Protoporphyria is an autosomal dominant disease that is characterized clinically by photosensitivity and occasionally by hepatobiliary disease. The most important clinical manifestation is liver cirrhosis leading to liver failure. The activity of the enzyme ferrochelatase is 15% to 25% of normal in all tissues of patients with protoporphyria. Ferrochelatase catalyzes the last step in the heme biosynthetic pathway, the chelation of ferrous iron and protoporphyrin to form heme. The precise nature of the genetic defect in protoporphyria is inknown. The primary goal of this research proposal is to isolate a cDNA clone that encodes for human ferrochelatase, which will provide a critical tool to determine the molecular defect in protoporphyria. The initial experiments will utilize mouse liver ferrochelatase, because of the availability of purified protein and antimouse liver ferrochelatase antiserum. A cDNA probe that encodes for mouse liver ferrochelatase will be isolated by using two approaches: screening a cDNA library with an oligonucleotide probe derived from the ferrochelatase amino acid sequence and screening a cDNA expression library with anti-ferrochelatase antiserum. A human cDNA library will be screened with the mouse liver ferrochelatase cDNA probe. The human ferrochelatase cDNA will be used in experiments with cultured human fibroblasts from patients with protoporphyria and normal controls. Southern blots using DNA extracted from fibroblasts and probed with human ferrochelatase cDNA will lead to the construction of restriction endonuclease maps of the chromosomal ferrochelatase gene. Northern blots using RNA extracted from fibroblasts will detect qualitative or quantitative differences in the ferrochelatase mRNA levels in protoporphyria versus normal controls. Further experiments will be determined by the above fundamental studies and might include DNA mediated gene transfer and cloning of ferrochelatase cDNAs derived from cultured fibroblasts from patients with protoporphyria.
