This project continues to focus on an analysis in cultured cells of clinically significant inborn errors of metabolism involving four enzymes of amino acid and organic acid metabolism: homocystinuria, due to cystathionine beta-synthase (CS) deficiency; methylmalonic acidemia, due to methylmalonyl CoA mutase apoenzyme (MUT) deficiency; hyperammonemia, due to ornithine transcarbamylase (OTC) deficiency; and propionic acidemia, due to propionyl CoA carboxylase (PCC) deficiency. The proposed research makes use both of fibroblast lines derived from patients with genetically determined defects in each of these enzymes and of cell lines expressing mutant proteins generated by in vitro mutagenesis of isolated cDNAs encoding these proteins. These studies are aimed at: 1) identifying naturally occurring mutations in MUT and PCC which result in abnormal mitochondrial uptake and processing of these nuclear- coded, cytoplasmically synthesized, mitochondrial enzymes; 2) analyzing in intact cultured cells the effects of artificial mutations in the OTC leader peptide and comparing these to the effects already described in vitro; 3) characterizing at the molecular level natural human mutations in CS, MUT, and PCC in order to understand their effects on mitochondrial transport, coenzyme binding, subunit interaction, or proteolytic activation; 4) addressing the possible relationship between CS overexpression (due to partial trisomy 21) and Down's syndrome, and between CS underexpression (due to heterozygosity for CS deficiency) and peripheral occlusive arterial disease; and 5) developing molecular tools which will permit early prenatal diagnosis and reliable heterozygote detection for each of these inborn errors of metabolism. Biochemical, cell biological, and molecular genetic approaches will be employed, including: radiolabeling and immunochemical recovery of specific enzymes in intact cells; calcium phosphate-mediated DNA transfection; DNA, RNA, and protein blotting with detection by (32P) labeled probes (DNA and RNA) or by specific antibodies and (125I) protein A (proteins); genomic cloning in bacteriophage vectors; mutant sequence identification by denaturing gradient (Lerman) gels; and DNA sequencing by Maxam-Gilbert or Sanger dideoxy techniques. These studies should provide new information on both the role of individual mutations in the pathogenesis of these inborn errors and on normal mechanisms of metabolism and homeostasis in man.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK012579-23
Application #
3224907
Study Section
Biochemistry Study Section (BIO)
Project Start
1977-07-01
Project End
1992-06-30
Budget Start
1991-07-01
Budget End
1992-06-30
Support Year
23
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520