Endocrine Abstracts

JOINT2837

Background: Our analysis of patients with 46,XY gonadal dysgenesis and 46,XX DSD (Disorders/Differences in Sex Development) has identified many genes involved in mammalian sex-determination (SD) and gonadal differentiation. Despite extensive exome studies >50% of all cases remain idiopathic. We hypothesize that unexplained cases are due to variants in gene regulatory elements (GREs) that may define gene regulatory networks (GRNs). Consistent with this hypothesis, recently we defined a novel SRY enhancer and identified SNVs in it, associated with a spectrum of phenotypes including 46,XY gonadal dysgenesis and under virilized male (PMID: 38555298). We have extended this work to develop a multi-omic genome-wide approach to identify functional GREs in human SD and predict GRNs.

Methods: Single-cell RNAseq and ATACseq data from human fetal gonad atlas (PMID: 35794482) was reanalysed by Scenic+, to define candidate GREs (cGREs). Transcription factor (TF) binding-site enrichment and mutational constraint in cGREs were determined using pycistarget and Gnocchi respectively. We defined cGREs corresponding to human accelerated regions (HARs, PMID: 27667684). Rare/novel variants from genome sequences of >160 individuals with unexplained DSD were mapped to cGREs and functional variants prioritized using CADD, FinSurf and Fabian.

Results: We defined 18,932 and 18,200 cGREs in Sertoli and pre-granulosa cells with a mean Gnocchi score of 7.90 and 7.88 respectively. Using our pipeline, we observed an enrichment of binding-sites for known SD TFs in cGREs: e.g: SOX9 (Normalized Enrichment Score (NES)=12.95), NR5A1 (NES=10.98) in Sertoli and LHX9 (NES=5.66) in pre-granulosa cells. As proof of principle, we identified a previously reported SOX9 GRE (eSR-A (PMID: 30552336)).

Conclusions: Our approach dissects the role of GREs and GRNs in SD providing a unique model to further understand developmental gene regulation. This approach also provides a unique pipeline to understand the genetic causes of unexplained DSD due to gene regulatory variants.