Enzyme Defects in Disorders of Protoporphyrin Metabolism

University Of Alabama At Birmingham, Birmingham AL

Investigators

Linked publications & trials

Abstract

DESCRIPTION (provided by applicant): Protoporphyria (EPP) is a genetic disorder in which deficient activity of the mitochondrial enzyme ferrochelatase (FECH) causes excessive accumulation of protoporphyrin. As a consequence patients have photosensitivity which is variable in degree, and some develop liver disease. Molecular studies from this and other laboratories have shown that most patients are heterozygous for FECH gene mutations which do not explain the phenotypic variation. The major objective of this proposal is to examine the hypothesis that the level of expression of the FECH allele which encodes normal protein (nonmutant allele), is also a critical factor. In the first specific aim families in which members have different phenotypic expression of EPP will be studied to determine whether inheritance of a low-expressing nonmutant FECH "allele, together with the mutant FECH allele, has occurred in those with symptomatic EPP. Haplotype analysis will distinguish mutant from nonmutant alleles, and the level of normal FECH mRNA will be specifically assessed in cultured lymphoblasts. The presence of an intron polymorphism associated with aberrantly spliced FECH mRNA that is rapidly degraded will be examined for in the nonmutant allele. The promoter region and more extended 5'- untranslated region of the nonmutant FECH allele will also be sequenced and expressed in an in vitro system. In the second specific aim, a mouse model of EPP in which there is a heterozygous deletion of exon 10 will be backcrossed with inbred strains of mice which are selected for different levels of FECH gene expression on the basis of FECH activity and FECH mRNA levels in liver and bone marrow. It is anticipated that the level of FECH activity, and hence degree of protoporphyrin accumulation, will vary in heterozygous offspring according to the level of FECH gene expression in the wild-type parent. Liver and bone marrow will be assessed for FECH activity, FECH mRNA, and levels of normal and mutant FECH proteins. Liver mitochondria will be examined for the presence of heterodimers consisting of the normal and mutant FECH proteins. Phenotype will be assessed by the protoporphyrin levels, and degree of photosensitivity and liver damage. The proposal thus is designed to define a critical relationship between FECH gene expression and phenotype in EPP which will lead to better genetic counseling and therapy.