P39 - Pathogenic TBX3 variants disrupt GnRH neuron development: functional evidence from CHH patients and Zebrafish models

Congenital hypogonadotropic hypogonadism (CHH) is a rare reproductive disorder caused by defective production, secretion, or action of gonadotropin-releasing hormone (GnRH). While over 60 genes have been implicated in CHH pathogenesis, approximately 50% of cases remain genetically unexplained. Through next-generation sequencing in a cohort of CHH patients, we identified three heterozygous variants in the TBX3 gene: S190R, c.857_858delCT, and R249W. To elucidate their pathogenicity, we performed in vitro studies in HEK293 cells. Western blot analyses confirmed protein expression for all variants. The c.857_858delCT variant results in a truncated TBX3 protein, as demonstrated by western blot, which likely compromises its functional capacity. Interestingly, this truncated form retains correct nuclear localization, as shown by immunofluorescence analysis. Moreover, immunofluorescence revealed that S190R and R249W mutants mislocalise predominantly in the cytoplasm compared to wild-type TBX3. Additionally, the S190R variant displayed significantly reduced protein levels, which we attributed to an accelerated turnover as demonstrated by analysis of protein half-life. To investigate the in vivo relevance of TBX3 in GnRH neuron development, we employed zebrafish (Danio rerio) model using the tg(GnRH3:EGFP) line. Morpholino-mediated knockdown of the zebrafish orthologs tbx3a and tbx3b, both individually and in combination, resulted in a significant reduction of GnRH3-positive neurons and profound disruption of the GnRH3 neuronal network. Given the known interaction between TBX3 and the Wnt/β-catenin signalling pathway, we explored Wnt-related gene expression and observed significant downregulation of canonical Wnt components in tbx3a/tbx3b double morphants. In conclusion, our findings identify TBX3 as a novel candidate gene in CHH and suggest that its pathogenic variants impair GnRH neuron development and function, potentially through dysregulation of Wnt signalling. These results expand the genetic and mechanistic landscape of CHH and may have future diagnostic and therapeutic implications.