MCM8 variants in two patients with primary ovarian insufficiency: clinical findings and in vitro defective DNA repair revealed by an MCM8 double-mutant construct.

Pediatric-onset primary ovarian insufficiency (POI) presents distinct clinical challenges, including delayed puberty or growth retardation. Previously, we reported a Chinese family with POI harboring compound heterozygous variants (p.C242R and p.S445*) in MCM8 gene, a critical factor for DNA repair and gonadal homeostasis. This study describes clinical findings of pediatric-onset POI patients with compound heterozygous MCM8 variants and investigates the in vitro functional consequences of an artificial double-mutant MCM8 construct harboring both p.C242R and p.S445*. Using this overexpression model, we assessed DNA damage response, protein interactions, and transcriptomic changes associated with the double-mutant MCM8. HeLa cell models were used to overexpress double-mutant (p.C242R and p.S445*) or wild-type MCM8. Functional consequences were evaluated using immunofluorescence staining, flow cytometry, and western blotting to assess DNA damage repair, cell cycle and apoptosis. Co-immunoprecipitation coupled with mass spectrometry (CoIP-MS/MS) was used to screen the disrupted protein interactome. Cut&Tag and mRNA-seq analyses were used to examine chromatin-binding-associated changes and transcriptomic alterations. Ovarian tissues from POI patients exhibited reduced MCM8 expression and increased apoptosis. Cells expressing the artificial double-mutant MCM8 construct showed delayed resolution of DNA damage, as indicated by persistent γ-H2AX accumulation, and altered recruitment of DMC1 and RAD51. These changes were accompanied by S-phase accumulation and increased apoptosis. CoIP-MS/MS analysis identified altered interaction between double-mutant MCM8 and MCM6, suggesting possible changes in replication-associated protein interactions. Integrated CUT&Tag and RNA-seq analyses identified MCM8-associated chromatin-binding changes and transcriptomic alterations involving candidate genes enriched in ovarian survival and metabolic pathways, including the PI3K/AKT signaling pathway. These specific MCM8 variants are clinically associated with pediatric-onset POI. In vitro, expression of double-mutant MCM8 construct was associated with impaired DNA damage resolution, altered MCM8-associated protein interactions, transcriptomic alterations, cell-cycle disturbance, and increased apoptosis. These findings provide cellular observations that may help generate hypotheses regarding how MCM8 dysfunction could contribute to ovarian insufficiency.