Red Cell Biology

National Human Genome Research Institute

Investigators

Linked publications & trials

Abstract

There are three related projects in this program: Project 1: Genome sequencing to identify DBA mutations. Project 2: Novel reporter cells for screen on compounds that increase ribosomal protein expression Project 3. Mouse models of red blood cell function. DBA is an inherited bone marrow failure syndrome in which ribosome defects specifically block erythropoiesis while cells of the other hematopoietic lineages are normal in both number and function. In addition to anemia, DBA shares several clinical features with other bone marrow failure syndromes, e.g. Fanconi anemia and Schwachman Diamond syndrome, including: 1) a high frequency of birth defects, 2) a predisposition to cancer (both solid tumors and AML) and 3) variable penetrance and clinical course, ranging from asymptomatic to severe. DBA is inherited as an autosomal dominant with incomplete penetrance and 60% of DBA mutations arise de novo. Despite the phenotypic variation observed among patients with DBA, only a few confirmed genotype/phenotype correlations have been established. Project 1. Mutations in at least 24 different ribosomal protein (RP) genes have been identified in 80% of DBA patients. These mutations lead to aberrant rRNA processing, impaired production of either the small or large ribosomal subunit and reduced numbers of mature 80S ribosomes. Targeted sequencing of the 80 ribosomal protein genes in >75 DBA patients without a molecular diagnosis failed to identify causative mutations in 35% of DBA patients. Exome sequencing of DBA patients showed that 80% have mutations in one of the 80 RP genes. Identifying and characterizing the remaining 20% of DBA mutations is the goal of Project 1. We are using whole genome sequencing to screen DBA patients for small deletions in and around ribosomal protein genes. In 15 patients we have identified 8 mutations, including deletions which are too small to be detected by other methods, which we have demonstrated to cause DBA in these patients. These mutations, which are in noncoding regions of the RP genes, interfere with the normal transcription and/or splicing of the RP mRNA, validating them as causative mutations We will be submitting a paper describing these results shortly. (Vlachos et al, in preparation). Project 2. We are using CRISPR to introduce RP mutations into the erythroid cell line Bel-A. We have successfully targeted three integrin loci whose expression changes during erythroid differentiation. The marker fluoresce when the gene is activated, allowing small molecule screens to be performed in collaboration with NCATS. This project will terminate on Sept 30, 2022. The cell lines will be turned over to NCATS for further analysis. Project 3. We are investigating two novel hypotheses about erythrocyte production and function and red cell metabolism. Mammalian red cell deformability and metabolism changes between low oxygen (tissues) and high oxygen (lung) environments. In hypoxic conditions red cells show increased deformability, increased ATP release and pentose pathway activity, while in normoxic conditions red cells revert to normal deformability and activate the glycolysis pathway to produce ATP. A candidate for the oxygen sensor is band 3, the protein encoded by the Slc4a1 gene. Band 3 is the most abundant protein in the mammalian erythrocyte membrane and functions as an anion exchange channel. In vitro, the N-terminus of the cytoplasmic domain of band 3 reversibly binds glycolytic enzymes in normoxic conditions and deoxyhemoglobin in hypoxic conditions and we have shown that this leads to specific changes in ATP levels, and either glycolytic or pentose pathway intermediates depending on whether the mutation is a gain or loss of Hb binding. In sickle cell disease (SCD), polymerization of deoxyhemoglobin S is the fundamental lesion underlying the multi-organ damage that characterizes the disease. DeoxyHbS forms long insoluble polymers that distort the red cell into the characteristic sickle shape. A paradox in the study of SCD is that the polymerization of HbS within red blood cells occurs at 10-fold higher concentrations of oxygen than the polymerization of HbS in vitro. We have observed that our Band3 mutations alter the rate of HbS polymerization in the Townes mouse model. We have shown that the rate of sickling correlates with the levels of glycolytic intermediates in the cell. We have used an oral available drug that activates the rate limiting step in erythroid glycolysis to treat SS mice. We showed that drug treatment reduces the levels of glycolytic intermediates in SS erythrocytes with concomitant increases in red cell production. These studies will be continued by the drug company. This project will terminate on Sept. 30, 2022. The red blood cells of an average adult human male store collectively about 2.5 grams of iron, representing about 75 percent of the total body iron. The continuous production of new red blood cells requires iron, some supplied by the diet and the remainder by macrophages which engulf senescent red blood cells and recycle iron to maturing red cells. Iron metabolism models predict that the iron required for heme synthesis (and therefore hemoglobin assembly) is imported into erythroid cells as transferrin and subsequently incorporated into heme by a series of enzymes associated with the mitochondrial membrane. To keep the levels of heme and globin chains balanced (excess heme is toxic), immature red cells express a heme exporter, FlvcR, which is expressed at its highest levels at the CFU-E stage before declining during terminal erythroid maturation (basophilic erythroblast to reticulocyte). However, the translation of globin mRNA peaks during the most terminal stages of erythroid maturation (orthochromatic erythroblast and reticulocyte) between which the nucleus is extruded and most of the heme producing mitochondria degrade. How then do reticulocytes generate enough heme to complete hemoglobin assembly? We have knocked out a novel heme transporter, Mrp5 to answer this question and found that Mrp5 deficient mice have reproductive disorders and normal erythropoiesis. This project will terminate on Sept. 30, 32022. The animals have been transferred to collaborators.