This project addresses the genetic factors responsible for the clinical expression of the most common form of porphyria in humans, namely porphyria cutanea tarda (PCT). Subnormal activity of hepatic uroporphyrinogen decarboxylase (URO-D) is a consistent finding in all patients with PCT. In some families the enzyme deficiency is inherited as an autosomal trait. This form of the disease is known as familial PCT. In other cases inherited factors are not obvious and the disease is called sporadic PCT. We have cloned and sequenced URO-D cDNA. We have identified a URO- D point substitution mutation in familial PCT which appears to affect protein stability. We plan to evaluate multiple pedigrees with familial PCT in order to catalogue the spectrum of URO-D mutations. Experimental methods will include cloning and sequencing of mutant cDNA's, oligonucleotide hybridization, and synthesis of mutant URO-D's in a bacterial expression system. The effects on the function and stability of URO-D resulting from the mutations will be studied. We have constructed cosmid libraries in order to search for polymorphisms in the flanking regions of the URO-D gene. We expect to define key structural elements in URO-D responsible for substrate binding, catalysis and protein stability. This will be accomplished using a variety of biochemical methods and site directed mutagenesis. Our approach to sporadic PCT will explore the hypothesis that patients with this disease have a mutation at the URO-D locus that renders the gene product sensitive to inhibitors generated in the liver. URO-D protein polymorphisms will be brought using electrophoresis over a wide pH range and URO-D cDNA's will be sequencer. Mutant proteins will be produced in a bacterial expression system and function effects of the mutations will be determined. This project focusses on one form of porphyria but information about structural features affecting protein stability and enzyme activity is applicable to many areas of human biology.
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