Epidermolysis bullosa with pyloric atresia (EB-PA: OMIM 226730), also known as Carmi syndrome, is a rare autosomal recessive genodermatosis that manifests with neonatal mucocutaneous fragility associated with congenital pyloric atresia. The disease is frequently lethal within the first year, but nonlethal cases have been reported. Mutations in the genes encoding subunit polypeptides of the ␣64 integrin (ITGA6 and ITGB4) have been demonstrated in EB-PA patients. To extend the repertoire of mutations and to identify genotype-phenotype correlations, we examined seven new EB-PA families, four with lethal and three with nonlethal disease variants. DNA from patients was screened for mutations using heteroduplex analysis followed by nucleotide sequencing of PCR products spanning all 4 integrincoding sequences. Mutation analysis disclosed 12 distinct mutations, 11 of them novel. Four mutations predicted a premature termination codon as a result of nonsense mutations or small out-of-frame insertions or deletions, whereas seven were missense mutations. This brings the total number of distinct ITGB4 mutations to 33. The mutation database indicates that premature termination codons are associated predominantly with the lethal EB-PA variants, whereas missense mutations are more prevalent in nonlethal forms. However, the consequences of the missense mutations are position dependent, and substitutions of highly conserved amino acids may have lethal consequences. In general, indirect immunofluorescence studies of affected skin revealed negative staining for 4 integrin in lethal cases and positive, but attenuated, staining in nonlethal cases and correlated with clinical phenotype. The data on specific mutations in EB-PA patients allows prenatal testing and preimplantation genetic diagnosis in families at risk. Abbreviations EB-PA, epidermolysis bullosa with pyloric atresia BMZ, basement membrane zone CSGE, conformation-sensitive gel electrophoresis PTC, premature termination codon TEM, transmission electron microscopy ITGB4, 4 integrin gene Epidermolysis bullosa (EB) is a group of genodermatoses manifesting with blistering and erosions of the skin and mucous membranes as a result of mechanical trauma (1, 2). The clinical severity of EB is highly variable. In the mildest cases, blistering occurs primarily on the hands and feet with relatively little morbidity, whereas, in the most severe cases, the disease can be lethal perinatally or during the early postnatal period. Traditionally, EB has been divided into three broad categories based on clinical findings and ultrastructural demonstrations of