R-spondin2, a novel target of NOBOX: identification of variants in a cohort of women with primary ovarian insufficiency

BACKGROUND: R-spondin2 (Rspo2) is a secreted agonist of the canonical Wnt/β-catenin signaling pathway. Rspo2 plays a key role in development of limbs, lungs and hair follicles, and more recently during ovarian follicle development. Rspo2 heterozygous deficient female mice become infertile around 4 months of age mimicking primary ovarian insufficiency (POI). The study aimed to investigate the regulation of RSPO2 and its potential involvement in pathophysiology of POI. METHODS: We cloned the RSPO2 promoter and performed transcriptional assays to determine if RSPO2 can be regulated by NOBOX, an ovarian transcription factor. Then, we evaluated 100 infertile women after obtaining a detailed history of the disease and follicle-stimulating hormone measurements, besides karyotype determination and fragile-X premutation syndrome investigation. All exons, intron-exon boundaries and untranslated regions of the RSPO2 gene were identified by sequencing, and the results were statistically analyzed. RESULTS: We found that RSPO2 can be regulated by NOBOX via the presence of NOBOX Binding Element in its promoter. Among 9 identified variants in POI women, 4 of them were equally homozygous, 4 have never been described (c.-359C > G, c.-190G > A, c.-170 + 13C > T and c.-169-8 T > A), only one c.557 T > C was predicted to alter a single amino acid in the RSPO2 protein (p.Leu186Pro). CONCLUSIONS: RSPO2 is a novel target gene of the NOBOX key transcription factor, confirming its important role during the follicular growth in ovary. However, RSPO2 mutations are rare or uncommon in women with POI.