Identification of variables causing different clinical expression of inherited c1-inh deficiency (hereditary angioedema)

C1-inhibitor (C1-inh) is a member of serine protease inhibitor (serpin) family which contains a common tertiary structure composed of three beta-sheets and several α-helices. Genetic variants of C1-inh lead to plasma deficiency, causing Hereditary Angioedema (HAE). HAE is a life-threatening autosomal dominant disorder characterized by episodic local subcutaneous edema and submucosal edema involving the upper respiratory and gastrointestinal tracts. Despite in HAE patients C1-inh plasma levels are low without significant variations, frequency and severity of symptoms are highly variable among patients and even in the same patient from time to time. Nothing is known on the variables responsible for such different clinical expressions. Moreover, the detailed molecular mechanism for the pathogenicity of most variant C1-inh molecules is not known. 492 HAE patients belonging to 196 unrelated families are regularly followed at our Department and characterized for clinical phenotype. Genotyping of these patients has been completed in 155. C1-inh gene expression has been evaluated. C1-inh mRNA of patients harboring different mutations are compared with healthy controls (1). The value is similar in HAE type I and II (40 and 47%). 74% of HAE I patients carrying small mutations present significant amount of mutated mRNA, suggesting that both allelics mRNA amount are reduced to approximately 50%. In 4 patients carrying large deletion, C1-inh mRNA is 23% on average respect control suggesting that normal mRNA is strongly unexpressed. This data, togheter with the previous described increased consumption, may explain the lower contribution of normal C1-inh allele, respect the expected 50% tipical of dominant diseases. Mutations affecting residues primarily involved in the unique suicide substrate-like inhibitory mechanism of serpins have been selected for expression in Pichia pastoris. This expression system provides an average yield of 80mg per liter of culture media with wild type C1-inh. We present the results on the function/structure characterization of R378C variant. The patient carrying this mutation presents unusual features compared to other HAE patients: absence of typical subcutaneous angioedema, recurrent short lasting (few hours) abdominal cramps, but no major abdominal symptoms with severe pain vomiting and/or dyarrhea, variable C1-inh plasma levels with spontaneous normalization. The expressed protein has a very low yield, most likely due to an impaired secretory profile, typical of multimerization inside the cells. The inhibitory capacity of the active form is preserved, the second order inhibition constant found in progress curves was comparable to wild-type recombinant C1-inh. Based on the known structure of other serpins together with molecular modeling, we expect that Arg378 in the native protein forms a salt bridge with Glu429. In alpha(1)-antitrypsin Z variant, it is predicted that loss of this salt bridge would have an effect on the rate of folding. Analogously, we speculate that the low plasma level of this variant could be due to retarded protein folding, that may promote protein aggregation by accumulation of aggregation-prone folding intermediates. In accordance, our results suggest that the HAE phenotype is due to a failure in general folding and structural stability. The R378C C1-inh variant could undergo different conformations (native, latent, polymer), that result in different degrees of impaired secretion and/or function, depending on specific environmental conditions