背景:能动纤毛功能障碍,包括呼吸道纤毛和精子鞭毛,通常导致原发性纤毛运动障碍和男性不育或人类生育力低。由于纤毛轴突的超微结构异常,LRRC6的遗传缺陷与原发性纤毛运动障碍和弱精子症有关。
目的:鉴定与精子鞭毛的多种形态异常和男性不育相关的LRRC6基因的新突变,并探讨其潜在的分子机制。
方法:通过全外显子组测序鉴定LRRC6突变,并用Sanger测序证实。巴氏染色,扫描,用透射电镜观察精子的形态和超微结构特征。进行进一步的串联质量标记蛋白质组学分析以探索突变的作用并通过免疫染色和蛋白质印迹证实。卵胞浆内单精子注射用于具有双等位基因LRRC6突变的男性的辅助生殖治疗。
结果:在这项研究中,我们在一个近亲家族中发现了一个新的纯合LRRC6突变,以弱精子症和原发性纤毛运动障碍为特征。进一步的精液参数和形态学分析表明,新的LRRC6突变导致精子鞭毛长度显著减少,精子进行性运动性参数的降低,精子超微结构异常。具体来说,缺乏外部动力蛋白臂和内部动力蛋白臂,透射电镜观察到鞭毛中段线粒体鞘不完整。此外,串联质量标记蛋白质组学分析显示,从携带LRRRC6突变的患者获得的精子表现出与动力蛋白轴突臂的组装和功能相关的蛋白质表达水平显着降低。功能分析显示,这种新的LRRC6突变破坏了富含亮氨酸的重复序列6蛋白的功能,进而影响动力蛋白臂蛋白和富含亮氨酸重复序列的6相互作用蛋白CCDC40,SPAG1和ZMYND10的表达。最后,我们报道了先证者的女性伴侣通过辅助生殖技术用卵胞浆内单精子注射成功怀孕。
结论:这项研究强调了近亲家族中一种新的纯合LRRC6突变的鉴定及其对精子进行性运动性的影响,形态学,和精子动力学参数,这可以促进弱精子症的遗传诊断,并为未来的遗传咨询工作提供有价值的观点。
BACKGROUND: Dysfunction of motile cilia, including respiratory cilia and sperm flagella, typically leads to primary ciliary dyskinesia and male infertility or low fertility in humans. Genetic defects of LRRC6 have been associated with primary ciliary dyskinesia and asthenozoospermia due to abnormal ultrastructure of ciliated axonemes.
OBJECTIVE: To identify novel mutations of the LRRC6 gene related to multiple morphological abnormalities of the sperm flagella and male infertility and investigate the underlying molecular mechanisms involved.
METHODS: The LRRC6 mutations were identified by whole exome sequencing and confirmed with Sanger sequencing. Papanicolaou staining, scanning, and transmission electron microscopy were performed to investigate the morphological and ultrastructural characteristics of spermatozoa. Further tandem mass tagging proteomics analyses were performed to explore the effect of mutations and confirmed by immunostaining and western blotting. Intracytoplasmic sperm injection was applied for the assisted reproductive therapy of males harboring biallelic LRRC6 mutations.
RESULTS: In this study, we identified a novel homozygous LRRC6 mutation in a consanguineous family, characterized by asthenozoospermia and primary ciliary dyskinesia. Further Semen parameter and morphology analysis demonstrate that the novel LRRC6 mutation leads to a significant reduction in sperm flagella length, a decrease in sperm progressive motility parameters, and abnormalities of sperm ultrastructure. Specifically, the absence of outer
dynein arms and inner
dynein arms, and incomplete mitochondrial sheath in the flagellar mid-piece were observed by transmission electron microscopy. In addition, tandem mass tagging proteomics analysis revealed that spermatozoa obtained from patients harboring the LRRC6 mutation exhibited a significant decrease in the expression levels of proteins related to the assembly and function of
dynein axonemal arms. Functional analysis revealed that this novel LRRC6 mutation disrupted the function of the leucine-rich repeat containing 6 protein, which in turn affects the expression of the
dynein arm proteins and leucine-rich repeat containing 6-interacting proteins CCDC40, SPAG1, and ZMYND10. Finally, we reported a successful pregnancy through assisted reproductive technology with intracytoplasmic sperm injection in the female partner of the proband.
CONCLUSIONS: This study highlights the identification of a novel homozygous LRRC6 mutation in a consanguineous family and its impact on sperm progressive motility, morphology, and sperm kinetics parameters, which could facilitate the genetic diagnosis of asthenozoospermia and offer valuable perspectives for future genetic counseling endeavors.