Balkan Journal of Medical Genetics

ANDROGEN INSENSITIVITY SYNDROME DUE TO NON-CODING VARIATION IN THE ANDROGEN RECEPTOR GENE: REVIEW OF THE LITERATURE AND CASE REPORT OF A PATIENT WITH MOSAIC C.-547C>T VARIANT
Noveski P, Plaseski T, Dimitrovska M, Plaseska-Karanfilska D
*Corresponding Author: Dijana Plaseska-Karanfilska, MD, PhD, Research Centre for Genetic Engineering and Biotechnology ‘Georgi D. Efremov’, Macedonian Academy of Sciences and Arts, Krste Misirkov 2, 1000 Skopje, Republic of Macedonia, Tel. +389 2 3235 410, E-mail: dijana@manu.edu.mk
page: 51

METHODS

Patient presentation A twenty-four year old patient with a disorder of sexual development and 46,XY karyotype was referred to our laboratory for genetic testing. According to the previous medical records, the patient was born with ambiguous external genitalia, described as a hypoplastic penis (resembling hypertrophic clitoris), partially covered with bifid scrotum (resembling oedematous labia), and in whom gonadal structures were identified after ultrasound examination. Urethrocystography showed the presence of a male urethra. Blind ending structure resembling a vagina was also observed. No uterus was detected. A human chorionic gonadotropin (hCG) stimulation test showed a positive response, resulting in increased testosterone production. At the age of seven months, cytological gonadal punction was performed, and, according to the chromatin status, cells resembling the Sertoli cells were observed. Although the parents were advised to raise the child as a female and a feminizing genitoplasty was performed at 6 years of age, they have reared him as a male and he decided to undergo surgical gender reassignment at 24 years of age. Genetic analysis We have analysed in total 4 DNA samples: 3 DNA samples from the patient and one DNA sample from his mother. Initially, DNA from the patient’s blood (peripheral white blood cells) was isolated using standard phenol/chloroform protocol. In the second patient’s admission, DNA was isolated from the blood sample of patient’s mother as well as from a new blood sample and a buccal swab from the patient. This time the extraction of the DNA was performed using MagCore Super automated nucleic acids extractor (RBC Bioscience Corp., Taiwan). Since the patient’s phenotype in combination with the results of the hCG test and 46XY karyotype was indicative for the presence of AIS, Sanger sequencing targeting the exons and exon/intron boundaries of the androgen receptor (AR) gene was performed. Exon 1 sequencing was performed with the inclusion of the 5’UTR region, which is a standard practice in our laboratory. We then performed a multiple ligation-dependent probe analysis (MLPA) for detection of the exon copy number changes in the AR gene, using the AR P074-A3 Androgen insensitivity syndrome kit (MRC-Holland, Amsterdam, The Netherlands). To confirm the biological relatedness between the patient and patient’s mother, a comparison was performed of the allelic profiles of 15 polymorphic short tandem repeat (STR) loci using previously the published multiplex PCR of fluorescently labelled primers [10]. We have used also another multiplex PCR of fluorescently labelled primers [11] which previously have been used to detect mosaic loss of sex chromosome in blood cells [12]. Both multiplex PCR reactions also contain primers amplifying the SRY gene. The Sanger sequencing, MLPA and fluorescent multiplex PCR reactions were analyzed on the ABI PRISM 3500 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). For the MLPA data analysis, Coffalyser.Net software (https://www.mrcholland.com/technology/software/ coffalyser-net) was used. Electropherograms from the Sanger sequencing were analyzed with Sequencing Analysis v5.4 (Thermo Fisher Scientific, Waltham, MA, USA). ImageJ software (https://imagej.nih.gov/ij/index. html) was used to compare area ratios of the normal and mutant allele’s fluorescence peaks from the electropherograms of the Sanger sequencing. UTRannotator [13], a plugin to the Ensembl VEP analysis software [14], was used for in silico prediction of the possible impact of the 5’UTR variant.

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