128. STRUCTURAL ANALYSIS OF GDF-9 MUTATIONS ASSOCIATED WITH PREMATURE OVARIAN FAILURE AND TWINNING
C. M. Simpson A B , K. L. Walton A , P. G. Stanton A B and C. A. Harrison A BA Prince Henry’s Institute of Medical Research, Clayton, VIC, Australia.
B Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia.
Reproduction, Fertility and Development 22(9) 46-46 https://doi.org/10.1071/SRB10Abs128
Published: 6 September 2010
Abstract
Premature Ovarian Failure (POF) is a disorder associated with female infertility affecting 1% of women under the age of 40 years [1]. It is characterised by amenorrhea associated with an increase in plasma gonadotrophins and decreased estrogen [2, 3]. The major consequence of POF is the loss of fertility, however women also have increased risk of osteoporosis [4] and cardiovascular disease [5]. The causes of POF are unknown; in recent times genetic factors have been considered as etiological components. Genetic screening of women with POF has identified three mutations in Growth and Differentiation Factor-9 (GDF-9), an oocyte-secreted factor critical for folliculogenesis. These are S186Y, V216M and T238A, which are all located within the GDF-9 prodomain [2]. Mutations in GDF-9 may also contribute to the earlier menopause observed in mothers of dizygotic twins, suggestive of a direct relationship to women with POF [6]. The GDF-9 mutations associated with dizygote twinning (DZ) are; P103S located in the prodomain, and P374L and R454C located within the mature domain [6, 7]. The aim of this study is to understand the consequence of these mutations on the synthesis, secretion and biological activity of GDF-9. Utilising site-directed mutagenesis, all six mutations were individually introduced into GDF-9 cDNA and the impact on protein synthesis and secretion was assessed following transfection in HEK293T cells. Mutants S186Y and T238A appeared to have no affect on mature GDF-9 secretion; mutant R454C resulted in misfolded GDF-9; while mutants V216M, P103S and P374L resulted in a significant decrease of mature GDF-9. As the prodomain is necessary for correct folding, dimerisation and secretion of the biologically active mature protein [8], this decrease is most likely due to the impaired posttranslational processing of the prodomain. In vitro biological activity of these mutants is currently being assessed using a HEK293T cell luciferase reporter assay. Preliminary data suggests that mutants V216M and T238A lead to increased biological activity. It is concluded that GDF9 mutations associated with POF and DZ alter synthesis, secretion and bioactivity of GDF-9. It is anticipated that this study will provide structural basis for the impact of heterozygous GDF-9 mutations on ovarian insufficiency in humans.
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