Functional and transcriptomic insights into 46,XY disorders of sex development associated with NR5A1 gene variants.

内容摘要

NR5A1 encodes a transcription factor essential for adrenal and gonadal development. Gene variants are a known cause of heterogeneous 46,XY disorders of sex development (DSD), but the mechanisms underlying the phenotypic variability remain unclear. We investigated how different NR5A1 variants affect downstream gene regulation and contribute to DSD pathogenesis. We analyzed four naturally occurring NR5A1 variants identified in patients with 46,XY DSD-two novel (p.Cys65Ser, p.His310Arg) and two previously reported (p.Cys30Ser, p.Gln329*). We performed protein and transcriptomic analyses to characterize variant effects and identify dysregulated and candidate target genes, validated by qPCR and luciferase assays. Transcriptomic and CUT&Tag analyses focused on the p.Gln329* truncating variant. All four variants occurred at conserved residues and resulted in reduced NR5A1 protein expression and impaired nuclear localization upon transfection in HEK293T cells. Transcriptomic analysis using the p.Gln329* variant revealed broad downregulation of genes involved in steroidogenesis, including CYP11A1, STAR, and CYP17A1. Notably, AMHR2 and STARD8 were significantly downregulated and showed reduced CUT&Tag signal in variant-transfected cells. Promoter assays confirmed that all variants diminished CYP11A1 and AMHR2 promoter activity. Only the p.Gln329* variant affected STARD8 promoter activity. These findings indicate that NR5A1 variants impair protein expression and localization, leading to transcriptional dysregulation of genes involved in steroid hormone biosynthesis and sexual development. Based on analysis of the p.Gln329* truncating variant, AMHR2 and STARD8 are strong candidate novel downstream targets of NR5A1, offering further insight into the mechanisms driving 46,XY DSD. Differences in sex development can occur when the genetic instructions for building male reproductive organs are disrupted before birth. This study focuses on a key gene called NR5A1, which acts like a master switch to turn on other genes essential for testis development and hormone production. We investigated four variants in this gene found in children with 46,XY differences in sex development. Using cell models, we showed that these variants cause the NR5A1 protein to be produced at lower levels and prevent it from reaching the cell’s nucleus, where it normally works. As a result, the variant proteins fail to properly switch on several crucial target genes involved in making male hormones. We identified two new candidate genes controlled by NR5A1, called AMHR2 and STARD8, which may play important roles in this process. Our research helps explain how changes in a single gene can lead to a broad range of developmental outcomes and provides a clearer picture of the molecular steps involved in human sex development.