Challenges in clinical and laboratory
diagnosis of androgen insensitivity
syndrome: a case report
Caroline O.A. Melo, Daniela M. Silva, and Aparecido D. da Cruz
AIS Overview
 AIS is an X-linked disorder caused by mutations in the Androgen
Receptor gene
 May caused a wide range of phenotypes from male infertility to
completely normal female external genitalia
 Complete AIS is relatively rare
 Subject with normal male karyotype (46, XY) presents with female
external genitalia, absence/thinning of pubic hair, and absence of a
uterus
 Most individuals not diagnosed during childhood are diagnosed
after puberty due to primary amenorrhea
 http://www.youtube.com/watch?v=ETIxoQGVjos
Background: Androgens
 Androgens are a group of sex steroid hormones
 primarily produced by a male’s testes
 Responsible for male sex differentiation during embryogenesis
 The sex-determining region on the Y chromosome (SRY)
directs androgens in male differentiation
 This differentiation occurs between the 9th and 13th weeks of
pregnancy
 Both testosterone and DHT are required for this differentiation
to occur, and these androgens, in turn, require the presence of
functional androgen receptors to exert their necessary effects
 Androgens are also responsible for male secondary sex
characteristics by triggering puberty
Background: AR gene
 The AR gene codes for a protein that is a steroid-hormone- activated
transcription factor and is found on the X chromosome
 Protein-coding region consists of 8 exons
 AR = Androgen Receptor
 In the absence of a ligand, the AR resides in the cytoplasm. Upon
binding the receptor dimerizes and translocates to the nucleus where
it exerts its effects on transcription
 Consists of four main regions:




N-terminal domain
DNA-binding domain
Hinge region – involved in DNA binding and AR dimerization
Ligand-binding domain
Figure 1
Background: Causes of AIS
 Loss-of-function mutations in the AR gene cause AIS by
leading to androgen unresponsiveness, which affects proper
male sexual development
 Results from a reduced capacity of the AR to transactivate
androgen-responsive genes in target cells
Background: AIS
 The phenotypes vary according to the AR defect
 Three classified forms:
 Complete AIS – appear as normal females until puberty hits and menstruation does
not occur
 Partial AIS – heterogeneous condition with a variety of phenotypes depending on
different degrees of undervirilization
 Mild AIS – underdeveloped males with ranging fertility
 All forms are inherited as X-linked recessive disorders
 Affected patients have the normal male karyotype (46, XY) with
female external genitalia, blind vaginas, an absent uterus, normal
breast development, and abdominal or inguinal testes
 Usually come to attention during the neonatal period due to inguinal
hernia and/or ambiguous genitalia or at puberty due to primary
amenorrhea
Purpose of Investigation
 To provide a genetic diagnosis of a teenage girl with normal
male karyotype (46, XY) using fluorescence in situ
hybridization (FISH) and PCR in order to determine the
nature and the extent of the mutation that affected the AR
gene
Case Presentation
 15-year-old Caucasian girl referred for genetic testing due to
primary amenorrhea
 Medical history of removal of an abdominal mass as a
newborn
 Mass referred to as umbilical hernia
 G-band karyotype revealed diploid set of chromosomes,
including 22 pairs of homologous autosomes and one pair of
sex chromosomes—compatible with a 46, XY
 Geneticists at Brazil lab concluded that the mass withdrawal
was in fact, testes, and the patient had a condition known as
cryptorchidism
 It is reproductive change characterized by a failure of the movement
of one or both testes from the abdominal cavity to the scrotum.
Case Presentation (Continued)
 PCR verified mutations of the exons 1, 4, 6, 7, and 8
on the AR gene
 Primers were used for selected exons of AR
 FISH was used to detect the AR gene
 Culture of T lymphocytes in RPMI 1640 medium,
supplemented with 20% calf serum and 2%
phytohemagglutinin
 Slides of good quality—in terms of metaphase—were
selected by phase contrast microscope and were subjected
to FISH using the LSI Androgen Receptor SpectrumOrange
(Xq12) probe.
Case Presentation (Continued)
 In situ hybridization with the LSI AR probe indicated presence
of the gene in all analyzed cells
 Genomic DNA extracted from peripheral blood leukocytes
assessed by PCR revealed coding sequence abnormalities for
the AR gene, which lacked exons 1 to 7 indicating a large
deletion spanning the proximal region of the gene.
Figure 2
Figure 3
Figure 4
Conclusions
 FISH alone was not able to properly diagnose the
patient, despite the proximal deletion within the AR
observed on PCR
 Explanation: the probe was 380 kb, which was bigger than
the AR Gene (90 kb)—indicating that the deletion of some
exons within the gene was not large enough to prevent
probe hybridization
 The PCR assay confirmed the diagnosis for the
patient having a chromosomally normal male
karyotype
Conclusions (Continued)
 Mutations affecting the AR gene may cause either complete or
partial AIS
 The patient reported is consistent with CAIS, misdiagnosed at
birth, and consequently raised as female.
Discussion
 It is critical that health care providers understand the
importance of properly diagnosing a new born with
ambiguous genitalia
 Evaluations of clinical and genetic findings is crucial to determine proper
gender assignment and detection of life threatening condition
 A child with pseudohermaphrodite phenotype should
undergo adequate endocrine and genetic testing for a
definitive diagnosis before gender is assigned or surgeries
performed
 Inadequate investigation may result in inappropriate gender assignment
in infancy with possible inferences on outcome. A patient with abnormal
genital development represents a difficult diagnostic and therapeutic
challenge
 A patient with abnormal genital development represents a
difficult diagnostic and therapeutic challenge
References
 Melo et al. Challenges in clinical and laboratory diagnosis of
androgen insensitivity syndrome: a case report. Journal of
Medical Case Reports 2011 5:446
 Galani, Angeliki et al. Androgen insensitivity syndrome:
clinical features and molecular defects. Hormones 2008,
7(3):217-229.