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Notch signaling controls spatial patterning and cell-fate decisions in all metazoans. Mutations in JAG1, one of the five Notch ligands in man, have been associated with Alagille syndrome and with a familial form of tetralogy of Fallot. A specific G274D mutation in the second epidermal growth factor repeat of the Jagged-1 was found to correlate with tetralogy of Fallot symptoms but not with usual Alagille syndrome phenotypes. To investigate the effects of this mutation, we studied the in vitro oxidative folding of the wild-type and mutant peptides encompassing the second epidermal growth factor. We found that the G274D mutation strongly impairs the correct folding of the epidermal growth factor module, and folding cannot be rescued by compensative mutations. The 274 position displays very low tolerance to substitution because neither the G274S nor the G274A mutants could be refolded in vitro. A sequence comparison of epidermal growth factor repeats found in human proteins revealed that the pattern displayed by the second epidermal growth factor is exclusively found in Notch ligands and that G274 is absolutely conserved within this group. We carried out a systematic and comprehensive analysis of mutations found in epidermal growth factor repeats and show that specific residue requirements for folding, structural integrity and correct post-translational processing may provide a rationale for most of the disease-associated mutations.