Functional characterization of two new variants in the bone morphogenetic protein 7 prodomain in two pairs of monozygotic twins with hypospadias

Abstract

Context: Variants in bone morphogenetic protein 7 (BMP7) have been reported in patientswith hypospadias. Here we report and analyze two variants in the BMP7 prodomain in monozygotic twins with hypospadias. Materials and Methods: Patients with hypospadias were prospectively recruited. After informed consent was obtained, DNA was extracted from blood. The coding regions of 1034 genes [including 64 known diagnostic genes and candidate genes for disorder/difference of sex development (DSD)] were sequenced using a targeted capture approach (HaloPlex, Agilent, Santa Clara, CA), combined withmassively parallel sequencing. The resulting variants were filtered for rarity in the general population (,1%) and in our screen. Quality, depth of the reads, and predicted pathogenicitywere also considered. The consequences of the identifiedmutations onBMP7 expression was determined byWestern blot analysis on culturemedia from transfected cells, and activity measured using a SMAD 1/5-responsiveness luciferase assay. Results: We analyzed DNA from 46 patients with hypospadias. Two variants in BMP7 were identified in two pairs of monozygotic concordant twins exhibiting proximal hypospadias. Both variants are heterozygous, nonsynonymous, and affect highly conserved amino acids in the prodomain of BMP7 in regions predicted to be important for BMP7 assembly/folding. Functional analyses demonstrated that both variants disrupt BMP7 synthesis or secretion. Conclusion: Through our targeted DSD panel we have identified two variants in the prodomain of BMP7 in hypospadias. By decreasing BMP7 synthesis, these variants are likely to limit BMP7 bioavailability during closure of the urethral plate.Further analysis of patients with hypospadias may uncover additional variants that cause this DSD.