The role of cytoplasmic pre-assembly of axonemal components in primary ciliary dyskinesia

原发性纤毛运动障碍（PCD; MIM#242650）是一种由纤毛运动功能障碍引起的遗传、功能和超微结构水平异质性的罕见疾病。由于粘膜纤毛清除率降低，PCD个体遭受慢性破坏性下呼吸道和上呼吸道感染，从而导致支气管扩张和慢性肺衰竭。其他典型的临床症状包括新生儿呼吸窘迫、慢性中耳炎、体轴侧边缺损、先天性心脏缺损、不孕症和罕见的脑积水。动力臂是纤毛运动所必需的。动力蛋白臂的成分首先在纤毛/鞭毛细胞的细胞质中预先组装成多蛋白复合物，然后在纤毛发生期间传递给轴突。这个过程是由进化上保守的蛋白质，即动力蛋白轴突组装因子（DNAAFs）调节的。在第一个资助期，我们发现了两种新型DNAAFs （pihdh3 /DNAAF6和C11orf70/CFAP300）的遗传缺陷，这些缺陷导致动力蛋白臂的细胞质预组装缺陷，导致纤毛运动障碍，气道粘膜纤毛清除紊乱，左右体不对称随机化和男性不育。此外，我们发现动力蛋白臂细胞质预组装缺陷影响精子鞭毛长度。通过检测导致PCD的PIH1D3/DNAAF6突变，我们能够描述第一个非综合征性x连锁PCD变体。由于x染色体失活的随机化，我们观察到在女性杂合子pihh1d3 /DNAAF6突变携带者中约有。50%的呼吸细胞缺乏动力蛋白臂。然而，到目前为止，对女性杂合突变携带者的临床状况一无所知。就治疗方法而言，重要的是要找出有多少呼吸细胞必须有效地清除气道的纤毛，以及哪些其他因素参与了动力蛋白臂的细胞质预组装。因此，本提案的目标如下：1)鉴定携带pihd3 /DNAAF6或其他DNAAF基因突变的其他家族2)分子，PIH1D3/DNAAF6基因突变在女性携带者和半合子突变体以及其他dnaaf中引起的纤毛缺陷的细胞和临床特征3)由动力蛋白臂的细胞质预组装受到干扰引起的纤毛和鞭毛长度缺陷的特征4)新型DNAAF缺陷的分子和细胞特征5)新发现的和已知的dnaaf之间的蛋白质相互作用的特征这一建议的结果将i)扩大对临床表型和病程，ii)进一步解读呼吸细胞和精子细胞中动力蛋白臂的细胞质预组装过程，iii)导致新的DNAAF基因的鉴定。该项目的成果将改善PCD的诊断和临床护理。

Primary Ciliary Dyskinesia (PCD; MIM#242650) is a genetically, functionally and on ultrastructural level heterogeneous group of rare diseases caused by dysfunction of motile cilia. Due to reduced mucociliary clearance PCD individuals suffer from chronic destructive lower and upper airway infections, which cause bronchiectasis and chronic lung failure. Other typical clinical symptoms include respiratory distress of the neonate, chronic otitis media, laterality defects of the body axis, congenital heart defects, infertility and rarely hydrocephalus. Dynein arms are necessary for ciliary motility. The components of dynein arms are first pre-assembled to a multi-protein complex in the cytoplasm of ciliated/ flagellated cells, and then delivered to the axoneme during ciliogenesis. This process is regulated by evolutionarily conserved proteins referred to as dynein axonemal assembly factors (DNAAFs).During the first funding period, we identified genetic defects in two novel DNAAFs (PIH1D3/DNAAF6 and C11orf70/CFAP300) that result in defective cytoplasmic pre-assembly of dynein arms causing ciliary immotility, disturbed mucociliary clearance of the airway, randomization of the left/right body asymmetry and male infertility. Additionally, we found that defective cytoplasmic pre-assembly of dynein arms affects sperm flagella length. By detecting mutations in PIH1D3/DNAAF6 that cause PCD, we were able to describe the first non-syndromic X-linked PCD variant. Due to randomization of X-chromosomal inactivation, we observed in female heterozygous PIH1D3/DNAAF6 mutation carriers that approx. 50% of respiratory cells show absence of dynein arms. However, so far nothing is known about the clinical status of female heterozygous mutation carriers. In terms of therapeutic approaches, it is important to find out how many respiratory cells have to be functional for effective ciliary clearance of the airways and which additional factors are involved in the cytoplasmic pre-assembly of dynein arms. Therefore, the objectives of this current proposal are as follows:1) Identification of additional families carrying mutations in PIH1D3/DNAAF6 or in other DNAAF genes2) Molecular, cellular and clinical characterization of the ciliary defect caused by mutations in PIH1D3/DNAAF6 in female carriers and hemizygous mutants and in other DNAAFs3) Characterization of the ciliary and flagellar length defects caused by disturbed cytoplasmic preassembly of dynein arms 4) Molecular and cellular characterization of novel DNAAF defects 5) Characterization of protein interactions between newly identified and known DNAAFsThe results of this proposal will i) expand knowledge of the clinical phenotype and disease course, ii) further decipher the process of cytoplasmic pre-assembly of dynein arms in respiratory as well as sperm cells, and iii) lead to the identification of new DNAAF genes. The results of this project will improve diagnostics as well as clinical care for PCD.