ENZYME DEFECTS IN DISORDERS OF PROTOPORPHYRIN METABOLISM

University Of Alabama At Birmingham, Birmingham AL

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Abstract

The ongoing effort of this research project is to define the pathogenesis of the biochemical and clinical manifestations in the porphyrias. The focus is on protoporphyria, in which a deficiency of ferrochelatase (FC) activity causes an excess accumulation and excretion of protoporphyrin. The FIRST AIM is to identify the FC gene mutations which have occurred in human subjects with protoporphyria, correlating genotype with phenotype. The hypothesis is that CRIM-positive mutations will cause the greatest reduction in FC activity because the functional state of FC in mitochondria is that of a dimer. A large number of patients with a broad range of elevated protoporphyrin levels is available to examine this hypothesis. Lymphoblastoid cell lines will be established on the patients and used to measure FC activity and the level of immunoreactive FC protein. FC cDNA will be amplified by a specific reverse transcriptase-PCR method, and the PCR products will be sequenced to define the gene mutation. FC mRNA will also be quantified. Mutant FC cDNA clones will be examined in an expression vector in order, to confirm that the FC protein has lost enzyme activity. This analysis should define CRIM-positive and CRIM-negative mutations, in most situations identifying the FC gene defects which have occurred, and allow correlation of genotype with phenotype. The SECOND AIM is to identify the FC gene mutation in bovine protoporphyria. This is a naturally occurring animal model of protoporphyria which is expressed only in the homozygous state. Using a radiolabeled fragment of human FC cDNA, positive clones have been identified in a bovine placenta FC cDNA library and are being purified for sequencing. The expected homology of bovine FC with human FC should determine if bovine FC cDNA has been cloned. When the sequence for bovine FC cDNA has been determined, the FC gene defect in bovine protoporphyria will be examined in cells from homozygous animals. Once it has been defined, a rapid genetic test will be developed so that heterozygous animals can be identified. Most cattle with protoporphyria in the United States should have the same FC gene mutation as a result of common ancestry. The THIRD AIM is to identify a putative porphyrin-peptide conjugate in the bile of patients with variegate porphyria. Variegate porphyria shares the biochemical feature of excess biliary and fecal protoporphyrin excretion with protoporphyria, yet the clinical manifestations are very different. The bile of patients with variegate porphyria has shown the presence of a porphyrin moiety which has a retention time on HPLC intermediate to that of protoporphyrin and coproporphyrin. This may be a protoporphyrinpeptide conjugate. The compound will be isolated and partially purified from bile by HPLC, with structural analysis by mass spectrometry and NMR. The fluorescence spectrum of the compound will also be compared with the plasma porphyrin fluorescence marker which is unique for variegate porphyria. The identification and study of this compound may provide an understanding as to the differences in clinical manifestations between protoporphyria and variegate porphyria.