ESPE Abstracts

Keywords: Primary ovarian insufficiency; Microsomal maintenance complex 8; Cell apoptosis; Cell cycle; PI3K/AKT

Background: Primary ovarian insufficiency (POI) with a chromosome karyotype of 46,XX in children, compared to adults, is difficult to diagnose and often seek medical attention due to delayed puberty or short stature. We have reported the two novel pathogenic mutations p.C242R and p.S445*of MCM8 gene in a pedigree of POI, characterized by a gradual decline of ovarian function in childhood.

Purpose: To explore the effects of MCM8 mutations on DNA damage repair, apoptosis, and cell cycle function, and clarifying the signal pathway mechanism related to POI induced by Mutant MCM8.

Methods: Construction overexpression plasmids of compound mutant C242R-S445* of MCM8 gene in Hela cells. Screening differential interacting proteins by immunoprecipitation (CoIP) combined with mass spectrometry (LC-MS/MS). Differential interaction genes were screened through Cut&tag-mRNA-seq cross-linking analysis. The expression of meiotic homologous recombinant repair proteins, the apoptosis and cell cycle proteins were detected by Western blotting. The flow cytometry was used to detect the cell cycle and cell apoptosis. Positive and negative COIP were used to verify MCM8 differential protein interaction.

Results: The delayed recovery of expression of meiotic repair proteins RAD51 and DMC1 suggests that the C242R-S445* mutation interferes with the meiotic homologous recombination repair pathway of DMC1-RAD51. Forward and reverse COIP validation confirmed that the interaction between mutant MCM8 and MCM6 is enhanced during the DNA repair phase. During the DNA repair phase, the G1 phase was significantly decreased, the S phase was significantly increased, the expression of CyclinD1 was downregulated, while the expression of CyclinA2 and CyclinB1 was sustained, suggesting that the S phase was stagnant. The downregulation of Bax and Capase3 expression was delayed compared with wild type, indicating the persistence of apoptosis. Cut&tag-mRNA-seq cross-linking analysis revealed the PI3K/AKT pathway may be inhibited. The genes involved in signal pathways such as PI3K/AKT/Bcl-2 is related to the mechanism of cell apoptosis, and PI3K/AKT/Cyclins is related to cell cycle.

Conclusion: Mutant MCM8 interferes with the meiotic recombination repair DMC1-RAD51 pathway, delaying DNA damage responses, which interacts with MCM6 to block the S phase of the cell cycle, and continuously activate the Bcl-2/Bax/Caspase 3 mitochondrial apoptosis pathway. It is speculated that mutant MCM8 interferes with the PI3K/AKT pathway, inducing cell cycle arrest and apoptosis.