Westerich Kim Joana, Reinecke Solveig, Emich Jana, Wyrwoll Margot Julia, Stallmeyer Birgit, Meyer Matthias, Oud Manon S, Fietz Daniela, Pilatz Adrian, Kliesch Sabine, Reichman-Fried Michal, Tarbashevich Katsiaryna, Limon Tamara, Stehling Martin, Friedrich Corinna, Tüttelmann Frank, Raz Erez
Institute of Cell Biology, Center for Molecular Biology of Inflammation, University of Münster, Münster, Germany.
Institute of Reproductive Genetics, University of Münster, Münster, Germany.
Hum Reprod. 2023 Apr 3;38(4):655-670. doi: 10.1093/humrep/dead031.
Is the vertebrate protein Dead end (DND1) a causative factor for human infertility and can novel in vivo assays in zebrafish help in evaluating this?
Combining patient genetic data with functional in vivo assays in zebrafish reveals a possible role for DND1 in human male fertility.
About 7% of the male population is affected by infertility but linking specific gene variants to the disease is challenging. The function of the DND1 protein was shown to be critical for germ cell development in several model organisms but a reliable and cost-effective method for evaluating the activity of the protein in the context of human male infertility is still missing.
STUDY DESIGN, SIZE, DURATION: Exome data from 1305 men included in the Male Reproductive Genomics cohort were examined in this study. A total of 1114 of the patients showed severely impaired spermatogenesis but were otherwise healthy. Eighty-five men with intact spermatogenesis were included in the study as controls.
PARTICIPANTS/MATERIALS, SETTING, METHODS: We screened the human exome data for rare, stop-gain, frameshift, splice site, as well as missense variants in DND1. The results were validated by Sanger sequencing. Immunohistochemical techniques and, when possible, segregation analyses were performed for patients with identified DND1 variants. The amino acid exchange in the human variant was mimicked at the corresponding site of the zebrafish protein. Using different aspects of germline development in live zebrafish embryos as biological assays, we examined the activity level of these DND1 protein variants.
In human exome sequencing data, we identified four heterozygous variants in DND1 (three missense and one frameshift variant) in five unrelated patients. The function of all of the variants was examined in the zebrafish and one of those was studied in more depth in this model. We demonstrate the use of zebrafish assays as a rapid and effective biological readout for evaluating the possible impact of multiple gene variants on male fertility. This in vivo approach allowed us to assess the direct impact of the variants on germ cell function in the context of the native germline. Focusing on the DND1 gene, we find that zebrafish germ cells, expressing orthologs of DND1 variants identified in infertile men, failed to arrive correctly at the position where the gonad develops and exhibited defects in cell fate maintenance. Importantly, our analysis facilitated the evaluation of single nucleotide variants, whose impact on protein function is difficult to predict, and allowed us to distinguish variants that do not affect the protein's activity from those that strongly reduce it and could thus potentially be the primary cause for the pathological condition. These aberrations in germline development resemble the testicular phenotype of azoospermic patients.
LIMITATIONS, REASONS FOR CAUTION: The pipeline we present requires access to zebrafish embryos and to basic imaging equipment. The notion that the activity of the protein in the zebrafish-based assays is relevant for the human homolog is well supported by previous knowledge. Nevertheless, the human protein may differ in some respects from its homologue in zebrafish. Thus, the assay should be considered only one of the parameters used in defining DND1 variants as causative or non-causative for infertility.
Using DND1 as an example, we have shown that the approach described in this study, relying on bridging between clinical findings and fundamental cell biology, can help to establish links between novel human disease candidate genes and fertility. In particular, the power of the approach we developed is manifested by the fact that it allows the identification of DND1 variants that arose de novo. The strategy presented here can be applied to different genes in other disease contexts.
STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the German Research Foundation, Clinical Research Unit, CRU326 'Male Germ Cells'. There are no competing interests.
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脊椎动物蛋白Dead end（DND1）是否是人类不育的致病因素，斑马鱼体内新的检测方法能否有助于对此进行评估？
将患者基因数据与斑马鱼体内功能检测相结合，揭示了DND1在人类男性生育中的可能作用。
约7%的男性人口受不育影响，但将特定基因变异与该疾病联系起来具有挑战性。DND1蛋白的功能在几种模式生物中已被证明对生殖细胞发育至关重要，但仍缺少一种可靠且经济高效的方法来评估该蛋白在人类男性不育背景下的活性。
研究设计、规模、持续时间：本研究检查了男性生殖基因组队列中1305名男性的外显子组数据。共有1114名患者精子发生严重受损，但其他方面健康。85名精子发生正常的男性作为对照纳入研究。
参与者/材料、环境、方法：我们在人类外显子组数据中筛选DND1的罕见、截短增益、移码、剪接位点以及错义变异。结果通过桑格测序验证。对鉴定出DND1变异的患者进行免疫组织化学技术检测，并在可能的情况下进行分离分析。在斑马鱼蛋白的相应位点模拟人类变异中的氨基酸交换。利用活斑马鱼胚胎生殖系发育的不同方面作为生物学检测方法，我们检测了这些DND1蛋白变异的活性水平。
在人类外显子组测序数据中，我们在5名无亲缘关系的患者中鉴定出DND1的4个杂合变异（3个错义变异和1个移码变异）。在斑马鱼中检测了所有变异的功能，其中一个变异在该模型中进行了更深入的研究。我们证明了斑马鱼检测作为一种快速有效的生物学读数，可用于评估多个基因变异对男性生育的可能影响。这种体内方法使我们能够在天然生殖系背景下评估变异对生殖细胞功能的直接影响。聚焦于DND1基因，我们发现表达在不育男性中鉴定出的DND1变异直系同源物的斑马鱼生殖细胞未能正确到达性腺发育的位置，并在细胞命运维持方面表现出缺陷。重要的是，我们的分析有助于评估单核苷酸变异，其对蛋白质功能的影响难以预测，并使我们能够区分不影响蛋白质活性的变异和强烈降低其活性的变异，因此后者可能是病理状况的主要原因。生殖系发育中的这些异常类似于无精子症患者的睾丸表型。
局限性、谨慎原因：我们提出的流程需要获得斑马鱼胚胎和基本成像设备。基于斑马鱼的检测中蛋白质的活性与人类同源物相关这一观点得到了先前知识的有力支持。然而，人类蛋白质可能在某些方面与其在斑马鱼中的同源物不同。因此，该检测应仅被视为将DND1变异定义为不育致病或非致病的参数之一。
以DND1为例，我们表明本研究中描述的方法，依赖于临床发现与基础细胞生物学之间的桥梁作用，有助于在新的人类疾病候选基因与生育力之间建立联系。特别是，我们开发的方法的强大之处在于它能够识别从头出现的DND1变异。这里提出的策略可应用于其他疾病背景下的不同基因。
研究资金/利益冲突：本研究由德国研究基金会临床研究单元CRU326“男性生殖细胞”资助。不存在利益冲突。
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