OR02-01 Gene Dosage Changes in RBFOX-2 Can Affect Upper and Lower Tract Genitourinary Development

Searching for genetic and genomic alterations in patients with genitourinary (GU) birth defects can aid in the identification of genes that are important for normal GU development. Using data in DECIPHER, RBFOX-2, located at 22q12 was identified as one such candidate gene and the effect of haploinsufficiency in humans and a mouse model (Rbfox-2(α/+)) assessed. RBFOX2 is an RNA-binding protein regulating alternative splicing. It also impacts steroid receptor transcriptional activity. RBFOX family members are thought to affect different developmental programs via isoform-specific localization and splicing activities. RBFOX-2 plays a vital role in multiple developmental processes including heart, brain, muscle and testis development. “Non-syndromic” patients with congenital GU anomalies were more likely to harbor RBFOX-2 CNVs (2.4%; hypospadias, 21% cryptorchidism, 29% vesicoureteral reflux and 29% ureteropelvic junction obstruction) relative to the general population (0.11%) indicating that 22q12 is a hotspot genetic locus for GU anomalies. CNVs (microdeletions and microduplications) spanning RBFOX-2 were identified in twenty-seven humans with a wide range of birth defects(intellectual disability, CNS, cardiac, genitourinary, facial, hearing, vision, growth and stature anomalies) and the phenotype of the haploinsufficient Rbfox-2(α/+)mouse model closely correlated with the human phenotype, although Rbfox-2(α/+ and)Rbfox-2(α/α) are neonatal lethal. By E18.5 day of development 100% of the Rbfox-2(α/+) and null fetuses exhibited hypospadias, megacystis (50%) and bilateral hydroureteronephrosis. RNA Seq was performed on genital tubercles from three Rbfox2fl/fl, Gdf9 icre+ and three Rbfox2fl/fl P1 males. 377 genes were differentially expressed between the two groups including 212 genes which were upregulated and 165 genes which were downregulated. Hallmark gene set analysis revealed downregulation of Wnt/β-catenin signaling and hedgehog signaling, both pathways which are well defined in genital development. Gene ontology (GO) analysis further defined downregulation of pathways (fibroblast growth factor, retinoic, androgen, estrogen and G-protein receptor signaling) including those involving negative regulation of embryonic development, epithelial development, morphogenesis of epithelial tube, renal, ureter, limb and several other developmental pathways. These results support the hypothesis that loss of Rbfox2 during development leads to hypospadias through impaired development of the urethral epithelium.