In this revised application, Dr. Beutler proposes a comprehensive study of various red cell enzyme deficiencies, including polymorphisms of the TPI gene (AIM3 of original proposal).
Two specific aims are now described:
In AIM1 PI proposes to deduce structure/function relationships underlying deficiencies of enzyme function in the most common genetic abnormalities of erythrocyte metabolism: Mutations affecting glucose-6-phosphate dehydrogenase (G6PD), pyruvate kinase (PK), glucose phosphate isomerase (GPI), and triosephosphate isomerase (TPI). The specific gene mutation underlying each case of red cell enzyme deficiency will be identified by restriction digest (in the case of known recurring mutations) and by direct sequencing of genomic DNA, where recurring mutations are unlikely. The mutant proteins will then be produced in E. coli or in K-562 cells using recombinant tag-expression systems, in order to circumvent various problems associated with purification from red cell. Wild type and mutant enzymes will then be compared with respect to Km for substrate and enzyme stability. Where possible, reference to the crystal structure of the enzyme (or, related structure) will be made. The crystal structure of PK will be pursued through collaboration with Dr. Varughese.
In AIM2, Dr. Beutler proposes to identify the TPI mutation that is common in the African-American population and believed to be lethal when homozygous. Preliminary data suggest deficiency is related to point mutation at positions -5,-8 and/or -24 from the transcriptional start site, although the impact of these identified polymorphisms on gene expression has not been confirmed. PI will continue to accumulated blood samples from Dr. Schneider (Chicago Medical School) for TPI enzyme assay, and for analysis of potential mutations in the 5' untranslated region of the gene that might affect transcription. Initial effort will focus on the known polymorphisms at -5, -8, and -24 nt from the transcriptional start site. The impact of these mutation on gene expression will be tested in vitro using reporter constructs. Should these point mutations fail to alter gene transcription, a search will be made for other potential mutations in linkage disequilibrium with the known -5,-8 and -24 polymorphisms, by sequencing upstream through the promotor.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL025552-21
Application #
6182897
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1979-09-01
Project End
2002-03-31
Budget Start
2000-04-01
Budget End
2001-03-31
Support Year
21
Fiscal Year
2000
Total Cost
$275,405
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
van Wijk, Richard; van Solinge, Wouter W; Nerlov, Claus et al. (2003) Disruption of a novel regulatory element in the erythroid-specific promoter of the human PKLR gene causes severe pyruvate kinase deficiency. Blood 101:1596-602
Corrons, Joan-Lluis Vives; Garcia, Estefania; Tusell, Joan J et al. (2003) Red cell adenylate kinase deficiency: molecular study of 3 new mutations (118G>A, 190G>A, and GAC deletion) associated with hereditary nonspherocytic hemolytic anemia. Blood 102:353-6
Beutler, Ernest; Vulliamy, Tom J (2002) Hematologically important mutations: glucose-6-phosphate dehydrogenase. Blood Cells Mol Dis 28:93-103
Herschel, Marguerite; Karrison, Theodore; Wen, Ming et al. (2002) Isoimmunization is unlikely to be the cause of hemolysis in ABO-incompatible but direct antiglobulin test-negative neonates. Pediatrics 110:127-30
Beutler, Ernest; Gelbart, Terri; Miller, William (2002) Severe jaundice in a patient with a previously undescribed glucose-6-phosphate dehydrogenase (G6PD) mutation and Gilbert syndrome. Blood Cells Mol Dis 28:104-7
Beutler, Ernest; West, Carol (2002) Polymorphisms in glucosylceramide (glucocerebroside) synthase and the Gaucher disease phenotype. Isr Med Assoc J 4:986-8
Lee, P L; Halloran, C; Trevino, R et al. (2001) Human transferrin G277S mutation: a risk factor for iron deficiency anaemia. Br J Haematol 115:329-33
Lee, P L; Halloran, C; Beutler, E (2001) Polymorphisms in the transferrin 5' flanking region associated with differences in total iron binding capacity: possible implications in iron homeostasis. Blood Cells Mol Dis 27:539-48
Herschel, M; Beutler, E (2001) Low glucose-6-phosphate dehydrogenase enzyme activity level at the time of hemolysis in a male neonate with the African type of deficiency. Blood Cells Mol Dis 27:918-23
Beutler, E (2001) Discrepancies between genotype and phenotype in hematology: an important frontier. Blood 98:2597-602

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