The Role of KCTD-13 in genitourinary development. - Project Summary Hypospadias and undescended testes (UDT, aka: cryptorchidism) are two of the most common congenital genitourinary (GU) defects, affecting 1 in 200 and 1 in 30 live male births, respectively. Proximal hypospadias, the most severe form, is associated with significant surgical morbidity and psychosocial consequences. UDT increases the risk of infertility and testicular cancer, and up to 40% of affected individuals present with both conditions, suggesting a shared molecular etiology. Despite their high prevalence, their molecular basis is poorly understood. Recent genomic studies, including our own, have identified copy number variants (CNVs) in KCTD13 in ~21% of boys with proximal hypospadias and/or UDT, implicating KCTD13 as a central regulator of male reproductive development. Kctd13 encodes a substrate-specific adapter of the BCR (BTB-CUL3-RBX1) E3 ubiquitin-protein ligase complex. We developed a Kctd13-knockout (KO) mouse that recapitulates features of human undervirilization, including micropenis, UDT, and subfertility. Kctd13 regulates both the androgen receptor (AR) and the Sertoli cell masculinization factor, SOX9. Kctd13 loss increases AR ubiquitination and degradation, resulting in lower levels of AR in the testis and penis. Replenishment of AR and SOX9 in the penises of tissue-specific knock-in mice reverts the micropenis and subfertility phenotype, suggesting that Kctd13 is, at least in part, acting via its regulation of AR and SOX9. Transcriptomic analysis of Kctd13-KO mice showed disruptions in other critical developmental pathways associated with UDT and hypospadias in the testis and penis, such as the Wnt/β-catenin and Fgfr2 signaling pathway. This project will test the hypothesis that KCTD13 coordinates AR, SOX9, Wnt/β-catenin, and FGFR2 signaling in a tissue-specific manner to regulate penile and testicular development. In Aim 1, we will initially use spatial transcriptomics to identify the precise cellular location of disrupted Kctd13 signaling in the genital tubercle, and the disrupted pathways will be validated in different cellular assays. In addition, we will determine the incidence and role of KCTD13 and associated proteins in proximal hypospadias in patients. In Aim 2, we will evaluate how KCTD13 regulates AR, SOX9, and Wnt/β- catenin pathways in the testis and use surgical orchidopexy to distinguish the effects of gene loss from testicular malposition. By integrating genetic, molecular, and spatial transcriptomic approaches, we will define the mechanistic role of KCTD13 in male reproductive development and inform future diagnostic and therapeutic strategies for hypospadias and UDT.
