HUMAN FERROCHELATASE - A NOVEL MUTATION IN PATIENTS WITH ERYTHROPOIETIC PROTOPORPHYRIA AND AN ISOFORM CAUSED BY ALTERNATIVE SPLICING

Erythropoietic protoporphyria (EPP), attributable to deficiency of fer rochelatase activity (FECH), is characterised mainly by cutaneous phot osensitivity. To define the molecular defect in two EPP-affected sibli ngs and their parents in a Swiss family, ferrochelatase cDNA was ampli fied by the polymerase chain reaction (PCR) and subjected to sequence analysis. A 5-bp deletion (T580-G584) was identified on one allele of the ferrochelatase gene in both patients and their mother. Screening o f the mutation among family members by RsaI digestion of PCR-amplified genomic DNA revealed autosomal dominant inheritance associated with a bnormal protoporphyrin concentration and enzyme activity. We also isol ated ferrochelatase cDNAs containing a 18-bp insertion (part of the in tron 2 sequence) between exons 2 and 3; this corresponded to six extra amino acids (YESNIR) inserted between Arg-65 and Lys-66 of the known ferrochelatase. This isoform was identified initially in mRNAs derived from both alleles of the ferrochelatase gene in one patient. Its exis tence was confirmed in six additional EPP patients, in five out of sev en controls, and in four different cell lines (fibroblast, muscle, hep atoma and myelogenous leukaemia). This isoform, roughly 20% of the tot al ferrochelatase mRNA, was generated through splicing at a second don or site in intron 2 and its presence was not linked to EPP.