Molecular studies of BMP15 variants associated with secondary amenorrhea and premature ovarian failure (POF)

Bone Morphogenetic Protein 15 (BMP15) is a member of TGFβ superfamily involved in follicular development producing its biological effects as homodimer or heterodimer. BMP15 is synthesized in the oocytes as pre-proprotein composed of signal, proregion and mature peptides. Animal and human findings indicate a critical role of this gene in female fertility. We previously reported the first mutation in the pro-region of BMP15 gene (Y235C) in two sisters with primary amenorrhea and ovarian dysgenesis. Y235C was associated with abnormal processing and dominant negative effect in a granulosa cell bioassay. Recently, a genetic screening of a large series of idiopathic POF women revealed 4 additional BMP15 variants all associated with onset of secondary amenorrhea at 15-29 years. All novel alterations are non-conservative and include one insertion of three nucleotides (p.262insL) and three missense substitutions (p.R68W, p.L148P and p.A180T), located in the initial part of the gene sequence encoding the pro-region. In order to test the biological impact and possibly explain the different clinical manifestations associated with these BMP15 variants we introduced these variations in a BMP15 expression vector and stably transfected HEK-293T cells. Wild-type and variant BMP15 were purified from cell lysates and culture media by affinity chromatography. By western blot analyses, we documented a normal processing and mature BMP15 secretion in the presence of ins262L variant. These biochemical data are in accord with the finding of this insertion in 5 control women. Similar in vitro data, were seen for A180T suggesting a minor role of this variant. In contrast, significant impairment of pro-protein processing and marked reduction of mature BMP15 protein secretion were seen in the case of R68W and L148P mutations. Consistently, in-silico analyses suggest that these substitutions cause significant modifications of charge (R68W) or hydrophobicity (L148P) in the pro-region that may alter folding. In conclusion, these in-vitro results indicate an impaired mature BMP15 secretion in the presence of R68W and L148P mutations which confirm the critical role of the pro-region in processing. The defects associated with these variations appear to be less pronounced than that of the original Y235C mutation and are consistent with the milder phenotype of the affected women (secondary amenorrhea).