Hereditary spherocytosis (HS), a common inherited hemolytic anemia, results from defects in the red blood cell (RBC) spectrin-based membrane skeleton, a multicomponent structure crucial to RBC mechanical stability. Defects in membrane skeleton components destabilize the membrane, leading to spherocytosis, splenic sequestration of damaged cells, and RBC dehydration. Heterogenous clinical presentation in humans results from both the specific protein defect and genetic diversity within the human population. An identical genetic lesion produces widely differing disease severity in human patients. Both the molecular interactions that underlie HS and the genetic loci that modulate its phenotype are ill defined. Deletion of the RBC integral membrane protein band 3 in mice causes profound HS without grossly affected membrane skeleton structure. Loss of the band 3 ligand, protein 4.2, produces mild HS and significantly alters RBC cation transport. Transfer of the band 3 null mutation to different inbred mouse strains lessens the severity of HS.
The specific aims of this study test the hypotheses that (1) interactions between the membrane-spanning domain of band 3 and plasma membrane lipids stabilize the red cell membrane. The ability of the membrane-spanning and cytoplasmic domains of band 3 to independently stabilize the RBC membrane in vivo will be assessed using targeted mutagenesis in embryonic stem cells. The investigator will replace the endogenous band 3 gene (knockin) with a cDNA expressing: A) the band 3 transmembrane domain only, B) the band 3 cytoplasmic domain only, and C) the cytoplasmic domain of band 3 attached to the transmembrane domain of the polytopic RBC glucose transporter, Glut1; (2) interactions between protein 4.2 and cation transporters, or components of cation transport regulatory pathways, are critical determinants of RBC hydration status. The investigator will: A) replace the endogenous protein 4.2 gene with a protein 4.2 cDNA containing a mutated band 3 binding site, and B) identify novel protein 4.2 binding proteins in RBCs; (3) interactions between genetic loci significantly modulate RBC membrane characteristics and production. The investigator will genetically map the chromosomal location of genetic modifiers of the band 3 null HS phenotype, establish minimal genetic intervals containing the modifying loci, and build physical contigs spanning the minimal genetic intervals.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL064885-04
Application #
6638650
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Qasba, Pankaj
Project Start
2000-06-01
Project End
2005-05-31
Budget Start
2003-06-01
Budget End
2005-05-31
Support Year
4
Fiscal Year
2003
Total Cost
$380,000
Indirect Cost
Name
Jackson Laboratory
Department
Type
DUNS #
042140483
City
Bar Harbor
State
ME
Country
United States
Zip Code
04609
Alper, Seth L; Vandorpe, David H; Peters, Luanne L et al. (2008) Reduced DIDS-sensitive chloride conductance in Ae1-/- mouse erythrocytes. Blood Cells Mol Dis 41:22-34
Alvarez, Bernardo V; Kieller, Dawn M; Quon, Anita L et al. (2007) Cardiac hypertrophy in anion exchanger 1-null mutant mice with severe hemolytic anemia. Am J Physiol Heart Circ Physiol 292:H1301-12
Peters, Luanne L; Lambert, Amy J; Zhang, Weidong et al. (2006) Quantitative trait loci for baseline erythroid traits. Mamm Genome 17:298-309
Rivera, Alicia; De Franceschi, Lucia; Peters, Luanne L et al. (2006) Effect of complete protein 4.1R deficiency on ion transport properties of murine erythrocytes. Am J Physiol Cell Physiol 291:C880-6
Peters, Luanne L; Zhang, Weidong; Lambert, Amy J et al. (2005) Quantitative trait loci for baseline white blood cell count, platelet count, and mean platelet volume. Mamm Genome 16:749-63
De Franceschi, Lucia; Rivera, Alicia; Fleming, Mark D et al. (2005) Evidence for a protective role of the Gardos channel against hemolysis in murine spherocytosis. Blood 106:1454-9
de Franceschi, Lucia; Turrini, Franco; Honczarenko, Marek et al. (2004) In vivo reduction of erythrocyte oxidant stress in a murine model of beta-thalassemia. Haematologica 89:1287-98
Peters, Luanne L; Swearingen, Rebecca A; Andersen, Sabra G et al. (2004) Identification of quantitative trait loci that modify the severity of hereditary spherocytosis in wan, a new mouse model of band-3 deficiency. Blood 103:3233-40
Johnson, Kenneth R; Gagnon, Leona H; Webb, Lisa S et al. (2003) Mouse models of USH1C and DFNB18: phenotypic and molecular analyses of two new spontaneous mutations of the Ush1c gene. Hum Mol Genet 12:3075-86
Mouro-Chanteloup, Isabelle; Delaunay, Jean; Gane, Pierre et al. (2003) Evidence that the red cell skeleton protein 4.2 interacts with the Rh membrane complex member CD47. Blood 101:338-44

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