Zeng Yang, Shi Juanzi, Xu Shiru, Shi Rong, Wu Tonghua, Li Hongyan, Xue Xia, Zhu Yuanchang, Chen Biaobang, Sang Qing, Wang Lei
Institute of Pediatrics, Children's Hospital of Fudan University, the Institutes of Biomedical Sciences, and the State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China.
Reproductive Center, Northwest Women's and Children's Hospital, Xi'an, Shaanxi, China.
Hum Reprod. 2022 Mar 1;37(3):612-620. doi: 10.1093/humrep/deab281.
Are mutations in MOS (MOS proto-oncogene, serine/threonine kinase) involved in early embryonic arrest in infertile women?
We identified mutations in MOS that may cause human female infertility characterized by preimplantation embryonic arrest (PREMBA), and the effects of the mutations in human embryonic kidney 293T (HEK293T cells) and mouse oocytes provided evidence for a causal relation between MOS and female infertility.
MOS, an activator of mitogen-activated protein kinase, mediates germinal vesicle breakdown and metaphase II arrest. Female MOS knockout mice are viable but sterile. Thus, MOS seems to be an important part of the mammalian cell cycle mechanism that regulates female meiosis.
STUDY DESIGN, SIZE, DURATION: Whole-exome sequencing, bioinformatics filtering analysis and genetic analysis were performed to identify two different biallelic mutations in MOS in two independent families. The infertile patients presenting with early embryonic arrest were recruited from October 2018 to June 2020.
PARTICIPANTS/MATERIALS, SETTING, METHODS: The female patients diagnosed with primary infertility were recruited from the reproduction centres of local hospitals. Genomic DNA from the affected individuals, their family members and healthy controls was extracted from peripheral blood. We performed whole-exome sequencing in patients diagnosed with PREMBA. Functional effects of the mutations were investigated in HEK293T cells by western blotting and in mouse oocytes by microinjection and immunofluorescence.
We identified the homozygous missense mutation c.285C>A (p.(Asn95Lys)) and the compound heterozygous mutations c.467delG (p.(Gly156Alafs18)) and c.956G>A (p.(Arg319His)) in MOS in two independent patients. The mutations c.285C>A (p.(Asn95Lys)) and c.467delG (p.(Gly156Alafs18)) reduced the protein level of MOS, and all mutations reduced the ability of MOS to phosphorylate its downstream target, extracellular signal-regulated kinase1/2. In addition, the identified mutations reduced the capacity of exogenous human MOS to rescue the metaphase II exit phenotype, and the F-actin cytoskeleton of mouse oocytes was affected by the patient-derived mutations.
LIMITATIONS, REASONS FOR CAUTION: Owing to the lack of in vivo data from patient oocytes, the exact molecular mechanism affected by MOS mutations and leading to PREMBA is still unknown and should be further investigated using knock-out or knock-in mice.
We identified recessive mutations in MOS in two independent patients with the PREMBA phenotype. Our findings reveal the important role of MOS during human oocyte meiosis and embryonic development and suggest that mutations in MOS may be precise diagnostic markers for clinical genetic counselling.
STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (81725006, 81822019, 81771581, 81971450, 81971382,82001538 and 82071642), the project supported by the Shanghai Municipal Science and Technology Major Project (2017SHZDZX01), the Project of the Shanghai Municipal Science and Technology Commission (19JC1411001), the Natural Science Foundation of Shanghai (19ZR1444500 and 21ZR1404800), the Shuguang Program of the Shanghai Education Development Foundation and the Shanghai Municipal Education Commission (18SG03), the Foundation of the Shanghai Health and Family Planning Commission (20154Y0162), the Capacity Building Planning Program for Shanghai Women and Children's Health Service and the collaborative innovation centre project construction for Shanghai Women and Children's Health. The authors have no conflicts of interest to declare.
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丝裂原活化蛋白激酶原癌基因（MOS）中的突变是否与不育女性的早期胚胎停滞有关？
我们在MOS中鉴定出可能导致以植入前胚胎停滞（PREMBA）为特征的人类女性不育的突变，并且这些突变在人胚肾293T细胞（HEK293T细胞）和小鼠卵母细胞中的作用为MOS与女性不育之间的因果关系提供了证据。
MOS是丝裂原活化蛋白激酶的激活剂，介导生发泡破裂和中期II停滞。雌性MOS基因敲除小鼠是存活的但不育。因此，MOS似乎是调节雌性减数分裂的哺乳动物细胞周期机制的重要组成部分。
研究设计、规模、持续时间：进行全外显子组测序、生物信息学过滤分析和遗传分析，以在两个独立家族中鉴定MOS中的两种不同的双等位基因突变。2018年10月至2020年6月招募了出现早期胚胎停滞的不育患者。
参与者/材料、环境、方法：从当地医院的生殖中心招募被诊断为原发性不育的女性患者。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。我们对诊断为PREMBA的患者进行了全外显子组测序。通过蛋白质印迹法在HEK293T细胞中以及通过显微注射和免疫荧光法在小鼠卵母细胞中研究了突变的功能影响。
我们在两名独立患者中鉴定出MOS中的纯合错义突变c.285C>A（p.(Asn95Lys)）以及复合杂合突变c.467delG（p.(Gly156Alafs18)）和c.956G>A（p.(Arg319His)）。突变c.285C>A（p.(Asn95Lys)）和c.467delG（p.(Gly156Alafs18)）降低了MOS的蛋白质水平，并且所有突变均降低了MOS磷酸化其下游靶点细胞外信号调节激酶1/2的能力。此外，鉴定出的突变降低了外源性人MOS挽救中期II退出表型的能力，并且患者来源的突变影响了小鼠卵母细胞的F-肌动蛋白细胞骨架。
局限性、谨慎的原因：由于缺乏来自患者卵母细胞的体内数据，受MOS突变影响并导致PREMBA的确切分子机制仍然未知，应使用基因敲除或基因敲入小鼠进一步研究。
我们在两名具有PREMBA表型的独立患者中鉴定出MOS中的隐性突变。我们的研究结果揭示了MOS在人类卵母细胞减数分裂和胚胎发育过程中的重要作用，并表明MOS中的突变可能是临床遗传咨询的精确诊断标志物。
研究资金/利益冲突：本研究得到中国国家自然科学基金（81725006、81822019、81771581、81971450、81971382、82001538和82071642）、上海市科技重大专项（2017SHZDZX01）、上海市科委项目（19JC1411001）、上海市自然科学基金（19ZR1444500和21ZR1404800）、上海教育发展基金会和上海市教育委员会的曙光计划（18SG03）、上海市卫生和计划生育委员会基金（20154Y0162）、上海市妇女儿童健康服务能力建设规划项目以及上海妇女儿童健康协同创新中心项目建设的支持。作者声明无利益冲突。
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