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

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 17sHSDIII 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.