Hereditary angioedema (HAE) is an autosomal dominant disease resulting from mutations of the C1 inhibitor (C1INH) gene. The disease is characterized by recurrent attacks of angioedema. Disease severity among HAE patients encompasses a spectrum from patients who are virtually asymptomatic to those who have episodes of life-threatening angioedema. The factors that determine the severity of clinical presentation have not been identified. Recent work has established that the underlying C1INH mutations in HAE patients are extremely heterogeneous. We propose to characterize type I and type II C1INH mutations in order to further elucidate the molecular basis of HAE and assess potential factors that govern a given patient's disease severity. We have identified 9 different C1INH genomic mutations in 17 type I and type II HAE patients studied to date using single-strand conformation polymorphism analysis followed by cycle sequencing. Seven of the 9 mutations represent C1INH mutations that had not been previously identified. Extending the study to additional HAE patients will allow us to identify other C1INH mutations that are known to cause HAE. The identified mutations will be used as probes to explore the regulation of C1INH expression and function. The effect of the identified C1INH mutations on C1INH structure and function will be assessed. Steady-state mutant and wild-type C1INH mRNA levels will be determined in monocytes using a quantitative allele specific reverse transcription-polymerase chain reaction single nucleotide primer extension assay. Transcription rates will be determined by nuclear run-on assays, and specific mRNA stability will be determined using actinomycin inhibition. Translational processing of the mutant C1INH will be determined by expression in COS-7 cells using biosynthetic labeling and pulse-chase experiments. Sites of intracellular degradation will be identified using inhibitors of cell function and glycosidase digestion. Evidence of intracellular accumulation will be tested by immunofluorescent co-localization. The ability of the mutant protein to be secreted will be determined, and its functional activity assessed. The clinical variability of HAE may also be a consequence of differences in the expression or function of the """"""""wild-type"""""""" C1INH allele. We have found that a common polymorphism of codon 458 is associated with diminished C1INH functional activity and decreased levels of specific mRNA transcripts. The allelic frequency of this polymorphism will be determined across different ethnic and gender groups, and we will also analyze whether it is over-represented in groups of patients characterized by involvement of the complement and contact systems. These studies will contribute to our understanding of the molecular nature of HAE, and should begin to elucidate those factors that determine HAE patients' disease severity.
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