The goal of the proposed research is to understand the mechanisms of how genes are regulated in different tissues. This will be accomplished through the study of the human gene encoding debranching enzyme and molecular analysis of Type III glycogen storage disease. In this disease there are patients who lack debranching enzyme activity in both liver and muscle, and patients with enzyme deficiency confined to liver; yet the enzyme is a monomeric protein and appears to be identical in all tissues. The proposed research hypothesizes that debranching enzyme in liver and muscle is encoded by a single gene; its expression, however, is under separate genetic control. Patients with various clinical and enzymatic subgroups of Type III glycogen storage disease are being followed at Duke Medical Center; fibroblasts from these patients have been collected and stored. The debranching enzyme has been purified from porcine muscle and the antibody characterized. The cDNA coding for debranching enzyme from both human liver and muscle will be isolated by screening lambda gtll human cDNA libraries with antibody against porcine debranching enzyme. The debranching enzyme mRNA in these two tissues will be compared by Northern blot analysis, cDNA sequencing and/or sequencing of mRNA by primer extension. The chromosomal gene(s) coding for debrancher enzyme will be isolated, and the molecular structure and number of copies of the genes will be determined. Information about the number of debrancher genes and the sequences of the mRNA from liver and muscle will be used to design strategies to investigate the mechanism responsible for control of tissue-specific debrancher gene expression. This may involve sequencing of the 5' flanking region for two possible transcription initiation sites, or in the case of two different mRNA sequences encoded by a single gene, sequencing the regions of the gene that specify the mRNA unique to liver or muscle. The defective chromosomal debrancher genes from patients with disease confined to liver will also be cloned and the mutations characterized. Molecular classification of Type III glycogen storage disease will be performed at the protein, mRNA, and gene levels. These molecular findings will be correlated to the type and severity of clinical phenotypic expression. Information gained on molecular structure of glycogen debrancher gene and human mutants lacking debrancher activity may lead to identifying DNA regions important in the control of tissue-specific gene expression, and may provide a model for mechanisms responsible for clinical variability in other genetic disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK039078-01
Application #
3238740
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1987-08-01
Project End
1992-07-31
Budget Start
1987-08-01
Budget End
1988-07-31
Support Year
1
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Duke University
Department
Type
Schools of Medicine
DUNS #
071723621
City
Durham
State
NC
Country
United States
Zip Code
27705
Matern, Dietrich; Seydewitz, Hans Hermann; Bali, Deeksha et al. (2002) Glycogen storage disease type I: diagnosis and phenotype/genotype correlation. Eur J Pediatr 161 Suppl 1:S10-9
Hadjigeorgiou, G M; Comi, G P; Bordoni, A et al. (1999) Novel donor splice site mutations of AGL gene in glycogen storage disease type IIIa. J Inherit Metab Dis 22:762-3
Matern, D; Starzl, T E; Arnaout, W et al. (1999) Liver transplantation for glycogen storage disease types I, III, and IV. Eur J Pediatr 158 Suppl 2:S43-8
Shen, J; Liu, H M; McConkie-Rosell, A et al. (1998) Prenatal diagnosis and carrier detection for glycogen storage disease type III using polymorphic DNA markers. Prenat Diagn 18:61-4
Shen, J; Bao, Y; Chen, Y T (1997) A nonsense mutation due to a single base insertion in the 3'-coding region of glycogen debranching enzyme gene associated with a severe phenotype in a patient with glycogen storage disease type IIIa. Hum Mutat 9:37-40
Shen, J; Liu, H M; Bao, Y et al. (1997) Polymorphic markers of the glycogen debranching enzyme gene allowing linkage analysis in families with glycogen storage disease type III. J Med Genet 34:34-8
Bao, Y; Yang, B Z; Dawson Jr, T L et al. (1997) Isolation and nucleotide sequence of human liver glycogen debranching enzyme mRNA: identification of multiple tissue-specific isoforms. Gene 197:389-98
McConkie-Rosell, A; Wilson, C; Piccoli, D A et al. (1996) Clinical and laboratory findings in four patients with the non-progressive hepatic form of type IV glycogen storage disease. J Inherit Metab Dis 19:51-8
Shen, J; Bao, Y; Liu, H M et al. (1996) Mutations in exon 3 of the glycogen debranching enzyme gene are associated with glycogen storage disease type III that is differentially expressed in liver and muscle. J Clin Invest 98:352-7
Bao, Y; Dawson Jr, T L; Chen, Y T (1996) Human glycogen debranching enzyme gene (AGL): complete structural organization and characterization of the 5' flanking region. Genomics 38:155-65

Showing the most recent 10 out of 26 publications