A Novel Homozygous BMP15 Mutation Causes Ovarian Dysgenesis and Primary Amenorrhea

Abstract

Context: Despite a growing number of studies, the genetic etiology in many cases of ovarian dysgenesis is incompletely understood.

Objectives: This work aimed to study the genetic etiology causing absence of spontaneous pubertal development, hypergonadotropic hypogonadism, and primary amenorrhea in 2 sisters.

Methods: Whole-exome sequencing was performed on DNA extracted from peripheral lymphocytes of 2 Palestinian sisters born to consanguineous parents. Following a BMP15 variant identification, confirming genetic segregation studies were performed in family members. Three-dimensional (3D) modeling for BMP15 dimer and BMP15-GDF-9 heterodimer were followed by functional studies in human ovarian COV434 granulosa cells cotransfected with plasmid harboring either the variant or a wild-type (WT) control, and a second plasmid harboring a luciferase-reporter-gene with a BMP-responsive element.

Results: A novel homozygous c.G959A/p.C320Y BMP15 mutation was identified in both sisters, and segregated with the disease in the family. By 3D-structure modeling, the mutations were predicted to damage a cysteine-knot motif, disrupt BMP15 dimerization, and severely impair activation of the BMP pathway. The homologous mutation C53Y occurring and identified spontaneously in sheep results in sterility in homozygotes, mimicking the human phenotype here. A 3.8-fold decrease in BMP15 signaling was observed in vitro in cells expressing the homozygous BMP15 mutant when compared to the WT control.

Conclusion: The novel homozygous missense C320Y mutation is the first homozygous human BMP15 variant causing impaired signaling ability, which correlates with the predicted 3D-structural changes leading to ovarian dysgenesis. The homologous mutation in sheep mimics the human phenotype by infertility. Beyond genetic counseling, and considering ovarian preservation, the ovine model enables further elucidation and interventions in the BMP signaling.

Keywords: BMP15; infertility; ovarian dysgenesis; primary amenorrhea; primary ovarian insufficiency.

Figure 1.

BMP15 in a family with ovarian dysgenesis. A, Segregation of BMP15 variants in the studied family. Solid black symbols indicate affected family members and solid gray symbols indicate prepubertal family members likely to be affected. Segregation is consistent with X-linked recessive inheritance. Genotype is indicated underneath tested family members, X(V) denotes the variant and X(N) the normal gene. To the right is the BMP15 Sanger sequencing result. Normal control wild-type (WT) shows reference sequence. The probands are homozygous for a missense mutation in locus NM_005448:exon2:c.G959A in the BMP15 gene on chromosome X. The mother is a carrier and the father is hemizygous for the mutation. B, Clinical characteristics of probands with primary ovarian insufficiency (POI). C, Clustal alignment analyses of BMP15 sequence over different species commonly used as an animal model for POI. C320 residue is highlighted in light blue.

Figure 2.

A, Predicted structural effect of C320Y variant in BMP15. G959A mutation substitutes a highly conserved cysteine residues in position 320 with tyrosine; left—full BMP15 protein structure includes signal peptide (green), propeptide (blue), and mature BMP15 peptide (magenta). Right—enlargement of the cysteine-rich region. Wild-type (WT) marks the normal protein sequence and structure; C320Y marks the predicted change in the mutant BMP15. Orange marks the cysteine residues and bonds within the cysteine motif, yellow marks C389 that bind WT C320 and form a cysteine bond. Red marks the C320Y substitute in the mutant BMP15. This structural change within a cysteine motif is disrupting a highly conserved cysteine bond and predicted to impair the protein structure and folding. B, Shown in the center are the ribbon diagrams of cumulin, a heterodimer consisting of one bone morphogenetic protein-15 (BMP15) subunit, and one growth differentiation factor-9 (GDF9) subunit, bound to the ectodomains of its receptor, the bone morphogenetic protein receptor (BMPR), a tetramer consisting of 2 BMPRI subunits, and 2 BMPRII subunits. The model is based on the structures of the BMP2/BMPR1 complex (PDB ID 1ES7) which serve as a scaffold for that of cumulin/BMPRI, and the structure of the activin A/activin receptor II complex (PDB ID 1NYU), which serves as a scaffold for that of cumulin/BMPRII. The C320Y mutation, indicated by a red arrow, disrupts the cysteine knot-motif (sticks representation), deforms the heterodimer interface of BMP15, and prevents cumulin formation. The R329H mutation, indicated by a second red arrow, exerts no effect on cumulin formation, but instead disrupts the interaction of cumulin with BMPRI, and prevents receptor activation. The model and figure were prepared using Pymol.

Figure 3.

The c.959G > A (p.C320Y) variant of BMP15 impairs the activation of the BMP pathway. The transcriptional activities of BMP15 WT and of its variant 320Y were studied through a luciferase assay using a BMP-responsive element (BRE)-luciferase reporter in COV434 granulosa cells. Recombinant human BMP4 (rhBMP4) was used as an exogenous stimulus of the BMP pathway. Results are shown as mean of fold changes vs wild-type (WT) ± SEM of 8 independent experiments, each performed in triplicate. **P less than .001; ****P less than .0001. RLU, relative light units.

Figure 4.

Heterozygous and homozygous carriers of BMP15 mutations. Shown are the different effect of mutation: A, In C320Y heterozygosity, the BMP15 gene is expressed as wild-type (WT) (50%) and mutant (50%). Only the WT dimerizes with GDF9 to produce cumulin, which can bind to the receptor, and exerts all activity (100%). The mutant cannot dimerize, cannot bind to the receptor, and exerts no effect on the receptor (0%). B, In R329H heterozygosity, the BMP15 gene is expressed as WT (50%) and mutant (50%). Both the WT and the mutant can dimerize into cumulin, and bind to the receptor. WT cumulin (50%) is a receptor agonist, whereas mutant cumulin (50%) is an antagonist, and their combination reduces receptor activity. C, In C320Y homozygosity, the BMP15 gene is expressed as mutant only (100%). The mutant cannot dimerize, cannot bind to the receptor, and exerts no effect on the receptor (0%). D, In R329H homozygosity, the BMP15 gene is expressed as mutant only (100%). The mutant dimerizes into a cumulin antagonist and can bind to the receptor. However, the antagonist does not activate the receptor (0%).