Abstract：
TTC12 is a cytoplasmic and centromere-localized protein that plays a role in the proper assembly of dynein arm complexes in motile cilia in both respiratory cells and sperm flagella. This finding underscores its significance in cellular motility and function. However, the wide role of TTC12 in human spermatogenesis-associated primary ciliary dyskinesia (PCD) still needs to be elucidated. Whole-exome sequencing (WES) and Sanger sequencing were performed to identify potentially pathogenic variants causing PCD and multiple morphological abnormalities of sperm flagella (MMAF) in an infertile Pakistani man. Diagnostic imaging techniques were used for PCD screening in the patient. Real-time polymerase chain reaction (RT‒PCR) was performed to detect the effect of mutations on the mRNA abundance of the affected genes. Papanicolaou staining and scanning electron microscopy (SEM) were carried out to examine sperm morphology. Transmission electron microscopy (TEM) was performed to examine the ultrastructure of the sperm flagella, and the results were confirmed by immunofluorescence staining. Using WES and Sanger sequencing, a novel homozygous missense variant (c.C1069T; p.Arg357Trp) in TTC12 was identified in a patient from a consanguineous family. A computed tomography scan of the paranasal sinuses confirmed the symptoms of the PCD. RT-PCR showed a decrease in TTC12 mRNA in the patient\'s sperm sample. Papanicolaou staining, SEM, and TEM analysis revealed a significant change in shape and a disorganized axonemal structure in the sperm flagella of the patient. Immunostaining assays revealed that TTC12 is distributed throughout the flagella and is predominantly concentrated in the midpiece in normal spermatozoa. In contrast, spermatozoa from patient deficient in TTC12 showed minimal staining intensity for TTC12 or DNAH17 (outer dynein arms components). This could lead to MMAF and result in male infertility. This novel TTC12 variant not only illuminates the underlying genetic causes of male infertility but also paves the way for potential treatments targeting these genetic factors. This study represents a significant advancement in understanding the genetic basis of PCD-related infertility.
摘要：
TTC12是一种细胞质和着丝粒定位蛋白，在呼吸细胞和精子鞭毛的活动纤毛中动力蛋白臂复合物的正确组装中起作用。这一发现强调了其在细胞运动性和功能中的重要性。然而,TTC12在人类精子发生相关原发性纤毛运动障碍(PCD)中的广泛作用仍有待阐明.进行了全外显子组测序（WES）和Sanger测序，以鉴定导致巴基斯坦不育男子PCD和精子鞭毛（MMAF）多种形态异常的潜在致病性变异。诊断成像技术用于患者的PCD筛查。进行实时聚合酶链反应（RT-PCR）以检测突变对受影响基因mRNA丰度的影响。进行巴氏染色和扫描电子显微镜（SEM）检查精子形态。透射电子显微镜（TEM）检查精子鞭毛的超微结构，结果经免疫荧光染色证实。使用WES和Sanger测序，一个新的纯合错义变体(c.TTC12中的C1069T；p.Arg357Trp）在一个近亲家庭的患者中被鉴定。鼻旁窦的计算机断层扫描扫描证实了PCD的症状。RT-PCR显示患者精子样本中TTC12mRNA减少。巴氏染色，SEM,和TEM分析显示，患者精子鞭毛的形状发生了显着变化，轴突结构混乱。免疫染色分析表明，TTC12分布在整个鞭毛中，并且主要集中在正常精子的中段中。相比之下,来自缺乏TTC12的患者的精子对TTC12或DNAH17（外部动力蛋白臂成分）的染色强度最小。这可能导致MMAF并导致男性不育。这种新型TTC12变体不仅阐明了男性不育的潜在遗传原因，而且为针对这些遗传因素的潜在治疗铺平了道路。这项研究代表了在理解PCD相关不孕症的遗传基础方面的重大进展。
