Challenges in clinical and laboratory diagnosis of androgen insensitivity syndrome: a case report

1. 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

2. 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

3. 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

4. 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

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6. 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

7. 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

8. 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

9. 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.

10. 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.

11. 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.

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15. 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

16. 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.

17. 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

18. 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.