Identification and characterization of novel genes causative for primary ciliary dyskinesia

Project Summary Primary Ciliary Dyskinesia (PCD) is a rare genetic disorder that affects multiple tissues leading to increased risk for situs inversus, infertility, hydrocephalus and respiratory disease. In the lung, PCD phenotypes are driven by the failure of multi-ciliated cells (MCCs) to generate proper mucociliary flow leading to chronic respiratory infections. To date the combined efforts of many groups have identified over 50 genes causative for PCD. However, the diagnostic tools for identifying PCD patients are time consuming, costly, and phenotypically constrained, likely leading to significant underdiagnosis. Cilia are complex molecular machines consisting of over a thousand proteins that contribute to their motility and function. To function properly, MCCs must: 1.) generate over 100 centrioles that will become the basal bodies of their motile cilia; 2.) dock those centrioles with the apical surface; 3.) nucleate the proper number and length of cilia; 4.) generate proper cilia motility; and 5.) coordinate the timing and orientation of cilia motility. Defects in any of these steps will lead to a decrease in mucociliary flow yet the diagnosis of PCD has been primarily focused on cilia motility. We have developed the ciliated epithelia of the Xenopus embryonic skin as a powerful system to address numerous aspects of ciliary function. Importantly, we have developed quantifiable assays for each step of MCC formation and can identify the underlying cause of a decrease in mucociliary flow. Our combinatorial approach will facilitate a detailed analysis of multiple genes that are either linked to PCD without a clear mechanism (e.g. AK7) or have only been predicted to cause PCD (e.g. Saxo2, Meig1, Rsph14). Additionally, we will perform a tiered CRISPR/Cas9 based genetic screen to identify and functionally characterize novel PCD causative genes. Our goal is to generate an extended list of PCD causing genes that can be included in genetic testing to facilitate the diagnosis of PCD. Additionally, we hope to develop clearly defined subclasses of the disease, marked by loss of motility, loss of cilia number or loss of cilia polarity.