application) Familial porphyria cutanea tarda [PCT] and hepatoerythropoietic porphyria [HEP] are related diseases caused by inherited defects in the gene for the heme biosynthetic enzyme, uroporphyrinogen decarboxylase [UROD]. Patients with these diseases accumulate massively increased tissue levels of porphyrins, and consequently develop a variety of clinical symptoms. Patients with HEP sometimes die in the first year or two of life, and those that survive are often severely photomutilated. Efforts to better understand familial PCT and HEP have been hampered because mammalian models for the inherited forms of these diseases have not been reported. So, the long-term objective of this study is to produce mammalian models which will be used to better understand the pathogenesis, and improve the treatment, of familial PCT and HEP. To accomplish this objective, directed homologous recombination was used to cause a gene disruption in which a normal UROD gene was replaced by a defective gene in pluripotent mouse embryonic stem [ES] cells. This was accomplished by: 1) transfecting a targeting construct into ES cells; 2) selecting for colonies that had undergone homologous recombination with G418 and gancyclovir; and 3) testing surviving cell lines with Southern blots. Further confirmation that the selected ES cell lines had undergone homologous recombination was done by demonstrating: 1) decreased UROD activities, and 2) increased uroporphyrin accumulation after exposure to the porphyrin precursor, delta-aminolevulinic acid. UROD 'knockout' mouse lines will be established by microinjecting ES cells with the defective UROD gene into a host blastocyst to generate chimeric mice; breeding these chimeras to produce mice that are heterozygous for the defective UROD gene (and a model for familial PCT); and interbreeding these mice in an attempt to produce viable mice that are homozygous for the defective UROD gene (and a model for HEP). These knockout mouse lines will ultimately be used to study the roles of various chemicals (including iron and delta-aminolevulinic acid) in modulating porphyrin accumulation, and the relationship of uroporphyrin accumulation to the incidence of hepatic tumor formation and other severe liver disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Small Research Grants (R03)
Project #
1R03DK056665-01
Application #
6027651
Study Section
Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
Program Officer
Serrano, Jose
Project Start
2000-06-01
Project End
2002-05-31
Budget Start
2000-06-01
Budget End
2001-05-31
Support Year
1
Fiscal Year
2000
Total Cost
$77,966
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
660735098
City
Worcester
State
MA
Country
United States
Zip Code
01655
Lambrecht, Richard W; Bonkovsky, Herbert L (2002) Hemochromatosis and porphyria. Semin Gastrointest Dis 13:109-19