY individuals.
AIS has a wide spectrum of phenotypes: in the complete form
(CAIS) the patients have a female phenotype, whereas in the
incomplete forms (PAIS) under-masculinization with genital
ambiguity is observed. In order to establish genotype-phenotype
correlations in AIS it is necessary to characterize the mutations
affecting the X-linked AR gene. In this work we used PCR-SSCP
(polymerase chain reaction and single strand conformation
polymorphism) and DNA sequencing to study the AR gene in three
patients with PAIS.
METHODS
Patients 1 (MOS, 03/23/81), 2 (MNP, 10/03/93) and 3 (LBS, 06/
05/98) were born with ambiguous genitalia, undescended testes,
severe hypospadia and micropenis, and had a 46XY karyotype.
Testosterone levels (basal and after HCG stimulation test) were
fairly high and the testosterone/ dihidrotestosterone ratio was
normal. Histopathological examination showed normal testes in
patients 2 and 3 and left atrophic testis and right normal testis in
patient 1. No Müllerian remnants were found.
Exons 1 to 8 of the androgen receptor gene were amplified by
polymerase chain reaction (PCR) using the primers described by(1).
Single-strand conformational polymorphism (SSCP) analyses were
carried out using an automated electrophoresis system coupled
with silver staining (GenePhor apparatus – Amersham Bioscience).
Exons whose PCR products demonstrated mobility shifts were reamplified using the same primers mentioned above. After column
purification with CONCERT™ Rapid PCR Purification System
(Gibco BRL), samples were sequenced with the Cy™5 Thermo
Sequenase™ Dye Terminator kit (Amersham Bioscience) according
to the manufacturer’s instructions.
* Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo & Divisão de Endocrinologia Pediátrica, Departamento de Pediatria, Unifesp, São Paulo, SP.
1
2
3
4
5
MSc, Instituto de Ensino e Pesquisa Albert Einstein, São Paulo, SP.
PhD, Departamento de Imunologia, Instituto de Ciências Biomédicas da USP.
MD, MSc, Divisão de Endocrinologia Pediátrica, Departamento de Pediatria, Unifesp.
MD, PhD, Associate Professor, Divisão de Endocrinologia Pediátrica, Departamento de Pediatria, Unifesp.
PhD, Coordinator, Centro de Pesquisa Experimental, Instituto de Ensino e Pesquisa Albert Einstein, and Professor, Instituto de Ciências Biomédicas da USP.
Correspondence to: Carlos A. Moreira-Filho - Instituto de Ensino e Pesquisa - HIAE - Av. Albert Einstein, 627/701 - Prédio Joseh Feher (Bloco A) - Piso Chinuch - Morumbi - CEP 05651-901 - São Paulo
- SP - Tel.: 55-11-3747-1540 - Fax: 55-11-3474-0302. e-mail: [email protected]
Recebido para publicação em 7/5/2003 – Aceito em 14/8/2003
einstein 2003; 1:1-2
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Tobo PR, Barbosa AS, Barbosa LG, Spínola-Castro AM, Moreira-Filho CA
Fig. 1. Chromatograms of the mutations detected in patients 1 (a), 2 (c) and 3 (e) and of the wild type sequences (b, d, f). Arrows point to mutation position.
RESULTS
CONCLUSION
Patient 1 has a A to G transition relative to the CAC codon of exon
2 of the androgen receptor gene leading to the amino acid exchange
histidine to arginine at position 570 of the protein (H570R).
Patient 2 has a G to A transition in the first codon of exon 3
leading to the amino acid exchange glycine to glutamate at position
589 of the protein (G589E).
Patient 3 has a T to A transversion relative to the TCC codon
of exon 5 leading to the amino acid exchange serine to threonine
at position 759 of the protein (S759T).
Mobility shifts were detected when the PCR products
encompassing exons 2, 3 and 5 of the patients were analyzed by
SSCP (data not shown). These mobility shifts differed from normal
controls, as well as from the patients´ mothers, who are
heterozygous carriers for the mutations.
In patients 1 and 2 we have demonstrated novel single base
substitutions within the exons 2 and 3 respectively (fig. 1a, 2c). These
exons code for the DNA-binding region of the receptor. Patient 3
was shown to have a single base substitution in exon 5, which codes
for the hormone-binding region. Two other mutations (Ser ® Pro759
and Ser ® Phe 759) within this same codon have already been
described(2-3). Our patient 3, however, carries a T to A transversion
relative to the first nucleotide of the codon leading to the amino
acid exchange serine to threonine at position 759 (fig. 3e).
The molecular characterization of AR mutations is important for
heterozygous detection and for genetic counseling but it is not
decisive for the management of patients with PAIS. One must also
consider the degree of genital ambiguity and the growth response
of the penis to therapeutic doses of testosterone. The growing
knowledge derived from structure-function studies on the AR
protein(8-9). and on the role of AR transcriptional cofactors in
different tissues(10) will certainly contribute to the development of
new individualized hormonal therapies for PAIS(9).
DISCUSSION
5. Gottlieb B, Pinsky L, Beitel LK, Trifiro M. Androgen insensitivity. Am J Med
Genet 1999;89:210-7.
6. Gottlieb B, Beitel LK, Trifiro, MA. Variable expressivity and mutation databases:
the androgen receptor gene mutations database. Hum Mutat 2001;17:382-8.
7. Sultan C, Paris F, Terouanne B, Balaguer P, Georget V, Poujol N et al. Disorders
linked to insufficient androgen action in male children. Hum Reprod Update
2001;7:314-22.
8. Poujol N, Lobaccaro JM, Chiche L, Lumbroso S, Sultan C. Functional and
structural analysis of R607Q and R608K androgen receptor substitutions
associated with male breast cancer. Mol Cell Endocrinol 1997;130:43-51.
Mutations in AR can disturb its ability to regulate target genes
transcription but no clear correlation can yet be established
between altered sequences and receptor function. In some familial
PAIS cases the same mutation causes different phenotypes(4) while
very different molecular defects, as major deletions and point
mutations, can produce the same phenotypic effects(5-7).
All mutations reported here are associated with a partial AIS
phenotype, what means that the amino acid substitutions did not
completely abolish the receptor function. Moreover, different
mutations in the DNA-binding or in the hormone–binding domains
of the AR gene exerted almost the same phenotypic effect, namely
severe hypospadia and micropenis.
einstein 2003; 1:1-2
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