Challenges in clinical and laboratory diagnosis of androgen insensitivity

syndrome: a case reportCaroline 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

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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.