Institute of Pediatrics, Children's Hospital of Fudan University, The Institutes of Biomedical Sciences, The State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China.
Department of Assisted Reproduction, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Hum Reprod. 2022 Jun 30;37(7):1394-1405. doi: 10.1093/humrep/deac102.
Are there new genetic factors responsible for male infertility with normal sperm quantity and morphology?
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).
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.
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.
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LIMITATIONS, REASONS FOR CAUTION: The exact molecular mechanism of KCNU1 and pathways need to be further explore in the future.
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.
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是否存在导致精子数量和形态正常的男性不育的新遗传因素？
我们在 KCNU1 中鉴定出双等位基因变异体，并证实其主要由于精子顶体反应（AR）受损导致男性不育的一种新的致病基因。
到目前为止，大量表现为精子数量和形态正常的男性不育个体的潜在遗传决定因素仍知之甚少。钾/钙激活通道亚家族 U 成员 1（KCNU1）是一种精子特异性钾通道。在雄性小鼠中，Kcnu1 缺失突变导致精子的渐进性运动和 AR 受损，从而引起不育。
研究设计、大小和持续时间：我们招募了 126 名具有典型弱精症或受精失败的男性不育患者，并于 2015 年至 2020 年期间重点关注了来自两个血缘关系家族的两名不育男性；进行了全外显子测序和纯合子作图。我们在 KCNU1 中鉴定出一个纯合错义变异（c.2144A>G，p.His715Arg）和一个纯合供体位点变异（c.1295+3A>C，p.Val405Glyfs*8）。然后，我们于 2020 年 9 月构建了 KI 小鼠模型，并进行了功能研究和可能的治疗策略。
参与者/材料、设置、方法：从上海交通大学附属第九人民医院招募患有不育症的个体，提取外周血中的个体基因组 DNA。使用全外显子测序、纯合子作图和计算机分析筛选和鉴定 KCNU1 变异体，并通过 Sanger 测序进行验证。我们使用 C57BL/6N 小鼠构建 KI 小鼠模型，以模拟体内生殖表型。通过 Western blot、AR 测定和免疫荧光染色进行功能实验。最后，我们进行了体外受精和卵胞浆内单精子注射，以探索治疗策略。
我们在两名不育男性中鉴定出 KCNU1 中的一个纯合错义变异（c.2144A>G，p.His715Arg）和一个纯合供体位点变异（c.1295+3A>C，p.Val405Glyfs*8）。我们证明，供体位点变异影响了 KCNU1 的正常剪接，从而导致 KCNU1 功能丧失。同时，错义致病性变异降低了个体和 KI 小鼠模型精子中的 KCNU1 蛋白水平，导致 AR 受损和男性不育。胞质内精子注射能够挽救这些缺陷。
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局限性、谨慎的原因：KCNU1 的确切分子机制和途径需要进一步探索。
这是第一个建立 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。我们的研究结果扩展了我们对在人类精子 AR 中起关键作用的基因的认识，并为不育症提供了一个新的遗传标志物。
研究基金/利益冲突：这项工作得到了上海精准医学研究所 SHIPM-pi 基金（来自上海交通大学医学院第九人民医院）、国家自然科学基金（81725006、81771649、81822019、81771581、81971450、81971382、82001538 和 82071642）的支持。作者声明没有利益冲突。
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