Effects of mutations within the coding region of C1-INH gene on disease manifestation and protein function in families with HAE

C1 esterase inhibitor (C1-INH) is a serine protease inhibitor (serpin) that controls activation of complement and contact systems, its inherited deficiency causes hereditary angioedema (HAE). The peculiar circumstances of symptoms recurrence in this disease remain fairly undisclosed. Hence, identifying mutations and their molecular effect is relevant for genotype/phenotypes as well as for structure/function correlates. We are now screening our case list of 448 HAE patients belonging to 166 independent families carefully characterized for the clinical phenotype. At present we have screened 85 unrelated patients: 69 have HAE type I and 16 HAE typeII. The screening of genomic DNA was performed using FAMA (Fluorescent-Assisted Mismatch Analysis). We found 65 different mutations (18 “de novo”) in 80families and none in five patients (currently checked forsplicing defects). Mutations consisted in: 7 large deletions/duplications, 1gene deletion, 1 point mutation in the promoter region, 28 missense, 4 nonsense, 15 frameshift, 4 splicing defects,1 missense/splicing defect and 4 insertion/deletion of 1–2codons. In order to define structure-function correlates in C1-INH, we selected mutations affecting residues primarily involved in the unique suicide substrate-like inhibitory mechanism of serpins. Such mutations were present in four different families, one of them, with R378C mutated protein, presenting with an extraordinary biochemical good response to danazol treatment. We expressed the mutated proteins (delT280, Y308N,R378C, L427P/R) in Pichia Pastoris expression system and functional/structural characterization is now being completed. The structural mobility of serpins required for inhibitory activity renders these inhibitors extremely sensitive to point mutations, resulting in conformational instability and polymerization. The deleterious effects of serpins polymerization are well known, while effects on minor conformational changes are just now revealing to work as signals for a range of pathological responses. The structural and functional properties of these targeted mutants will contribute to elucidate the biological activities of serpins in general, providing useful insights into the pathogenesis of conformational diseases and possibly revealing new therapeutic strategies.