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闻星星, 柴梦晗, 张倩楠, 等. 范科尼贫血通路FANCM基因纯合突变导致早发性卵巢功能不全的致病机制[J]. koko体育app 学报(医学版), 2024, 55(3): 559-565. DOI:
引用本文: 闻星星, 柴梦晗, 张倩楠, 等. 范科尼贫血通路FANCM基因纯合突变导致早发性卵巢功能不全的致病机制[J]. koko体育app 学报(医学版), 2024, 55(3): 559-565. DOI:
WEN Xingxing, CHAI Menghan, ZHANG Qiannan, et al. Homozygous Variant of FANCM of the Fanconi Anemia Pathway Causes Premature Ovarian Insufficiency: Investigation of the Pathogenic Mechanism[J]. Journal of Sichuan University (Medical Sciences), 2024, 55(3): 559-565. DOI:
Citation: WEN Xingxing, CHAI Menghan, ZHANG Qiannan, et al. Homozygous Variant of FANCM of the🎉 Fanconi Anemia Pathway Causes Premature Ovarian Insufficiency: Investigation of the Pathogenic Mechanism[J]. Journal of Sichuan🌠 University (Medical Sciences), 2024, 55(3): 559-565. DOI:


Homozygous Variant of FANCM of the Fanconi Anemia Pathway Causes Premature Ovarian Insufficiency: Investigation of the Pathogenic Mechanism

  • 摘要:
    目的 探究范科尼贫血(Fanconi anemia, FA)通路中的FANCM(FA complementation group M)基因及其突变在早发性卵巢功能不全(premature ovarian insufficiency, POI)发生中的致病机制。
    方法  对1例POI患者进行了全外显子组测序,并通过Sanger测序对突变位点进行验证。构建含有野生型和突变型FANCM基因的质粒,分别转染至293T细胞中,转染野生型人类 FANCM质粒、突变型人类FANCM 质粒、pEGFP-C1空载体质粒的293T细胞分别命名为EGFP FANCM-WT组、EGFP FANCM-MUT组和EGFP组。对于截短蛋白的验证,3组质粒转染48 h后提取细胞蛋白,使用GFP抗体进行验证。对于DNA损伤修复影响的研究,免疫荧光实验在EGFP FANCM-WT组、EGFP FANCM-MUT组293T细胞质粒转染48 h后进行,以研究该突变是否会影响FANCM定位于染色质的能力;利用丝裂霉素C(mitomycin C, MMC)在体外诱导EGFP FANCM-WT组、EGFP FANCM-MUT组293T细胞链间交联(interstrand crosslinks, ICLs)损伤的形成,然后使用γ-H2AX抗体验证其对ICLs损伤修复的影响。
    结果  在1例近亲婚配家系的POI患者中,koko体育app 发现了FANCM基因c.1152-1155del:p.Leu386Valfs*10的纯合突变。Western blot结果表明,该突变导致截短的FANCM蛋白产生。在丝裂霉素C处理后,与EGFP FANCM-WT组相比,EGFP FANCM-MUT组293T细胞中γ-H2AX水平升高(P<0.01)。免疫荧光结果提示,突变FANCM蛋白的细胞定位只存在于细胞质,在细胞核中缺失,突变的FANCM定位于染色质的能力受损。
    结论 FANCM基因c.1152-1155del:p.Leu386Valfs*10纯合突变导致截短的FANCM蛋白产生,并且影响FANCM蛋白在细胞核的定位,抑制其应对DNA损伤修复的能力,从而引起女性不孕。
    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 (FANCM) is a group of genes involved in the damage repair of DNA interstrand crosslinks (ICLs), including FANCA-FANCW. Abnormalities in the FANCM genes are associated with female infertility and FANCM 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 FANCM. This study aims to explore the pathogenic mechanisms of the FANCM 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 FANCM genes were constructed and introduced into 293T cells. The 293T cells transfected with wild-type and mutant human FANCM plasmids and pEGFP-C1 empty vector plasmids were designated as the EGFP FANCM-WT group, the EGFP FANCM-MUT 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 FANCM-WT group and the EGFP FANCM-MUT group to examine whether the variant affected FANCM's ability to localize on chromatin. Mitomycin C was used to induce ICLs damage in vitro in both the EGFP FANCM-WT group and the EGFP FANCM-MUT 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 FANCM 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 FANCM 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. In vitro 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 (P<0.01), indicating a statistically significant impairment of DNA damage repair capability caused by this variant.
    Conclusions The homozygous variant in the FANCM 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.


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