In this report, 14 heterozygous mutations in the FI gene (CFI, complement factor I), previously identified by different groups 4, 7, 8, 31, 32, have been studied to determine their effects on protein expression, secretion and function. To date, only the locations

of these CFI mutations and the clinical descriptions of patient symptoms have been reported. At the molecular level, the functional effects of only three of the currently analyzed 14 mutations have been investigated previously using eukaryotic expression system; one of these three was not secreted and therefore not amenable to functional analysis 9. It is important to understand how the complement system is regulated in these patients, especially with a view to developing therapeutic options. We found that the presence of pre-mature stop codons affected mainly protein secretion, whereas the amino acid Rapamycin in vivo substitutions affected either the secretion or the function of the FI protein. Thus, mutations in CFI lead to impaired regulation of the complement alternative pathway because of either impaired secretion or impaired function of FI, in turn predisposing patients to aHUS. In this study, we have investigated the functional effects of 14 CFI mutations identified in aHUS patients 4, 7, 8, 31, 32. These mutations are present in different domains: the FIMAC, CD5, LDLr1, region of unknown

homology and SP domain (Fig. 1A). Of the mutations, 11 are point mutations, eight

resulting in amino acid changes, and another three generate pre-mature stop codons. Another two of the mutations are MK-2206 manufacturer deletions, (del C or del CYTH4 CACTT) and the final mutation was due to the insertion of an AT dinucleotide. These last three mutations also generated pre-mature stop codons (Fig. 1A, Table 1). Transient transfections were performed to determine how the mutations affect the expression and secretion of FI. Human embryonic kidney (HEK) 293 cells were transfected with different FI constructs and the FI concentrations in the cell lysates and supernatants were analyzed by ELISA. The C25F, P32A, M120V, H165R, R299W, W468x and D501N mutants were all expressed as efficiently as the WT FI, but the remaining seven mutants were expressed at significantly decreased levels (Fig. 1B). Only three of the mutants (P32A, H165R and D501N) were secreted at similar levels as WT. The mutants M120V, A222G and R299W were secreted, but at significantly lower levels compared with WT FI (Fig. 1C). The remaining eight mutants (C25F, W127x, N133S, L289x, R456x, W468x, T520x and W528x) were not secreted (Fig. 1C). The ratio of FI concentrations between the supernatant and cell lysate for each mutant shows that the P32A, H165R and R299W mutants were secreted as efficiently as WT FI from the HEK 293 cells (Fig. 1D). The remaining mutants were secreted less efficiently than WT FI.