FBXO43 variants in patients with female infertility characterized by early embryonic arrest
FBXO43 variants in patients with female infertility characterized by early embryonic arrest
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DOI：
10.1093/humrep/deab131
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发表时间：
2021-05-30
影响因子：
6.1
通讯作者：
Sang, Qing
中科院分区：
文献类型：
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作者：
Wang, Weijie;Wang, Wenjing;Sang, Qing
研究问题：是否可以确定导致不孕患者早期胚胎停滞的新遗传因素以及致病变异的机制？简要答复：我们在不孕症患者中鉴定了F-box蛋白43基因（FBXO 43）的三种纯合变体，并对HEK 293 T细胞和小鼠卵母细胞中这些变体的影响进行了研究，为FBXO 43与女性不孕症之间的因果关系提供了证据。FBXO 43是一种后期促进复合物/细胞周期体的抑制剂，作为卵母细胞中细胞生长抑制因子的一种成分介导中期II停滞。雄性和雌性Fbxo 43基因敲除小鼠都能存活但不育。因此，FBXO 43似乎是调节雄性和雌性减数分裂的哺乳动物细胞周期机制的重要组成部分。到目前为止，只有一篇文章报道了一个纯合子FBXO 43变异与畸形精子症相关，但没有建立因果关系的功能证据。研究设计，规模，持续时间：对24例早期胚胎停止家系先证者进行全外显子组测序和纯合性定位，在两个独立的家族中鉴定出FBXO 43的两种不同的纯合变体。WES数据从另外950不孕妇女与早期胚胎停滞筛选纯合子和复合杂合子变异体FBXO 43，并与一个额外的纯合子变异体FBXO 43的第三个人被确定。方法：选择2016 - 08/2020 - 05诊断为早期胚胎停止的不孕症患者。对象/材料、地点、方法：原发性不孕症患者均来自当地医院生殖中心。从外周血中提取受影响个体、其家庭成员和健康对照的基因组DNA样本。使用WES、纯合性作图、计算机模拟分析和变体筛选鉴定FBXO 43变体。通过桑格测序确认所有的变体，并且通过蛋白质印迹在人胚肾（HEK）293 T细胞中和通过互补RNA注射在小鼠卵母细胞中研究变体的作用。（Glu500Serfs*2）），c.1747C>T（p.（Gln 583 *））和c.154delG（p.（Asp 52 Thrfs *30））-在三个独立的家庭。所有的纯合变体都降低了HEK 293 T细胞中FBXO 43的蛋白水平，并降低了其下游靶细胞周期蛋白B1的水平。此外，这些变异体降低了外源性人FBXO 43在FbxO 43敲除小鼠卵母细胞中挽救孤雌激活表型的能力。局限性，原因：由于缺乏来自患者卵母细胞的体内数据，确切的分子机制仍然未知，应使用敲除或敲入小鼠进一步研究。我们的研究已经确定了FBXO 43中涉及人类早期胚胎停滞的三种致病性变体。这些发现有助于我们理解FBXO 43在人类早期胚胎发育中的作用，并为女性不孕症提供了新的遗传标记。
STUDY QUESTION: Can any new genetic factors responsible for early embryonic arrest in infertile patients be identified, together with the mechanism of pathogenic variants?SUMMARY ANSWER: We identified three homozygous variants in the F-box protein 43 gene (FBXO43) in infertile patients and studies on the effects of the variants in HEK293T cells and mouse oocytes provided evidence for a causal relation between FBXO43 and female infertility.WHAT IS KNOWN ALREADY: FBXO43, an inhibitor of the anaphase-promoting complex/cyclosome, mediates Metaphase II arrest as a component of the cytostatic factor in oocytes. Both male and female Fbxo43 knockout mice are viable but sterile. FBXO43, therefore, appears to be an essential component of the mammalian cell-cycle machinery that regulates both male and female meiosis. Until now, only one article has reported a homozygous FBXO43 variant associated with teratozoospermia, but the causal relationship was not established with functional evidence.STUDY DESIGN, SIZE, DURATION: Whole-exome sequencing (WES) and homozygosity mapping were performed in 24 probands from consanguineous families who suffered from early embryonic arrest, and two different homozygous variants in FBXO43 were identified in two independent families. WES data from a further 950 infertile women with early embryonic arrest were screened for homozygous and compound heterozygous variants in FBXO43, and a third individual with an additional homozygous variant in FBXO43 was identified. The infertile patients presenting with early embryonic arrest were recruited from August 2016 to May 2020.PARTICIPANTS/MATERIALS, SETTING, METHODS: The women diagnosed with primary infertility were recruited from the reproduction centers of local hospitals. Genomic DNA samples from the affected individuals, their family members, and healthy controls were extracted from peripheral blood. The FBXO43 variants were identified using WES, homozygosity mapping, in silico analysis, and variant screening. All of the variants were confirmed by Sanger sequencing, and the effects of the variants were investigated in human embryonic kidney (HEK) 293T cells by western blotting and in mouse oocytes by complementary RNA injection.MAIN RESULTS AND THE ROLE OF CHANCE: We identified three homozygous variants in FBXO43 (NM_001029860.4)-namely, c.1490_1497dup (p.(Glu500Serfs*2)), c.1747C>T (p.(Gln583*)), and c.154delG (p.(Asp52Thrfs*30))-in three independent families. All of the homozygous variants reduced the protein level of FBXO43 and reduced the level of its downstream target Cyclin B1 in HEK293T cells. In addition, the variants reduced the ability of exogenous human FBXO43 to rescue the parthenogenetic activation phenotype in Fbxo43 knockdown mouse oocytes.LIMITATIONS, REASONS FOR CAUTION: Owing to the lack of in vivo data from the oocytes of patients, the exact molecular mechanism remains unknown and should be further investigated using knock out or knock in mice.WIDER IMPLICATIONS OF THE FINDINGS: Our study has identified three pathogenic variants in FBXO43 that are involved in human early embryonic arrest. These findings contribute to our understanding of the role of FBXO43 in human early embryonic development and provide a new genetic marker for female infertility.