ESPE Abstracts (2014) 82 FC6.2

aDepartment of Pediatrics, Christian-Albrechts-University Kiel and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany; bInstitute of Human Genetics, Christian-Albrechts-University Kiel and University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany; cUniversity Hospital Bonn, Dermatologie, Bonn, Germany; dDivision of Pediatric Endocrinology, Department of Pediatrics, Rotterdam, The Netherlands; eDepartment of Pediatrics, University Hospital Gent, Gent, Belgium; fDepartment of Pediatrics, University of Lübeck and University Hospital Schleswig-Holstein, Campus Lübeck, Lübeck, Germany; gDCCP–Clinique Pédiatrique de Luxembourg, Luxembourg, Luxembourg

Background: In a large fraction of patients with clinically presumed Androgen Insensitivity Syndrome, no mutation of the Androgen Receptor gene can be detected. However, established Sanger sequencing techniques of the AR gene are often limited to the coding region.

Objective and Hypotheses: To set up a next generation sequencing (NGS) approach of the entire AR locus (including UTRs, exons, introns, up- and downstream regions) for a comprehensive AR gene mutation analysis in patients with AIS.

Method: DNA was extracted from cultured genital skin fibroblasts (GSF=scrotum, foreskin, and labia) of 70 patients with known and presumed AIS, two patients with 17βHSDIII deficiency, four patients with 5α-reductase deficiency and ten control males. Patients were suspected to have AIS based on clinical findings, pathological androgen binding, reduced AR expression in GSF, a pathological APOD-assay (assay for AR function) 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 9 kb upstream and 5 kb 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).

Results: Targeted NGS confirmed AR mutations in all patients with mutations previously identified by Sanger sequencing. Additional mutations were detected by NGS, which affected, e.g. the UTR, and could be validated by Sanger sequencing. Normal male controls and patients having a different DSD diagnosis, e.g. 17βHSDIII deficiency, showed no mutations in the AR locus.

Conclusion: Targeted NGS is a valid method for AR-sequencing in presumed AIS and extends the diagnostic perspective beyond the coding regions.

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