ALBERT

Description: Abstract 3217 The zinc finger protein Erythroid Krüuppel-like factor (EKLF, KLF1) regulates definitive erythropoiesis and terminal differentiation of red blood cells. KLF1 facilitates transcription through high affinity binding to CACCC elements within its erythroid-specific target genes which include genes encoding erythrocyte membrane skeleton (EMS) proteins. Deficiencies of EMS proteins lead to the hemolytic anemia Hereditary Spherocytosis (HS). We have identified a new HS gene by studying the hemolytic anemia mouse mutant Nan (Neonatal Anemia). Here we report that a mutation, E339D, in the second zinc finger domain of KLF1 is responsible for HS in Nan mice. The causative nature of the E339D mutation was verified with an allelic test cross between Nan/+ and heterozygous Klf1+/− knockout mice. Homology modeling predicted Nan KLF1 binds CACCC elements more tightly, suggesting that Nan KLF1 is a competitive inhibitor of wild type KLF1. Competitive inhibition may help explain the apparent disconnect between the finding that Nan/+ heterozygous mice are anemic, whereas Klf1+/− heterozygous mice are normal and haplo-sufficient. This is the first direct association of a KLF1mutation with a disease in adult mammals. After examining a small population of HS patients, we also discovered one HS patient with a KLF1 mutation, which resulted in a significant amino acid substitution (T251I) in the activator/repressor domain, 28 amino acid residues upstream of the first zinc finger domain. This HS subject had no known mutations in the exons or intron/exon boundaries of EMS genes (SPTA1, SPTB, ANK1, SLC4A1) which comprise 85% of HS mutations in humans. The lack of a known genetic mutation in EMS genes leaves this patient's KLF1 mutation as the leading candidate defect. The identification of the gene causing the Nan mutation is significant because the Nan mutant has allowed discovery of a new HS gene which may also cause this disease in humans. In addition, the putative dominant/negative competitive inhibition of the Nan mutation makes the Nan mouse an excellent model system to study the function of KLF1. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3162 The Xla (X-linked anemia) mutant mouse was generated by N-ethyl-N-nitrosourea (ENU) mutagenesis and results in a severe and transient neonatal anemia. Xla/+ females exhibit severe anemia with 50% the level of red blood cell number, hematocrit and hemoglobin. Male Xla mice die in utero at 10.5 days gestation. The neonatal anemia observed in Xla/+ female pups is resolved by weaning age at 3 weeks by which time the mice present with a normal hematological phenotype. It is unknown how the neonatal anemia in Xla/+ females is alleviated. Previously, we mapped the Xla locus to the proximal end of the X chromosome near candidate gene Gata1 which showed no change in the coding sequence of GATA1 protein. Now we report the identification of a Gata1 mutation in Xla mice that results in an mRNA splicing defect. A nucleotide change (G to A) was identified 5 base pairs downstream of Exon 1E in intron 1 of the Xla Gata1 gene and results in the lack of incorporation of Exon 1E in the Gata1 mRNA expressed from the mutant locus. Therefore, in some erythroid lineage cells in Xla/+ mice, the normal 1E exon of Gata1 mRNA is replaced by Exon 1Eb/c which is known not to impact erythropoeisis since no GATA1 protein is made by this mRNA due to its inability to bind to ribosomes. These data show the Xla mouse results from a single nucleotide change impacting the normal splicing of the Gata1 gene. A second goal of this study was to understand why Xla/+ mice exhibit the neonatal transient anemia. A contributing factor is X chromosome inactivation which occurs in female mice during development. The short-term anemia in Xla mice was thought to be due to clonal selection of erythroid lineage cells characterized by the expression of GATA1 protein from the active X chromosome expressing only from the wild type Gata1 locus. Using an X-linked gene expressed in red blood cells (Pgk1, phosphoglycerate kinase 1) that varies between Xla mice and a wild derived strain, CAST/Ei, we examined the active state of the X chromosomes based on the expression of Pgk1 RNA in reticulocytes from hybrid Xla mice generated by breeding of these different strains. Examining expression of the X-linked Pgk1 SNP variant in the RNA of reticulocytes from hybrid Xla/+ mice reveals red blood cells are generated from two types of erythroid lineage cells. Pgk1 SNP RT-PCR analysis reveals that red blood cells not only derive from erythroid progenitors with the active X chromosome carrying the wild type Gata1 gene but also red blood cells are produced by erythroid lineage cells expressing the Xla mutant Gata1 mRNA on the active X chromosome (which does not make GATA1 protein). Therefore, some Xla erythroid cells derive from progenitors which express Gata1 transcripts using Exon 1Eb/c that does not stimulate erythropoiesis due to lack of GATA1 protein. The question is how these erythroid precursors generate normal red blood cells without the production of GATA1 protein. We hypothesize there is a developmentally expressed compensatory gene or pathway replacing GATA1 expression in GATA1-lacking erythroid precursors and required for the production of red blood cells in Xla mice. Analysis is underway to identify a potential novel gene or pathway impacting erythropoiesis in these mutant mice. Disclosures: No relevant conflicts of interest to declare.

