Bi-allelic variants in KCNU1 cause impaired acrosome reactions and male infertility
Bi-allelic variants in KCNU1 cause impaired acrosome reactions and male infertility
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DOI：
10.1093/humrep/deac102
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发表时间：
2022-05-11
影响因子：
6.1
通讯作者：
Kuang, Yanping
中科院分区：
文献类型：
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作者：
Liu, Ruyi;Yan, Zheng;Kuang, Yanping
研究问题：是否有新的遗传因素导致精子数量和形态正常的男性不育？我们发现了KCNU1的双等位基因变异，并证实它是一个主要由精子顶体反应受损（ARs）引起的男性不育的新致病基因。到目前为止，影响男性患者表现出正常精子数量和形态的潜在遗传决定因素在很大程度上是未知的。钾/钙激活通道亚家族U成员1 （KCNU1）是精子特异性钾通道。研究设计、大小、持续时间：研究人员招募了126例典型弱精子症或受精失败的雄性不育个体，研究对象为2015年至2020年来自两个近亲家庭的2例不育雄性；进行全外显子组测序和纯合子作图。我们在KCNU1中发现了一个纯合子错义变体（C . 2144a >G, p.His715Arg）和一个纯合子供体剪接位点变体（C .1295 + 3A>C, p.Val405Glyfs*8）。然后，我们在2020年9月创建了一个敲入（KI）小鼠模型，现在已经进行了功能研究和可能的治疗策略。对象/材料、环境、方法从上海交通大学附属上海第九医院招募不孕不育患者。从外周血中提取受影响个体的基因组DNA。利用全外显子组测序、纯合子作图和计算机分析筛选和鉴定KCNU1变异，并通过Sanger测序确认变异。我们利用C57BL/6N小鼠构建KI小鼠模型，模拟KI小鼠体内生殖表型。采用免疫印迹法、AR法和免疫荧光染色进行功能实验。最后，我们进行IVF和ICSI来探讨治疗策略。我们在2个不育男性的KCNU1基因中发现了一个纯合错义变异（C . 2144a >G, p.His715Arg）和一个纯合供体剪接位点变异（C .1295 + 3A>C, p.Val405Glyfs*8）。我们证明了剪接位点变异影响了KCNU1正常的选择性剪接，从而导致KCNU1功能的丧失。同时，错义致病变异降低了受影响个体和KI小鼠模型精子中的KCNU1蛋白水平，导致ARs受损和男性不育。胞浆内单精子注射能够弥补这些缺陷。KCNU1的确切分子机制和通路需要在未来进一步探索。这是第一份建立KCNU1缺乏与男性不育之间因果关系的报告，证实了KCNU1在人类生殖中的关键作用。我们的发现扩大了我们对在人类精子AR中起关键作用的基因的认识，并为不孕症提供了新的遗传标记。研究经费/竞争利益(S)本工作由SHIPM-pi基金（no. 5）资助。国家自然科学基金项目（81725006、81771649、81822019、81771581、81971450、81971382、82001538、82071642）。作者声明无利益冲突。
STUDY QUESTION Are there new genetic factors responsible for male infertility with normal sperm quantity and morphology? SUMMARY ANSWER We identified the bi-allelic variants in KCNU1 and confirmed it a novel pathogenetic gene for male infertility mainly due to impaired sperm acrosome reactions (ARs). WHAT IS KNOWN ALREADY Until now, the underlying genetic determinants for male affected individuals exhibiting normal sperm quantity and morphology have been largely unknown. Potassium/calcium-activated channel subfamily U member 1 (KCNU1) is a sperm-specific potassium channel. The Kcnu1 null mutation in male mice causes infertility due to the impaired progressive motility and AR. STUDY DESIGN, SIZE, DURATION We recruited a cohort of 126 male infertility individuals with typical asthenospermia or fertilization failure and focused on two infertile males from two consanguineous families from 2015 to 2020; whole-exome sequencing and homozygosity mapping were performed. We identified a homozygous missense variant (c.2144A>G, p.His715Arg) and a homozygous donor splice-site variant (c.1295 + 3A>C, p.Val405Glyfs*8) in KCNU1. Then, we generated a knock-in (KI) mouse model in September 2020 and have now carried out functional studies and possible treatment strategies. PARTICIPANTS/MATERIALS, SETTING, METHODS The affected individuals with infertility were recruited from the Shanghai Ninth Hospital affiliated to Shanghai Jiao Tong University. Genomic DNA from the affected individual was extracted from peripheral blood. Whole-exome sequencing, homozygosity mapping and in silico analyses were used to screen and identify KCNU1 variants, and the variants were confirmed by Sanger sequencing. We used C57BL/6N mouse to construct KI mouse model to mimic the reproductive phenotype in vivo. We performed functional experiments by western blotting, AR assay and immunofluorescent Staining. Finally, we performed IVF and ICSI to explore the treatment strategies. MAIN RESULTS AND THE ROLE OF CHANCE We identified a homozygous missense variant (c.2144A>G, p.His715Arg) and a homozygous donor splice-site variant (c.1295 + 3A>C, p.Val405Glyfs*8) in KCNU1 in two infertile males. We demonstrated that the splice-site variant affected normal alternative splicing of KCNU1, thus leading to the loss of function of KCNU1. Meanwhile, the missense pathogenic variant reduced the KCNU1 protein levels in sperm of both the affected individual and the KI mouse model, resulting in impaired ARs and male infertility. Intracytoplasmic sperm injection was able to rescue the deficiencies. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION The exact molecular mechanism of KCNU1 and pathways need to be further explore in the future. WIDER IMPLICATIONS OF THE FINDINGS This is the first report that establishes a causal relationship between KCNU1 deficiency and male infertility, confirming the critical role of KCNU1 in human reproduction. Our findings expand our knowledge of the genes that play critical roles in the human sperm AR and provide a new genetic marker for infertility. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the SHIPM-pi fund no. JY201801 from the Shanghai Institute of Precision Medicine, Ninth People's Hospital Shanghai Jiao Tong University School of Medicine, the National Natural Science Foundation of China (81725006, 81771649, 81822019, 81771581, 81971450, 81971382, 82001538 and 82071642). The authors declare no conflict of interest.