Defense Date


Document Type


Degree Name

Doctor of Philosophy


Human Genetics

First Advisor

Robert Z. Eanes


Propionic acidemia is an autosomal recessively inherited disorder of organic acid metabolism caused by deficient activity of propionyl CoA carboxylase. This enzyme is required for the catabolism of the odd-chain fatty acids, the side chain of cholesterol and the amino acids: isoleucine, methionine, and threonine. Although the clinical expression of this disorder is variable, affected children usually develop ketoacidosis and hyperammonemia which may progress to seizures, coma and possibly death. Some patients may exhibit vomiting, lethargy and hypotonia in the first few weeks of life whereas others may remain asymptomatic for months or even years.(1) The degree of residual enzyme activity in the tissues from these patients does not correlate to this apparent clinical heterogeneity. The relationship between the clinical variation observed among propionyl CoA carboxylase deficient patients and the corresponding genetic and biochemical make up, is not yet understood. The aim of this project is to study the biochemical and immunological properties of propionyl CoA carboxylase in liver homogenates and fibroblast samples from several patients with propionic acidemia, to gain a better understanding of the nature of the defect in this disorder.

Previous comparisons of propionyl CoA carboxylase from fibroblasts of patients with this enzyme deficiency and unaffected individuals have suggested that propionyl CoA carboxylase is structurally altered in the patients with propionic acidemia. The fibroblast lines from these patients can be categorized into two major genetic complementation groups, pccA and pccBC based on the increase in activity observed in heterokaryons formed by pairwise cell fusions.(2) In addition, the defective propionyl CoA carboxylase can be differentiated biochemically from the normal enzyme and from each compIementation group.(3) These differences suggest that the aIterations in propionyl CoA carboxylase structure in each complementation group represent mutations in different subunits. In addition, mutations within a single gene resulting slightly different enzyme structures, would explain the clinical variation within a complementation group. Therefore, biochemical differences among mutant propionyl CoA carboxylases from the same c0mp1ementation group were investigated.

To provide further evidence that propionic acidemia is the result of structural alterations in propionyl CoA carboxylase, immunologic techniques were used to determine if equal quantities of cross-reacting material (CRM) were present in liver and fibrobiast homogenates from propionyl CoA carboxyIase deficient patients from the various genetic complementation groups. Antiserum prepared against purified pig heart propionyl CoA carboxylase which cross-reacts with human propionyI CoA carboxyIase and another antiserum prepared against the human biotin-containing enzymes, were compared using immunotitration techniques. These tests demonstrated that there are equal quantities of cross-reacting material in the tissue homogenates of propionic acidemia patients.(4, 5)

Investigations were undertaken to biochemically characterize and evaluate the heterogeneity within the pccBC genetic complementation group. This group was chosen because previous complementation and biochemical studies with fibroblasts from the pccBC subgroups have suggested the existence of interallelic complementation in this group. (6) Specific biochemical differences among propionyl CoA carboxylases from cells belonging to patients in this group could identify heterogeneity and characterize the complementation pattern. Normal and mutant propionyl CoA carboxylase from the pccBC complementation group were highly purified and their biochemical properties were compared using their isoelectric paint, thermostabiilty, and enzyme affinity for substrates. The properties of the purified enzymes were then compared with propionyl CoA carboxylase from other mutants in this group. These comparisons demonstrated biochemical heterogeneity within the pccBC complementation group.(7)

The results provide compelling evidence that the defect in propionic acidemia represents a structural alteration of propionyl CoA carboxylase in the pccA and pccBC complementation groups. In addition, the biochemical heterogeneity demonstrated within the pccBC complementation group suggests that several different structural mutations, possibly of the same subunit, are involved that result in slightly different biochemical parameters for each mutant enzyme. These structural alterations may explain the complicated complementation map for the pccBC subgroups. Since interallelic complementation is based on the heteropolymer being slightly more active than the homopolymer, different structural alterations in the mutants may interfere with their ability to participate in interallelic complementation. Furthermore, biochemical differences within the group may be reflected in the clinical phenotype of the disease and may be an indicator of the clinical variation which has been observed in these patients.


Scanned, with permission from the author, from the original print version, which resides in University Archives.


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