Primary ovarian insufficiency (POI) is one of the major causes of female infertility, affecting about 3.7% of women before the age of 40. POI is associated with the premature loss of ovarian function and can manifest with primary amenorrhea (PA) or post-pubertal secondary amenorrhea (SA), with elevated gonadotropins and hypoestrogenism. Several evidence established a clear genetic component to POI, albeit heterogeneous. In search of novel causative genes, we screened 64 POI patients through a customized next generation sequencing panel, which includes known and novel candidate genes potentially involved in POI pathogenesis. Among the novel rare variants identified in genes belonging to pathways relevant in ovarian physiology, we focused our functional studies on a nonsense variant (c.1927C>T) in TP63 gene, that was identified in two sisters with PA, and a frameshift insertion (c.682_683insT) in SAMD11 gene, that was identified in 6 unrelated SA patients. The longest isoform of TP63 gene, TAp63α, is almost exclusively expressed in the nucleus of oocytes after meiotic double-stranded DNA break repair and protect germ line fidelity during meiosis. We investigated the contribution to POI of the TP63 variant through luciferase reporter assays in Saos-2 cell line using the firefly luciferase gene under the control of three different human promoters, direct targets of p63: BAX, involved in cell cycle and apoptosis; K14, regulating differentiation; and IRF6, required for cell cycle exit and tissue development. TP63 variant falls in the transactivation inhibitory domain, which inhibits the transactivation domain of the protein. The presence of this variant showed an increment of transcriptional activity on both K14 and IRF6 promoters but a slight reduction on BAX, which is partially recovered in presence of the wild-type counterpart. The pathogenic consequences of this variant could be the overexpression of dosage-sensible genes involved in differentiation and development of the ovarian follicle and the haploinsufficiency of TP63 in cell cycle regulation. SAMD11gene encodes for a transcriptional modulator that might slightly promote cell proliferation. We observed an enrichment of the frequency of SAMD11variant in the POI cohort with respect to the female population worldwide. We observed by immunoblotting that the frameshift generates a truncated protein which can still be produced and possibly further degraded. Moreover, proliferation assay in HEK293T showed a reduced cell growth both at homo- and heterozygous state. SAMD11has never been associated to ovarian phenotypes, however, our results suggest this variation might be considered as a POI predisposing factor.
21 May 2022 - 24 May 2022