OR25-2 RBFOX2 Copy Number Variation Disrupts Urethral Development Causing Hypospadias

Abstract Despite the frequency of hypospadias (1: 125 live male births) often the etiology is not known. The Lamb Laboratory previously determined that RNA Binding Fox-1 Homolog 2 (RBFOX2) copy number variation (CNV) is associated with clinical cases of upper and lower tract genitourinary (GU) anomalies, including hypospadias. RBFOX2 encodes a RNA binding protein that regulates mRNA splicing. The hypothesis was tested that altered RBFOX2 gene dosage impacts the developing penis transcriptome via mRNA splicing, specifically the mesenchymal to epithelial transition critical for penile urethra formation, resulting in hypospadias.Searches, in publically available and local clinical databases, centered on hypospadias occurance with gene CNV: identified the RBFOX2 locus as a gene dosage sensitve region. To identify developmental pathways regulated by RBFOX2 in the GU system, Rbfox2 haploinsufficient and null mouse models were developed, and penises were collected for RNA-seq, in situ hybridization, and micro-CT analysis of urethral development. In situ hybridization together with re-analysis of published scRNA-seq datasets were employed to define the anatomical regions of Rbfox2-specific expression in the developing mouse penis [Amato & Yao, PNAS 118(25): e2103856118, 2021; Armfield & Cohn, Dev Biol 477: 145-154.2021].Publicly available clinical data identified 17 patients (0.068% n=24,896) with pathogenic RBFOX2 CNVs: 4 were noted to have hypospadias. Results from our cohort of non-syndromic GU birth defect patient samples identified 11 patients (2.4% n=450) with RBFOX2 CNVs: 21% of whom have hypospadias. RNA immunoprecipitation agianst RBFOX2 in human kidney cells validated mRNA transcipt targets previously identified in cardiac cells [Verma 2016]. Micro-CT analysis on fetal Rbfox2-haploinsufficient mice validated the modeled clinical phenotype. Combined in situhybridization and scRNA-seq spatial analysis on mouse samples revealed, after penile sex determination (E15.5), Rbfox2 expression becomes restricted to the penile urethral mesenchyme. Differential gene expression and transcript usage analysis on the Rbfox2-loss mouse penis transcriptome identified mis-expression of gene products associated the estrogen signalling pathway.Alterations in RBFOX2 gene dosage clinically correlate with upper and lower tract genitourinary anomalies, specifically hypospadias. Data from human cell lines and mouse models seemingly indicate the phenotype seems is partially the product of an RBFOX2 role in directing the mesenchymal to epithelial transition. Specifically our data support the a direct role of RBFOX2 in balancing the competing male and female pathways at the level of estrogen and androgen signaling pathways. We are currently using immunofluorescence experiments to define the changes in androgen-receptor, estrogen-receptor, and RBFOX2 patterning throughout perinatal penis development. Presentation: Monday, June 13, 2022 11:15 a.m. - 11:30 a.m.