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

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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.