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 patients with 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 with massively 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 pathogenicity were also considered. The consequences of the identified mutations on BMP7 expression was determined by Western blot analysis on culture media 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.

Keywords: bone morphogenetic protein; disorders of sex development; hypospadias; massively parallel sequencing.

Figure 1.

BMP7 expression and function in the developing genital tubercle. When BMP7 is expressed in the urethral epithelium of the GT, it triggers mesenchymal proliferation and epithelial apoptosis to result in the closure of the urethral plate and formation of the median raphe. In the absence of BMP7 expression, there is no mesenchymal proliferation and no epithelial apoptosis. This results in a hypospadias phenotype with enlarged capillaries in the mesenchyme.

Figure 2.

BMP7 structure and variants reported in humans. Structure: DNA is presented in orange and include 7 exons. Blue represents the protein with its three components: Signal P, Prodomain, and BMP7 chain. Number of amino acids in each part is presented at the bottom line of the protein structure. Note the RSIR furin-like consensus cleavage site between the prodomain and the BMP7 chain.

Figure 3.

Variants affect highly conserved amino-acids in the BMP7 prodomain. (A) Variants in both pairs of twins (nucleic acid highlighted in orange) affect amino acids highly conserved across multiple species. (B) Sanger sequencing confirmation for both variants. Pair 1: exon 1. c.G265T; p.A89S. Pair 2: exon 3 c.G634A; p.D212N.

Figure 4.

BMP7 variants affect protein expression. (A) Homozygous and heterozygous variants’ expression is compared by Western Blot to the wild-type and standard BMP7 (Std). (B) Densitometry results for the two variants, at both homozygous and heterozygous states. Twin pair 1: G265T (A89S). Twin pair 2: G634A (D212N). (C) Total protein concentrations as determined by a BCA assay.

Figure 5.

BMP7 bioactivity was not affected in patient variants. The ability of the BMP7 variants to induce pSMAD 1/5 activation was assessed by (A) a luciferase bioassay and (B) analysis of endogenous pSMAD 1/5 levels, both in COV434 cells.