Molecular and functional characterization of BMP15 variants associated with secondary amenorrhea and Premature Ovarian Failure (POF)

BMP15 is an oocyte-derived growth factor belonging to TGF- superfamily involved in follicular development as a critical regulator of granulosa cell processes. BMP15 gene maps at Xp11.2 and is expressed throughout folliculogenesis. BMP15 is translated as a pre-proprotein consisting of signal peptide, pro-region and mature peptide. The pro-region has an important role in the processing driving the pro-protein dimerization and secretion of active mature dimers. We report the genetic screening of 250 unrelated idiopathic POF women affected by primary or secondary amenorrhea. Six heterozygous BMP15 variations have been identified in association with secondary amenorrhea and POF. All novel alterations are non conservative and include a 3bp insertion (262insLeu) and five missense substitutions in the proregion or signal sequence: S5R, R68W, R138H, L148P, A180T. Only the insertion was found in 100 control women with physiological menopause. To clarify the role of these variants, we characterized their molecular processing by Western blot and their biological activity by a luciferase-reporter assay using the COV434 granulosa cell line. We attest that 262insLeu is a polymorphic variant provided with normal processing and biological activity, consistent with its finding in controls. Deleterious effects in vitro were not detected also for A180T and S5R variants. In contrast, significant impairment of precursor processing and 5-20 fold reduction of mature BMP15 protein secretion were seen for the other mutations. Moreover, R68W, L148P and R138H biological activities were significantly impaired in comparison with the wild-type even if tested in co-transfection experiments which reproduce the heterozygous state seen in vivo. Our findings indicate that haploinsufficiency of BMP15 gene is associated with secondary amenorrhea and POF, and support the idea that BMP15 may be the first ovary determining gene on X chromosome whose haploinsufficiency may account for the ovarian defect in Turner syndrome. Partially supported by grant 2005.1055/104878 from Cariplo Foundation.