Overexpression of SOX3 due to an X chromosome inversion leading to ovotesticular difference in sex development

Structural variants (SVs) may increase SOX3 expression in the gonads and have been observed in individuals with ovotesticular differences in sex development (OT-DSD) and XX testicular differences in sex development (T-DSD). Most of the SVs found in OT-DSD individuals are whole-gene duplications, and to date, only one SV affecting SOX3 expression by a positional effect has been described. We report an individual raised as a female with SRY-negative OT-DSD. Karyotype analysis showed a pericentric inversion in one of the X chromosomes - 46,X, inv(X)(p22;q27). The breakpoints and fusion were mapped using optical genome mapping (OGM) and short-read whole genome sequencing. One of the breakpoints was mapped on Xq27.1 (genomic position chrX:140,420,874 - GRCh38), 82 kb downstream of the SOX3 gene. This breakpoint was predicted to interrupt a topological associate domain (TAD) affecting 24 enhancer-promoter interactions of SOX3. RNA sequencing (RNA-seq) of a formalin-fixed paraffin-embedded (FFPE) sample of the gonads confirmed increased SOX3 expression. The present study is the first to analyze gene expression in gonadal tissues from an OT-DSD individual, and the first reporting an inversion-based mechanism leading to XX OT-DSD. Additionally, an X-inactivation assay on DNA extracted from the gonads revealed random inactivation. These findings support the hypothesis that inappropriate SOX3 expression may result from the positional effects of SVs, leading to OT-DSD in 46,XX individuals.