Case Report: A novel exon 1 splicing mutation in the non-coding region of the NR5A1 gene causing 46,XY disorders of sex development

Background NR5A1 plays a critical role in sex differentiation and adrenal development. As a transcription factor, it regulates multiple genes involved in reproductive and endocrine processes. Mutations in NR5A1 are commonly associated with disorders of sex development and adrenal insufficiency, and most reported variants are located in coding regions. Methods A de novo NR5A1 c.−16 + 1G > A mutation was identified in a 2-year-old patient who was gender-ambiguous. To investigate the pathogenicity and underlying mechanism of this non-coding variant, in vitro constructs with exon 1, intron 1, and exon 2 regions, including the splice sites, were generated by a minigene assay. Reverse transcription quantitative PCR was used to compare transcript expression between wild-type and mutant-type. Results The c.−16 + 1G > A mutation caused aberrant alternative splicing, introducing a novel start codon within intron 1 that led to premature termination in exon 2, producing a truncated protein distinct from the canonical NR5A1 protein. RT-qPCR analysis demonstrated an approximately 75% reduction normal of NR5A1 mRNA expression. These findings indicate that this mutation alters protein structure and significantly decreases normal transcript levels. Conclusions The functional evidence suggests that the NR5A1 c.−16 + 1G > A non-coding region variant results in aberrant splicing and decreased expression. This variant may lead to loss of function, impairing testicular development while sparing adrenal function. These findings assist in the classification of this non-coding region NR5A1 mutation and may inform clinical management and decision-making.