Inborn errors of pyruvate metabolism are usually associated with severe developmental disability and death in infants and children. Deficiency of two enzymes, pyruvate dehydrogenase complex (PDC) and pyruvate carboxylase (PC) are the main focus of this project. By assaying cells or tissues from 99 patients with suspected defects, we have identified 13 cases of PDC deficiency, 6 cases of PC deficiency, and 2 previously unknown disorders. We found that PDC deficiency is expressed in lymphocytes as well as in skin fibroblasts, and that in some cases PDC deficiency is not expressed in fibroblasts. Using assays of activity and specific antibodies for measurement of the components of PDC, we found greatly reduced amounts of both the alpha and beta subunits of the F1 component of PDC in cells and/or tissues from 4 E1 deficient patients; 2 of which were shown to be systemic defects. In 3 other cases, activity is reduced but the subunit proteins appear antigenically normal in both size and amount. We have also found heterogeneity of PC deficiency, based on the presence or absence of specific mRNA. We plan to continue to collect and characterize defects of these enzymes at the protein and gene level in order to understand the molecular mechanisms involved. This effort will be enhanced by recent isolation in our laboratory of full-length human cDNAs for the 4 proteins involved in the catalytic components of PDC (E1 alpha, E1 beta, E2 and E3); the cDNA for E3 is now nearly completely characterized. We will complete the characterization of these PDC cDNA clones, which will provide the presently unknown complete primary amino acid sequences of the respective proteins. We also will isolate and characterize the structure and organization of these PDC genes. We have obtained previously cloned cDNAs for PC and PEP-carboxykinase. Using these specific cDNA clones, we plan to examine mutant cells for various possible gene modifications. We will determine the size and content of the specific mRNAs and look for major abnormalities of the respective genes by Southern analysis. More subtle changes in DNA will be examined by procedures designed to detect single base mutations, selected sequence analysis, and measurement of transcriptional rates. Techniques of identification of altered genes and gene products will be applied to family studies. Our multifaceted approach to analysis of defects of these enzymes of pyruvate metabolism are designed to enhance understanding of normal as well as abnormal molecular structure, function, and regulation.
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