As a type of severe asthenoteratospermia, multiple morphological abnormalities of the flagella (MMAF) are characterized by the presence of immotile spermatozoa with severe flagellar malformations. MMAF is a genetically heterogeneous disorder, and the known MMAF-associated genes can only account for approximately 60% of human MMAF cases. Here we conducted whole-exome sequencing and identified bi-allelic truncating mutations of the TTC29 (tetratricopeptide repeat domain 29) gene in three (3.8%) unrelated cases from a cohort of 80 MMAF-affected Han Chinese men. TTC29 is preferentially expressed in the testis, and TTC29 protein contains the tetratricopeptide repeat domains that play an important role in cilia- and flagella-associated functions. All of the men harboring TTC29 mutations presented a typical MMAF phenotype and dramatic disorganization in axonemal and/or other peri-axonemal structures. Immunofluorescence assays of spermatozoa from men harboring TTC29 mutations showed deficiency of TTC29 and remarkably reduced staining of intraflagellar-transport-complex-B-associated proteins (TTC30A and IFT52). We also generated a Ttc29-mutated mouse model through the use of CRISPR-Cas9 technology. Remarkably, Ttc29-mutated male mice also presented reduced sperm motility, abnormal flagellar ultrastructure, and male subfertility. Furthermore, intracytoplasmic sperm injections performed for Ttc29-mutated mice and men harboring TTC29 mutations consistently acquired satisfactory outcomes. Collectively, our experimental observations in humans and mice suggest that bi-allelic mutations in TTC29, as an important genetic pathogeny, can induce MMAF-related asthenoteratospermia. Our study also provided effective guidance for clinical diagnosis and assisted reproduction treatments.

中文翻译：

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TTC29中的双等位基因突变会导致人类和小鼠的睾丸无精子症导致男性不育。

作为一种严重的无精子症，鞭毛的多种形态异常（MMAF）的特征是存在不活跃的精子，伴有严重的鞭毛畸形。MMAF是一种遗传异质性疾病，已知的与MMAF相关的基因仅占人类MMAF病例的约60％。在这里，我们进行了全外显子组测序，在来自80名受MMAF影响的汉族人群中的三例（3.8％）无关病例中，确定了TTC29（四肽重复序列域29）基因的双等位基因截短突变。TTC29在睾丸中优先表达，TTC29蛋白包含四纤肽重复结构域，这些结构域在纤毛和鞭毛相关功能中起重要作用。携带TTC29突变的所有男性均表现出典型的MMAF表型，并在轴突和/或其他轴突周围结构中出现严重的组织紊乱。携带TTC29突变的男性精子的免疫荧光分析显示，TTC29缺乏，鞭毛内运输复合物B相关蛋白（TTC30A和IFT52）的染色显着降低。我们还通过使用CRISPR-Cas9技术生成了Ttc29突变的小鼠模型。值得注意的是，Ttc29突变的雄性小鼠还表现出精子活动力降低，鞭毛超微结构异常和雄性不育。此外，对Ttc29突变的小鼠和携带TTC29突变的男人进行的胞浆内精子注射始终获得令人满意的结果。总体而言，我们在人和小鼠中的实验观察表明，TTC29中的双等位基因突变，作为重要的遗传病原体，可诱发与MMAF相关的无精子症。我们的研究还为临床诊断和辅助生殖治疗提供了有效的指导。