Protoporphyria is characterized enzymatically by decreased ferrochelatase activity, chemically by elevated protoporphyrin levels, and clinically by photosensitivity and hepatobiliary disease. Although protoporphyria is generally inherited as an autosomal dominant disease, the enzymatic activity of ferrochelatase, which catalyzes the last step in the heme biosynthetic pathway, is reduced 15 percent to 30 percent of normal in all tissues of patients with protoporphyria. The present proposal is a direct continuation of our previous studies in which the murine ferrochelatase cDNA and gene were cloned, the human ferrochelatase cDNA and gene were cloned, mutations in the ferrochelatase genes in patients with protoporphyria were identified and correlated with clinical phenotypes, and the regulation ferrochelatase gene was characterized in cultured cells and in transgenic mice.
The Specific Aims of the present proposal are: 1. To study the regulation of ferrochelatase gene expression. 2. To assess the functional significance of novel mutations in the ferrochelatase gene in patients with protoporphyria. 3. To knock out the ferrochelatase gene in the mouse to develop a model of protoporphyria. 4. To assess therapeutic interventions for protoporphyria. The emphasis of the present proposal is to use advanced techniques to study the regulation of the expression of the ferrochelatase gene in order to gain new insights into the molecular pathogenesis of protoporphyria. To optimize studies in gene regulation, homologous recombination will be used to place either ferrochelatase mutations into the normal chromosomal organization in knock out mice or reporter genes driven by the ferrochelatase promoter (either wild-type or mutant) in the context of erythroid and non-erythroid cells in culture. The novel reagents developed in this proposal will be used to design and assess new therapeutic interventions including the delivery of wild-type ferrochelatase by adeno-associated virus gene delivery.