Description: Defects in iron absorption and utilization lead to iron deficiency and anemia. Although iron transport in transferrin receptor-mediated endocytosis is well understood, it is not clear how iron is transported from the endosome to mitochondria where it is used to synthesize heme. We undertook a positional cloning project to identify the causative mutation for the hemoglobin deficit (hbd) mouse which suffers from a microcytic, hypochromic anemia associated with a defect in reticulocyte iron uptake. The hbd locus was previously mapped to Chromosome 19 in mouse. We established a mating of B6MOLDF1-+/hbd with C57BL/6J-hbd/hbd mice to generate a high resolution map from 2,454 backcross mice. The hbd critical region was defined by the flanking marker with 6 crossover backcross mice, Kif11, at 0.24 cM on the proximal side, and by the very informative marker with only 1 crossover backcross mouse, Fer1l3 at 0.04 cM on the distal side. The calculated size of the hbd critical region is 836 kb as determined by the sequence available in GenBank contig NT 039689. Our approach for mutation analysis was to amplify exons for each gene in the hbd critical region by genomic PCR using primers derived from flanking sequence. The results were examined for gene deletions, insertions, or large rearrangements as determined by the size of the PCR product generated from normal and mutant DNA. We now report the identification of a strong candidate gene for hbd: Sec15l1, a homologue to yeast SEC15 which has been shown to be a key protein in vesicle docking. The Sec15l1 gene was the only gene in this region with a defect and was shown to have a deletion of exon 8 in hbd genomic DNA. The Sec15l1 partial deletion was verified by Southern blotting. Sec15l1 RNA expression in hbd mice was examined by RT-PCR and subsequent sequencing of the amplicon product. The exon 8 deletion leaves the coding sequence in-frame and a truncated SEC15L1 protein (3 kDa smaller) is predicted to be produced in hbd mice. The deletion in Sec15l1 removes 23 amino acids which includes two amino acids important for tertiary structure: a cysteine residue at position 283 and a proline residue at position 287. Studies of mutant hbd reticulocytes have shown that the binding of transferrin to its receptor and the formation of the endosome is normal but accumulation of iron is deficient in these cells. Iron and transferrin enter the hbd cells but iron does not build up in the cell. The efficient transfer of iron from endosome to mitochondria in normal reticulocytes has been proposed to result from the endosome traversing the cell to dock onto the mitochondria in order to directly transfer iron to it ("kiss and run" hypothesis). In hbd reticulocytes iron may not be transferred efficiently from the endosome to mitochondria because vesicle docking is impaired. This is the first known mutation of a SEC gene homologue in mammals and our findings suggest that SEC15L1 plays a crucial role in the transport of iron in the endocytosis cycle and may involve iron targeting to mitochondria.

Description: Understanding iron metabolism has been enhanced by identification of genes for iron deficiency mouse mutants. We characterized the genetics and iron metabolism of the severe anemia mutant hea (hereditary erythroblastic anemia), which is lethal at 5 to 7 days. The hea mutation results in reduced red blood cell number, hematocrit, and hemoglobin. The hea mice also have elevated Zn protoporphyrin and serum iron. Blood smears from hea mice are abnormal with elevated numbers of smudge cells. Aspects of the hea anemia can be transferred by hematopoietic stem cell transplantation. Neonatal hea mice show a similar hematologic phenotype to the flaky skin (fsn) mutant. We mapped the hea gene near the fsn locus on mouse chromosome 17 and show that the mutants are allelic. Both tissue iron overloading and elevated serum iron are also found in hea and fsn neonates. There is a shift from iron overloading to iron deficiency as fsn mice age. The fsn anemia is cured by an iron-supplemented diet, suggesting an iron utilization defect. When this diet is removed there is reversion to anemia with concomitant loss of overloaded iron stores. We speculate that the hea/fsn gene is required for iron uptake into erythropoietic cells and for kidney iron reabsorption.