Kenneth Astrin

Research

Molecular Genetics of the Inherited Porphyrias

The biosynthesis of heme in man requires eight enzymatic steps to convert succinyl-CoA and glycine to the final product, heme. All eight enzymes are encoded by nuclear genes and four of the reactions occur in the cytosol, while four take place in the mitochondrion. Studies on the tissue-specific regulation of heme biosynthesis, and in particular the role of the first and rate-limiting enzyme in the pathway are discussed under the research description by Dr. D.F. Bishop. The focus of my research, in collaboration with Dr. R. J. Desnick, is the investigation of the inborn errors of heme biosynthesis, the inherited porphyrias. Each of these disorders results from the deficient activity of a particular heme biosynthetic enzyme. The inborn errors of heme biosynthesis provide the opportunity to investigate the effects of dominant mutations and the action of environmental factors, since most of these diseases are latent until exacerbated by an environmental or pharmacologic stress.

Past accomplishments of the porphyria laboratory in the study of the human porphyrias have included the following: 1) Development of specific assays for the first four enzymes in the pathway, ALA-synthase, ALA-dehydratase, HMB-synthase and URO-synthase, 2) purification to homogeneity and characterization of the physical and kinetic properties of human ALA-dehydratase, HMB-synthase and URO-synthase, 3) isolation and characterization of the full-length cDNAs encoding human ALA-synthase (erythroid and hepatic cDNAs, see Dr. Bishop's research description), ALA-dehydratase, HMB-synthase and URO-synthase, 4) regional chromosomal assignment of the genes encoding ALA-synthase (erythroid and hepatic), ALA-dehydratase, HMB-synthase and URO-synthase, 5) demonstration of normal isozymes for ALA-dehydratase and identification of their role in the susceptibility to lead poisoning, 6) demonstration of the genetic heterogeneity in unrelated families with acute intermittent porphyria (AIP) by the immunologic identification of different mutations resulting in the presence or absence of the non-functional enzyme proteins, and 7) characterization of the molecular defects causing congenital erythropoietic porphyria.

Current efforts in our laboratory are focused at: 1) identification of the molecular lesions in patients with six porphyrias: acute intermittent porphyria, congenital erythropoietic porphyria, porphyria cutanea tarda, hereditary coproporphyria, variegate porphyria and erythroprotoporhyria 3) expression of unique/interesting mutations found in any of the six porphyrias using an E. coli expression system and 4) establishment of a murine knock-out models for acute intermittent porphyria.