Society For Pediatric Urology

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RBFOX-2 Haploinsufficiency Alters Urethral Development Causing Hypospadias and Urinary Tract Obstruction
Jeffrey T. White, MD, PhD1, Marisol A. O'Neill, MS2, Kunj R. Sheth, MD1, Chester J. Koh, MD1, David R. Roth, MD1, Dolores J. Lamb, PhD, HCLD3.
1Baylor College of Medicine / Texas Children's Hospital, Houston, TX, USA, 2Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA, 3Departments of Urology and Genetic Medicine, Weill Cornell School of Medicine, New York, NY, USA.

BACKGROUND: Genitourinary (GU) anomalies are among the most common birth defects and their causes are often unknown. When copy number variants (CNVs), which are micro-deletions or micro-duplications of a chromosome, occur in a dosage-sensitive genetic region, birth defects can develop. An externally identifiable GU birth defect, hypospadias, is one of the common pathologies surgically corrected by a pediatric urologist. We hypothesized that, through a top-down approach utilizing CNVs from patients with hypospadias, novel genes important in penile development can be discovered.
METHODS: By searching chromosomal CNV databases for hypospadias phenotypes, several candidate genes were identified. Four hypospadic male patients were noted to have CNVs within the RNA binding FOX 2 (RBFOX-2) gene. TaqMan CNV assays were performed on control and GU phenotype patients to identify the incidence of RBFOX-2 CNVs. In situ hybridization was performed to study expression of RBFOX-2 and FgfR2 during penile development. By crossing Gdf9-Cre mice with conditional RBFOX-2-Flox mice, RBFOX-2Δ/+ mice were obtained, where a single copy of RBFOX-2 was removed. These mice were phenotyped using microCT and gene expression analyses were performed with in vitro and in vivo models.
RESULTS: Fifteen patients with RBFOX-2 pathogenic CNVs were identified in DECIPHER; four were noted to have hypospadias. RBFOX-2 CNV rates appear at a low rate in population-level database (Database of Genomic Variants, 0.11%, n=42,511, p=0.3). Results from local GU birth defect patient samples identified eleven patients (2.4%, n=450) with CNVs for RBFOX-2: 21% have hypospadias, 21% cryptorchidism, 29% vesicoureteral reflux and 29% ureteropelvic junction obstruction. When compared to control patient samples (0.0%, n=64), an enrichment in RBFOX-2 CNVs was discovered in GU birth defect patients (p<0.01). In situ hybridization for RBFOX-2 demonstrated ubiquitous expression throughout E14.5 embryos and restricted expression in the urethral mesenchyme undergoing mesenchyme-to-epithelial transitions (MET) in E16.5 and E18.5 embryos. Similarly, RBFOX-2 and FgfR2 were co-expressed in tissues undergoing MET along the urethra, demonstrating a possible mechanistic relationship based on spatiotemporal expression. In vitro knockdown and overexpression experiments as well as in vivo haploinsufficient experiments were performed to verify that RBFOX-2 gene dosage changes alters urethral MET to cause bilateral hydroureteronephrosis, urinary retention and hypospadias.
CONCLUSIONS: RBFOX-2 gene dosage changes are enriched in patients with GU birth defects. Expression of RBFOX-2 in the genital tubercle and co-expression with FgfR2 demonstrated a novel role for RBFOX-2 in regulating urethral MET. Cell culture experiments and conditional RBFOX-2 knockout mice were used to demonstrate that RBFOX-2 gene dosage changes alter normal urethral formation resulting in hypospadias and lower urinary tract obstruction due to urethral malformation.

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