BMP15 mutations associated with primary ovarian insufficiency cause a defective production of bioactive protein

Abstract

Bone morphogenetic protein-15 (BMP15) is selectively synthesized by oocytes as a pre-proprotein and is considered an ovarian follicle organizer whose adequate function is critical for female fertility. Missense mutations were reported in primary ovarian insufficiency (POI) but their biological impact remained unexplored. Here, screening of 300 unrelated idiopathic overt POI women with primary or secondary amenorrhea (SA) led to the identification of six heterozygous BMP15 variations in 29 of them. All alterations are nonconservative and include one insertion of three nucleotides (p.L262_L263insL) and five missense substitutions. Except for the p.S5R located in the signal sequence, the other variants (p.R68W, p.R138H, p.L148P, and p.A180T) localize in the proregion, which is essential for the processing and secretion of bioactive dimers. The mutations p.R68W, p.L148P, and the novel p.R138H lead to marked reductions of mature protein production. Their biological effects, evaluated by a novel luciferase-reporter assay in a human granulosa cell (GC) line, were significantly reduced. Cotransfection experiments of defective mutants with equal amounts of wild-type BMP15 cDNA, thus reproducing the heterozygous state seen in patients, did not generate a complete recovery of wild-type activity. No or minor deleterious effects were detected for the variants p.L262_L263insL, p.A180T, or p.S5R. In conclusion, heterozygous BMP15 mutations associated with the early onset of overt POI lead to defective secretion of bioactive dimers. These findings support the concept that an adequate amount of BMP15 secreted in the follicular fluid is critical for female fertility. We propose to consider the screening of BMP15 mutations among the analyses for the prediction of POI risk.

Figure 1

A: SDS-PAGE protein analysis. 10 micrograms of purified protein from conditioned media of the wild-type (wt), mutants, and not-transfected (nt) cells were analyzed. The BMP15 mature peptide is visible in the wt and p.S5R, p.A180T and p.262insL (p.L262_L263insL) lanes whereas only a small amount of it is detectable in the p.R68W, p.R138H and p.L148P mutant lanes. B: Analysis of α-tubulin expression in total cell lysates showing that control and conditioned media were obtained from a similar amount of cultured cells. C: SDS-PAGE protein analysis. 20 micrograms of proteins purified from the mutant media are compared with 10 micrograms of wt counterpart. Decrement in mature peptide bands of mutant proteins does not depend on the amount of protein loaded. D: Denaturant non-reducing Western blot analysis. 20 micrograms of proteins loaded. Faint bands corresponding to the mature peptide and precursor, as dimer or monomer, are present in the mutants conditioned media. No abnormal products of processing are revealed. E: Densitometric analysis (Scion Image software, IBM) of BMP15 mature peptide bands. Decreasing amounts of the wild-type preparation (10, 5, 2, 1, 0.5 micrograms) are compared with a fixed quantity of mutated proteins (10 micrograms). A reduction in mature BMP15 mutant protein secretion of 3-fold, 8-fold and 20-fold is present respectively for the p.R138H, p.R68W and p.L148P substitutions.

Figure 2

In vitro reporter luciferase assay. COV434 granulosa cells were transiently cotransfected with 400 ng of the different BMP15 vectors and 100 ng of the BRE-luciferase reporter vector. Results are expressed as the mean (±SD) of 12 determinations in four separate experiments. A: The cell system shows specific activation of the BMP-signaling pathway by incubation with 10 ng/ml of rhBMP-4 agonist or transfection with wild-type BMP15 vector. The defective p.C53Y mutation is responsible for the FecXL phenotype in Lacaune sheep [Bodin et al., 2007] and the vector carrying this mutation was associated with a complete loss of luciferase activity. Co-transfection with equal amounts of wild-type (200 ng) and p.C53Y mutation (200 ng) restores luciferase activity to 50% of the wild-type alone (400 ng). B: In presence of p.S5R, p.R68W, p.R138H and p.L148P mutations, the activity on the BMP pathway resulted significantly different from the stimulus produced by 200 or 400 ng of wild-type cDNA (P<0.05, Student’s t test). This impairment is only partially rescued when 200 ng of p.R68W, p.R138H and p.L148P mutant plasmids were co-transfected with equal amounts of the wild-type.