Application of molecular techniques and gene expression analysis to the study of ovariotesticular disorder of sex development

Abstract

Ovotesticular disorder of sex development (OT-DSD) is a rare condition characterized by ovarian and testicular parenchyma in the same subject, in separate gonads or in the same gonad (ovotestis), with variable degrees of genital ambiguity and both male and female secondary sex features. In most cases (about 60%) karyotype is 46,XX, in 10% is 46,XY and the remaining subjects have abnormal chromosomes (including mosaicism or chimerism). In some cases with normal karyotypes (XX or XY) a genetic etiology could be identified, but most have an unknown origin. The hypotheses include cryptomosaicism with a cell line with a different set of sex chromosomes and mutations in genes participating in gonadal differentiation, as NR5A1. There are also few studies on the expression of proteins which are specific of the gonadal tissue in these cases. The aim of this study is to widen knowledge on the etiology of OT-DSD. Studies on paraffin-embedded gonadal tissue from previous biopsies of patients with a 46,XX or 46,XY karyotype or chromosome abnormalities who were seen in the last three decades will be performed. These include: fluorescent in situ hybridization (FISH) with X- and Y- specific probes and SRY probes; DNA extraction to amplify this gene; RNA extraction for transcriptome analysis by RNA-seq; and immunohistochemistry for SRY, FOXL2 and SOX9. In 46,XX patients, sequencing of NR5A1 by Sanger will be performed in DNA samples from peripheral blood. Genome sequencing will be performed in two families with recurrence of XX DSD, in cases without NR5A1 mutations and in those without evidences of mosaicism or chimerism. (AU)