Exp Clin Endocrinol Diabetes 2015; 123 - P14_06
DOI: 10.1055/s-0035-1547779

Next generation sequencing and functional characterization of the androgen receptor (AR) gene in patients with androgen insensitivity syndrome (AIS) and controls

N Hornig 1, HU Schweikert 2, M Ukat 3, AE Kulle 4, M Welzel 3, G Wehner 5, R Werner 6, O Hiort 6, SLS Drop 7, M Cools 8, C de Beaufort 9, I Hughes 10, C van der Horst 11, C Seif 12, R Siebert 13, O Ammerpohl 13, PM Holterhus 4, AK Eckstein 14
  • 1Department of Pediatrics; Division of Pediatric Endocrinology and Diabetology; Christian-Albrechts-University Kiel & University Hospital Schleswig-Holstein, Campus Kiel
  • 2Department of Medicine III
  • 3Department of Pediatrics, Division of Pediatric Endocrinology and Diabetology; Christian-Albrechts-University Kiel & University Hospital Schleswig-Holstein, Campus Kiel
  • 4Universitätsklinikum Schleswig-Holstein; Klinik für Allg. Pädiatrie/Bereich Endokrinologie
  • 5Department of Medicine III, University Bonn
  • 6Sektion für Experimentelle Pädiatrische Endokrinologie und Diabetologie; Klinik für Kinder- und Jugendmedizin; Universität zu Lübeck
  • 75department of Pediatrics, Division of Pediatric Endocrinology
  • 8Department of Pediatrics, Division of Pediatric and Adolescent Endocrinology and Diabetology University Hospital Gent
  • 9Pediatric Clinic, Ch de Luxemburg
  • 10University of Cambridge, The Old Schools
  • 11Urologische Gemeinschaftspraxis Prüner Gang
  • 12Urologie Zentrum Kiel
  • 13Institute of Human Genetics, Christian-Albrechts-University Kiel & University Hospital Schleswig-Holstein, Campus Kiel
  • 14Praxisklinik Kronshagen GmbH & Co. Kg

Androgen insensitivity syndrome (AIS) is characterized by a partial to complete lack of genital virilization in genetically male individuals. It is classically caused by inactivating mutations in the coding region of the X-chromosomal androgen receptor (AR) gene. However, up to two-third of the patients with a clinically established diagnosis of AIS lack a detectable molecular cause to date.

As conventional sequencing is restricted to the AR coding exons, we set up a next generation sequencing (NGS) approach of the entire AR-gene locus for a comprehensive AR mutation analysis in patients with AIS. To this purpose, DNA was extracted from cultured genital skin fibroblasts (GSF = scrotum, foreskin, labia) of 80 patients with known and presumed AIS, two patients with 17ßHSDIII deficiency, six patients with 5alpha-reductase deficiency and 15 control males. Patients were suspected to have AIS based on clinical findings, pathological androgen binding, reduced AR expression in GSF or a combination of these.

The AR-sequencing library was produced using a capture-based method (Haloplex; Agilent). The target sequence included the coding region, the UTRs, 90% of the intron sequences as well as a 9kb upstream and 5kb downstream sequence. Sequencing was performed on a Miseq benchtop sequencer (Illumina). Alignment to the hg19 reference genome and single nucleotide polymorphism (SNP) calling was performed by the MiSeq-Reporter software (Illumina).

Targeted NGS confirmed AR mutations in all patients with mutations previously identified by Sanger sequencing. Additional SNPs were detected in and outside the AR-coding region. In order to understand the functional impact of the SNPs, they were further tested for the ability of the endogenous AR to induce transcription of the AR target gene Apolipoprotein D (APOD-Assay).

In conclusion we show that NGS is a valid method for AR-sequencing in presumed AIS and allows in combination with the APOD-Assay a refined classification of AIS.