Introduction: The dosage-sensitive gene, RBFOX2, encodes a RNA binding protein that is a regulator of alternative mRNA splicing. Copy number variations (CNVs) in RBFOX2 cause birth defects in the cardiac and nervous systems. A subset of patients were identified in public databases with genitourinary birth defects, including hydronephrosis and hypospadias, associated with RBFOX2 CNVs. RBOX2 CNVs are more common in patients with GU and CAKUT anomalies (2.4 % of non-syndromic patients with GU birth defects, 0% of our control patients with no GU anomalies and 0.11% of subjects in the general population (Database of Genomic Variants) and 0.06% of patients with anomalies in DECIPHER (p < 0.00001; ANOVA). Work by others posited that RBFOX2-mediated splicing is integral in the mesenchymal-to-epithelial transition pathway. In support of this hypothesis, our RNA-sequencing analysis with postnatal penises from Rbfox2-knockout mice, that exhibit CAKUT and/or hypospadias, identified disruption of this pathway. Sequencing analysis revealed evidence of RBFOX2 interaction with sex hormone signal pathways.
Methods: We tested the hypothesis that the developmental origins of a subset of hypospadias is due to RBFOX2 CNVs. Whole exome sequencing identified patients with RBFOX2 CNVs and SNPs. To define the cell type specific expression ofRbfox2, spatiotemporal expression was defined over a fetal time course of mouse penises before and after sex determination of the genital tubercle. Fluorescent in situ hybridization and immunofluorescent whole mount samples were imaged using confocal microscopy.
Results: Whole exome sequencing data confirmed in patients with RBFOX2 CNV and damaging SNPs, that these variants were a root cause for lower tract genitourinary anomalies, as well as CAKUT, cardiac defects, intellectual disabilities and head deformities. Imaging experiments defined Rbfox2 expression after sex determination of the murine male genital tubercle. Rbfox2 expression is first confined to the peri-urethral space of the developing penis. In this region, the most abundant urethral RBFOX2 protein signal travels distally starting at the base of the penis as a urethral ring of signal that halts at the junction of the penile body and glans. The glans is encased with an epithelial layer of cells that are strongly RBFOX2-positive. These cells aggregate at the site of glandular urethra formation. Most RBFOX2-positive glandular epithelial cells co-express androgen receptor. The preputial folds have a similar epithelial net of RBFOX2- and androgen receptor-positive cells.
Conclusions: The coincident locations of RBFOX2 signal during urethral formation and development of hypospadias when Rbfox2 is deleted, implicate RBFOX2 as a molecular player in the double zipper mechanism of urethral formation and closure. The more peripheral co-expression of RBFOX2 and androgen receptor in the glandular and preputial epithelium may be part of a RBFOX2-mediated balancing of androgen and estrogen signaling. The hypospadias that is observed when Rbfox2 is deleted would then, at least partly, be due to resultant favoring of the feminization pathway.
Source of Funding: AUA/UCF Research Scholar Award to JW; NIH-R01DK078121 and Frederick J. and Theresa Dow Wallace Fund of the New York Community Trust to DJL; NIH-T32DK007763 to DJL for MO.
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