ESPE Abstracts

The widespread availability of relatively inexpensive and rapidly evolving genetic screening by whole exome sequencing (WES) has transformed diagnosis in DSD. WES has identified many new genetic causes of DSD (e.g. ZFPM2, NR2F2, ZNRF3), several of which would not have been possible by classic candidate gene approaches (e.g. DHX37 or SART3). However, despite these advances, pathogenic variants in each gene affect only a comparatively small number of individuals (e.g. DMRT1, PBX1, ZNRF3). Consequently, the number of genes causing DSD continue to increase without a proportionate augmentation of the overall diagnostic yield. Approximately, 50% of all children with DSD still do not have a genetic diagnosis. Even when a genetic diagnosis is established, the considerable variability in the clinical presentation cannot be easily explained. In a WES study of 919 cases of DSD, we established a genetic etiology in 36.8% of a 46,XY cohort, whereas in a 46,XX DSD cohort the overall genetic diagnostic yield was 24.9%. In the entire DSD cohort, 67% of causal variants were novel (not reported in the literature and absent from public databases). Surprisingly, only 4.3% of individuals with 46,XY DSD (9.9% syndromic cases and 2% non-syndromic cases) showed evidence of either digenic or oligogenic etiology. The considerable variability in the clinical presentation of autosomal dominant forms of DSD may therefore be modulated by expression of the wild-type alleles of pathogenic variants. A significant proportion of the unexplained DSD cases may be due to variants in regulatory elements of genes required for gonad development. To test this hypothesis, we are performing whole genome sequencing of individuals with unexplained 46,XY DSD and correlating non-coding variation with extensive -omics data on gene regulation during human gonad formation. We identified unique single-base substitutions of highly conserved residues within an SF-1/NR5A1-binding enhancer element located approximately 5 kb upstream of the SRY gene. The phenotypic presentation is variable ranging from female with 46,XY gonadal dysgenesis, to male with atypical external genitalia. Deletion of this hemizygous SRY enhancer element by genome-editing, in a novel in vitro cellular model recapitulating human Sertoli cell formation, resulted in a significant and variable reduction in the expression of SRY. Since disease causing variants in enhancers are currently rare, the genomic analysis of DSD cases, offers a paradigm to understand gene regulatory mechanisms in a key developmental process as well as defining new genomic loci causing DSD.