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A4467 - Different Mutations in Cilia Preassembly Gene HEATR2 Are Associated with Variable Clinical Phenotypes and Defects in Proteostasis
Author Block: A. Horani1, T. Huang2, S. L. Brody2; 1Department of Pediatrics, Washington University School of Medicine, St. Louis, MO, United States, 2Department of Medicine, Washington University School of Medicine, St. Louis, MO, United States.
Rational: Primary ciliary dyskinesia (PCD) is an autosomal recessive pediatric disease that affects the motile cilia due to mutations in one of 40 different genes. The relation between mutations in the same gene and the clinical phenotype is still evolving. Methods: Primary nasal cells were cultured from subjects with known mutations in HEATR2 and assessed for motility. Native protein in primary airway cells and fluorescent-tagged proteins with patient-specific mutations introduced into cell lines, were evaluated for p62/SQSTM1 localization. Results: Three different mutations in the gene HEATR2 were identified in 4 subjects with sinopulmonary disease. HEATR2 is a cytoplasmic protein required for ciliary dynein motor protein preassembly. All subjects had electron microscopy showing absent outer and inner dynein arms, and low nasal NO supportive of the diagnosis of PCD. Mutation analysis identified: (1) a c.2384T>C (L795P) missense mutation and (2) c.2353-2356del AG (S785Cins) deletion mutation that leads to a premature stop codon, both within the C-terminal HEAT domain 10; and (3) a c.1499G>T (C500F) missense mutation between HEAT domains 6 and 7 mid protein. We observed significant variability in the clinical features among the subjects with different mutations; those with L795P and S785Cins mutations presented with classical symptoms of PCD including significant sinopulmonary disease and chronic cough. The subjects harboring a C500F mutation presented with mild otitis media and rare cough. Motile cilia from cultured nasal cells of individuals with severe disease were completely immotile, while those with mild disease had variable motility. To determine the stability of mutant proteins, we transfected cell lines with plasmids expressing normal or mutant HEATR2, and treated cells with cyclohexamide to halt new protein production. Mutant HEATR2 associated with the most severe phenotypes rapidly degraded, while the c.1499G>T (C500F) associated with mild symptoms demonstrated stability greater than even the normal HEATR2, by western blot. To assess the fate of the mutant HEATR2, we examined nasal cells for evidence of proteostasis. Compared to normal cells, those with mutations triggered p62/SQSTM1-positive aggregate formation containing HEATR2 proteins. Similar results were observed in cell lines expressing mutants, indicating that protein degradation was responsible for disease. Conclusion: These findings hint that different mutations affect rates of degradation, accounting for variability in clinical presentation and that ultimately, some mutations may be amenable to therapeutic rescue by altering folding or fate.