In Vitro Molecular And Functional Studies Of Bmp15 Mutations Associated With Premature Ovarian Failure

Bone morphogenetic proteins 15 (BMP15) is a Transforming Growth Factor beta (TGF) superfamily member selectively expressed by oocytes throughout folliculogenesis. BMP15 is synthesized as a dimeric pre-proprotein, consisting of a signal peptide, a large proregion and a mature peptide. The proregion is essential for the processes of dimerization and secretion of the active mature dimers. BMP15 gene is located at Xp11.2 and several recent studies report that mutations in BMP15 are associated with Premature Ovarian Failure (POF). Our genetic screening of a cohort of 250 unrelated idiopathic POF women affected by primary or secondary amenorrhea led to the identification of six novel heterozygous BMP15 variations. All novel alterations are non-conservative and include one insertion of three nucleotides coding the 262insLeu and five missense substitutions: S5R, R68W, R138H, L148P and A180T, located in the proregion or in the signal sequence. In order to investigate the biological impact of these variants on the protein function, we characterized their molecular processing by Western blot and biological activity by a luciferase-reporter assay using COV434 cells in comparison with BMP15 wild type. We show that 262insLeu is a polymorphic variant provided with normal processing, protein secretion and in vitro biological activity consistent with its finding in 5 of 95 menopausal women. No deleterious effects were also detected for the A180T and S5R, suggesting a minor role for these variants. In contrast, significant impairment of precursor processing and 5-20 fold reduction in mature BMP15 protein secretion were seen for the R68W and L148P mutations, respectively. Moreover, biological activities of R68W, L148P and R138H mutations resulted significantly impaired in comparison with wild-type BMP15 even in co-transfection experiments reproducing the heterozygous state seen in patients. In conclusion, our findings validate the relevance of human BMP15 proregion for the correct post-translational processing and firstly indicate that a mechanism of haploinsufficiency should be implicated for BMP15 heterozygous mutations associated with secondary amenorrhea and POF. These data support the idea that BMP15 may be the first X-linked gene whose haploinsufficiency would contribute to cause the ovarian defect in Turner Syndrome. Partially supported by grant 2005.1055/104878 from Cariplo Foundation.