Exp Clin Endocrinol Diabetes 2015; 123 - P11_01
DOI: 10.1055/s-0035-1547726

A new next generation sequencing panel for mutational screening of putative genes causing 46,XY disorders of sex development (DSD)

S Flieger 1, B Brix 1, D Braunholz 2, B Reiz 3, FJ Kaiser 2, O Hiort 1, R Werner 1
  • 1Sektion für Experimentelle Pädiatrische Endokrinologie und Diabetologie; Klinik für Kinder- und Jugendmedizin; Universität zu Lübeck
  • 2Sektion für Funktionelle Genetik Am Institut für Humangenetik; Universität zu Lübeck
  • 3Institut für Integrative und Experimentelle Genomik; Universität zu Lübeck

Disorders (or Differences) of Sex Development (DSD) are rare congenital conditions in which the development of chromosomal, gonadal, or anatomical sex is atypical. DSDs can be divided into 3 groups: 46,XX; 46,XY and chromosomal DSDs; especially 46,XY DSD is characterized by clinical and genetic heterogeneity. Currently the genetic causes in about 80% of 46,XY cases are unknown. Thus we have developed a customized AmpliSeq 46,XY next generation sequencing (NGS) gene panel that allows a rapid and cost-effective screening of mutations in 46,XY DSD.

Our “DSD gene panel” includes 83 genes, that are already described to be mutated in patients with 46,XY DSD or were selected as interesting candidate genes based on functional investigations in human or animal model systems. In addition to the coding regions of the genes the splice-sites were included. Using the AmpliSeq Designer Tool the gene panel was designed in two libraries of approx. 600 amplicons each, covering ˜95% of target regions. These amplicons were sequenced by an ION Torrent PGM (personal genome maschine). The sequences were aligned to a reference sequence and the Variant Caller Files were matched with dbSNP, 1000genomes or the Exome Sequencing Project 6500. The pathogenicity was determined with different mutation prediction tools. The coverage and validity of mutations were reviewed with Bam-Files in the Integrative Genome Viewer.

So far we have sequenced 24 samples within four panel experiments. Four of these patients with known causative mutations were included as controls and all their mutations were identified. The remaining 20 samples came from patients with unknown cause for 46,XY DSD. For four patients we found likely pathogenic mutations in CHD7, WWOX, DHH, MAMLD1 and LHCGR. All of these mutations were confirmed by Sanger-Sequencing.

We conclude that NGS panel sequencing allows a targeted mutational screening of many genes simultaneously in a short time with a high sensitivity, less cost and a small amount of DNA. This will benefit research investigations for better description of unusual phenotypes in 46,XY DSD and might be embedded into future diagnostics of these patients.