Homozygous missense variations of APC12 cause meiotic metaphase I arrest in oocytes and female infertility.

Background: Oocyte maturation arrest is a leading cause of female infertility. However, the genetic variables remain largely unknown. In oocytes, the activation of anaphase-promoting complex/cyclosome (APC/C) is a critical step in the transition from meiosis I to meiosis II. However, the causal relationship between variants in APC/C components and female infertility has not been completely investigated.

Objective: This study aims to find a novel gene and its pathogenic mutation as a cause for metaphase I arrest in oocytes, thus introducing a new APC/C component for screening causes of female infertility.

Study Design: Whole-exome sequencing was performed on 30 infertile women with recurrent oocyte maturation arrest without known gene variants. A homozygous missense mutation in the APC12 gene (p.R8H) was identified as a candidate for oocyte metaphase I arrest in a consanguineous family member. The experiment was conducted in vitro with HEK293T cells and mouse oocytes. Methods such as oocyte microinjection, western blotting, co-immunoprecipitation, and immunofluorescence were used. A knockdown mouse model was generated to verify the function of Apc12 in causing oocyte metaphase I arrest. About 100 wild-type C57BL/6J mice and 50 gene-edited mice were used in this study.

Results: APC12 p.R8H was identified in an infertile woman with oocyte metaphase I arrest. Microinjection of Apc12 mutant cRNA in mouse oocytes caused a considerably higher rate of metaphase I stage arrest (65.21±5.64% vs 30.86±1.74%, P<.01) with decreased APC12 protein expression and Securin accumulation compared to the control group, while oocytes injected with Apc12 cRNA showed comparable metaphase I arrest rate (31.51±3.05%). This fit the phenotype we identified in our case and suggested that Apc12 p.R8H was a loss-of-function mutation leading to oocyte metaphase I arrest. The in vitro experiments in HEK293T cells suggested that the APC12 p.R8H mutation disrupted the interaction between APC12 and APC6, as well as impaired APC/C activity by disrupting Securin ubiquitination. Knocking down APC12 with siRNA in mouse oocytes led to metaphase I arrest (41.65±6.10% vs 24.20±2.19%, P<.01). Oocytes from Apc12 mice exhibited metaphase I arrest compared with oocytes from wild-type mice (72.29±0.51% vs 23.33±5.82%, P<.01), which could be rescued by injecting Apc12 cRNA (53.44±1.20%).

Conclusion: We identified a pathogenic mutation in APC12 in a female patient and confirmed its relevance as a causative factor for metaphase I arrest in oocytes, implying its importance as an APC/C component in the pathophysiology of oocyte maturation arrest, which caused female infertility.