目的：原发性纤毛运动障碍（PCD）是一种重要的遗传性呼吸系统疾病，其病理特征是动纤毛的结构或功能异常，具有很高的遗传异质性和临床异质性。由于地区、种族及数据来源的不同，当前报道的发病率在1/40,000至1/10,000之间，由于漏诊率较高，实际发病率可能高于文献报道。虽然目前已报道了50多个PCD致病基因可以解释75%左右患者的发病原因，但仍有许多潜在的致病基因尚待发现和验证。本研究通过收集散发性PCD患者的病例队列，对患者的致病基因及其变异进行筛查和验证，旨在扩展中国人群中的PCD致病基因变异谱和表型谱。此外，本课题还将对新发现的潜在致病基因ANKRD42进行详细的功能鉴定和分子机制研究，以深入理解其在PCD中的作用。

方法：通过提取基因组DNA，并应用全外显子组测序（WES）与Sanger测序技术鉴定潜在致病变异，并进行生物信息学分析。此外，使用透射电镜和高速视频显微镜观察和分析纤毛的超微结构及摆动频率。通过体外细胞实验验证了DNAAF3和CCDC40基因的新变异的致病性。同时，通过建立小鼠原代气管上皮细胞（MBEC）气-液界面（ALI）培养模型，利用RNA荧光原位杂交、免疫荧光和实时荧光定量PCR技术评估Ankrd42基因对纤毛细胞分化的影响，并通过蛋白质印迹实验研究其在自噬调控中的作用。

结果：（1）本研究共收集了28例散发性PCD患者，通过WES检测找到了11个致病基因上的24个致病变异，其中有12个为新变异。（2）体外minigene实验发现DNAAF3：c.1364G>A（p.G455D）和CCDC40：c.1806+1G>T影响了转录本的剪接。（3）RNA荧光原位杂交技术显示Ankrd42具有显著的空间表达差异，主要分布纤毛细胞内。免疫荧光实验验证突变小鼠MBEC中纤毛细胞数量减少。实时荧光定量PCR提示Ankrd42的表达随着纤毛分化而逐渐升高。与野生型小鼠相比，突变小鼠MBEC分化9d、13d时Foxj1表达量下降，而Ccno和Mcidas的表达上升。免疫印迹技术实验验证ALI分化21d的突变小鼠MBEC LC3-II和p62的表达水平均显著增高，且在自噬溶酶体抑制剂处理后LC3-II 和 p62 的蛋白质水平无明显增加，显示自噬通量抑制。

结论：（1）本研究共发现了12个新PCD致病变异，扩展了中国PCD人群的基因突变谱。（2）DNAAF3:c.1364G>A（p.G455D）和CCDC40:c.1806+1G>T变异会导致剪接异常从而导致产生提前终止密码子，是PCD新的致病变异。（3）可疑致病新基因Ankrd42可能通过影响纤毛生成及分化过程的关键基因Ccno、Foxj1、Mcidas的表达，导致动纤毛的数量减少，引起呼吸道动纤毛粘液清除能力下降而导致呼吸系统症状。Ankrd42抑制自噬通量可能会阻碍纤毛的发生。

Objective: Primary ciliary dyskinesia (PCD) is a significant genetic respiratory disease characterized by structural or functional abnormalities in motile cilia, exhibiting considerable genetic and clinical heterogeneity. Due to regional, ethnic, and data source variations, the reported incidence rates range from 1/40,000 to 1/10,000, potentially underestimated due to high rates of misdiagnosis. Although over 50 PCD-causative genes have been identified, accounting for approximately 75% of cases, many potential pathogenic genes remain undiscovered and unverified. This study aims to expand the spectrum of genetic mutations and phenotypes in the Chinese population by collecting and analyzing data from sporadic PCD patients, focusing on identifying and validating the pathogenic genes and their variants. Additionally, the study will conduct detailed functional characterization and molecular mechanism investigations of the newly identified potential pathogenic gene, ANKRD42, to deepen understanding of its role in PCD.

Methods: Genomic DNA was extracted and subjected to Whole Exome Sequencing(WES) and Sanger sequencing to identify potential pathogenic variants. Additionally, transmission electron microscopy and high-speed video microscopy were utilized to observe and analyze the ultrastructure and motility frequency of cilia. The pathogenicity of new variants in the DNAAF3 and CCDC40 genes was validated through in vitro cellular experiments. Concurrently, an air-liquid interface (ALI) culture model of mouse tracheal epithelial cells (MBEC) was established to assess the impact of the Ankrd42 gene on ciliary cell differentiation using RNA fluorescence in situ hybridization, immunofluorescence and real-time quantitative PCR techniques. The role of Ankrd42 in autophagy regulation was further studied through Western blot assays.

Results:(1)This study collected data from 28 sporadic cases of PCD, identifying 24 pathogenic variants across 11 causative genes via Whole Exome Sequencing (WES), including 12 novel variants. In vitro minigene assays revealed that variants DNAAF3: c.1364G>A:p.G455D and CCDC40: c.1806+1G>T affected transcript splicing.(2)RNA fluorescence in situ hybridization demonstrated significant spatial expression differences of Ankrd42, with notably higher expression in the ciliated cells of the mouse trachea. Immunofluorescence experiments confirmed a reduction in the number of ciliated cells in mutant MBECs. Real-time quantitative PCR indicated that the expression of Ankrd42 increased with ciliary differentiation. Compared to wild-type mice, the expression of Foxj1 decreased while Ccno and Mcidas increased during the 9th and 13th days of differentiation in mutant MBECs. Western blot assays conducted on mutant MBECs at 21st days of differentiation showed a significant increase in LC3-II and p62 levels, with no noticeable change in LC3-II and p62 protein levels following treatment with autophagy-lysosome inhibitors, indicating inhibited autophagy flux.

Conclusions:(1)In this study, a total of 12 novel pathogenic variants of PCD were identified, expanding the spectrum of genetic mutations in the Chinese population. (2)Variants DNAAF3:c.1364G>A (p.G455D) and CCDC40: c.1806+1G>T lead to splicing anomalies resulting in premature stop codons, representing novel pathogenic variants for PCD. (3) The suspected pathogenic gene Ankrd42 may decrease the number of motile cilia by affecting the expression of key genes involved in ciliary generation and differentiation, such as Ccno, Foxj1, and Mcidas, leading to reduced mucociliary clearance in the respiratory tract and subsequent respiratory symptoms. The inhibition of autophagy flux by Ankrd42 might hinder ciliogenesis.