Primary ciliary dyskinesia (PCD) is an inherited disease which is characterized by various ultrastructural abnormalities in the cilia lining the respiratory tract. The defects in respiratory cilia are believed to result in impaired mucociliary clearance, and affected individuals suffer from recurrent respiratory infections, including rhinitis, sinusitis, bronchitis and pneumonia. In addition, many PCD patients also suffer from chronic otitis media, and males are frequently infertile. Currently, there is no curative treatment available for PCD, and the genetic basis of the disease is unknown. The long-term objectives of this proposal are to identify the genetic basis of PCD and to understand the pathogenesis of the disease. To achieve these goals, the following specific aims are proposed: 1: To identify protein(s) which are absent or altered in cilia isolated from cultured PCD cells. 2: To develop and characterize molecular and biochemical probes specific for the protein(s) which are altered in the cilia of PCD cells. 3: To determine if the gene coding for the protein(s) absent or altered in cilia from a PCD patient is mutated. Airway epithelial cells isolated from normal individuals and individuals with PCD will be cultured in vitro using techniques which allow the cells to differentiate into a well-ciliated epithelium. Cellular proteins will be radioactively labelled by the incorporation of labelled precursors and cilia will be isolated. The ciliary proteins will be compared using one- and two-dimensional polyacrylamide gel electrophoresis. Proteins which are altered or absent in the cilia from the PCD cells will be isolated and identified by mass-spectrometry. Antibodies and cDNA probes will be developed against the identified proteins. These probes will be used to determine if the gene coding for the protein is mutated, or if the protein appears altered in PCD cilia due to a mutation in another gene. These studies will identify defects in the cilia of PCD patients at the level of individual proteins. Ultimately, these studies will identify the mutation responsible for some cases of PCD, and will increase our understanding of how the mutation leads to disease. This information may lead to improvements in the diagnosis and therapy of this disease, including the possibility of gene therapy. Further studies of the role of these proteins in the assembly and function of cilia may also result in improved treatment for other air way diseases.
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