DECODING THE GENETIC LANDSCAPE OF CONGENITAL HYPOGONADOTROPIC HYPOGONADISM: FROM PATIENT ALLELIC VARIANTS TO FUNCTIONAL INSIGHTS IN ZEBRAFISH

Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disorder characterized by incomplete or absent puberty and infertility, due to deficient secretion or action of gonadotropin-releasing hormone (GnRH). Although more than 60 causative genes have been described, the genetic heterogeneity of CHH remains only partially understood and many patients still lack a molecular diagnosis. Among the novel candidates, WFS1 and TBX3 have recently emerged as potential candidate genes for isolated CHH. The central research question of this thesis is whether heterozygous variants in WFS1 and TBX3, identified in CHH patients, can disrupt GnRH neuronal development, migration, or regulatory pathways, contributing to the disease pathogenesis. In our cohort of CHH patients, genetic analysis revealed heterozygous WFS1 variants identified by whole-exome sequencing (WES) and TBX3 variants identified by next-generation sequencing (NGS) panel. To evaluate the functional impact of these variants, we performed site-directed mutagenesis followed by in vitro characterization in cellular models. Specifically, WFS1 wild-type and mutant constructs were evaluated in COS7 cells, while TBX3 constructs were tested in HEK293 cells. Functional assays included Western blotting, immunofluorescence, quantitative real-time PCR, and luminometric assays to investigate protein localization, expression stability, turnover and transcriptional activity. To validate these findings in vivo, we used the Tg(GnRH3:EGFP) zebrafish line and generated knockdown (KD) model via morpholino of wfs1b and tbx3a/b. This strategy allows direct visualization of GnRH3 neurons development, proliferation and migration steps. Knockdown of wfs1b in Tg (GnRH3:EGFP) zebrafish embryos disrupted GnRH3 neuronal architecture and downregulated isl1a, a key transcription factor involved in axon guidance. Our findings establish a mechanistic link between WFS1-mediated ER homeostasis, isl1a expression, and GnRH neuron development. In parallel, tbx3 knockdown revealed defective GnRH3 neurons and kisspeptin neuronal development, associated with a marked downregulation of the Wnt signaling pathway. These results suggest that TBX3 variants may affect both migration and differentiation of GnRH neurons. In conclusion, this thesis provides novel functional evidence that heterozygous variants in WFS1 and TBX3 converge on the disruption of GnRH and kisspeptin neuronal networks, through distinct but complementary mechanisms: WFS1 via ISL1-related pathways and axon guidance, and TBX3 through Wnt signaling and kisspeptin regulation. These findings enlarge the genetic and mechanistic landscape of CHH, propose zebrafish as a versatile model for functional validation of candidate genes and open new perspectives for targeted therapeutic approaches aiming to restore GnRH neuron function.