[Homozygous Variant of of the Fanconi Anemia Pathway Causes Premature Ovarian Insufficiency: Investigation of the Pathogenic Mechanism].

OBJECTIVE

Infertility affects approximately one-sixth of the people of childbearing age worldwide, causing not only economic burdens of treatment for families with fertility problems but also psychological stress for patients and presenting challenges to societal and economic development. Premature ovarian insufficiency (POI) refers to the loss of ovarian function in women before the age of 40 due to the depletion of follicles or decreased quality of remaining follicles, constituting a significant cause of female infertility. In recent years, with the help of the rapid development in genetic sequencing technology, it has been demonstrated that genetic factors play a crucial role in the onset of POI. Among the population suffering from POI, genetic studies have revealed that genes involved in processes such as meiosis, DNA damage repair, and mitosis account for approximately 37.4% of all pathogenic and potentially pathogenic genes identified. FA complementation group M () is a group of genes involved in the damage repair of DNA interstrand crosslinks (ICLs), including -. Abnormalities in the genes are associated with female infertility and gene knockout mice also exhibit phenotypes similar to those of POI. During the genetic screening of POI patients, this study identified a suspicious variant in . This study aims to explore the pathogenic mechanisms of the genes of the FA pathway and their variants in the development of POI. We hope to help shed light on potential diagnostic and therapeutic strategies for the affected individuals.

METHODS

One POI patient was included in the study. The inclusion criteria for POI patients were as follows: women under 40 years old exhibiting two or more instances of basal serum follicle-stimulating hormone levels>25 IU/L (with a minimum interval of 4 weeks inbetween tests), alongside clinical symptoms of menstrual disorders, normal chromosomal karyotype analysis results, and exclusion of other known diseases that can lead to ovarian dysfunction. We conducted whole-exome sequencing for the POI patient and identified pathogenic genes by classifying variants according to the standards and guidelines established by the American College of Medical Genetics and Genomics (ACMG). Subsequently, the identified variants were validated through Sanger sequencing and subjected to bioinformatics analysis. Plasmids containing wild-type and mutant genes were constructed and introduced into 293T cells. The 293T cells transfected with wild-type and mutant human plasmids and pEGFP-C1 empty vector plasmids were designated as the EGFP - group, the EGFP - group, and the EGFP group, respectively. To validate the production of truncated proteins, cell proteins were extracted 48 hours post-transfection from the three groups and confirmed using GFP antibody. In order to investigate the impact on DNA damage repair, immunofluorescence experiments were conducted 48 hours post-transfection in the EGFP WT group and the EGFP -MUT group to examine whether the variant affected FANCM's ability to localize on chromatin. Mitomycin C was used to induce ICLs damage in both the EGFP - group and the EGFP - group, which was followed by verification of its effect on ICLs damage repair using γ-H2AX antibody.

RESULTS

In a POI patient from a consanguineous family, we identified a homozygous variant in the gene, c.1152-1155del:p.Leu386Valfs*10. The patient presented with primary infertility, experiencing irregular menstruation since menarche at the age of 16. Hormonal evaluation revealed an FSH level of 26.79 IU/L and an anti-Müllerian hormone (AMH) level of 0.07 ng/mL. Vaginal ultrasound indicated unsatisfactory visualization of the ovaries on both sides and uterine dysplasia. The patient's parents were a consanguineous couple, with the mother having regular menstrual cycles. The patient had two sisters, one of whom passed away due to osteosarcoma, while the other exhibited irregular menstruation, had been diagnosed with ovarian insufficiency, and remained childless. Bioinformatics analysis revealed a deletion of four nucleotides (c.1152-1155del) in the exon 6 of the patient's gene. This variant resulted in a frameshift at codon 386, introducing a premature stop codon at codon 396, which ultimately led to the production of a truncated protein consisting of 395 amino acids. experiments demonstrated that this variant led to the production of a truncated FANCM protein of approximately 43 kDa and caused a defect in its nuclear localization, with the protein being present only in the cytoplasm. Following treatment with mitomycin C, there was a significant increase in γ-H2AX levels in 293T cells transfected with the mutant plasmid (<0.01), indicating a statistically significant impairment of DNA damage repair capability caused by this variant.

CONCLUSIONS

The homozygous variant in the gene, c.1152-1155del:p.Leu386Valfs*10, results in the production of a truncated FANCM protein. This truncation leads to the loss of its interaction site with the MHF1-MHF2 complex, preventing its entry into the nucleus and the subsequent recognition of DNA damage. Consequently, the localization of the FA core complex on chromatin is disrupted, impeding the normal activation of the FA pathway and reducing the cell's ability to repair damaged ICLs. By disrupting the rapid proliferation and meiotic division processes of primordial germ cells, the reserve of oocytes is depleted, thereby triggering premature ovarian insufficiency in females.