ALBERT

Description: We previously described a zebrafish mutant, frascati (frs), which exhibits profound hypochromic anemia and erythroid maturation arrest due to defects in mitochondrial iron uptake. Through positional cloning, we showed that the frs gene encodes a novel member of the vertebrate mitochondrial solute carrier family (SLC25), mitoferrin (mfrn, slc25a37). Mfrn, which is highly expressed in fetal and adult hematopoietic tissues of zebrafish and mouse, functions as the major mitochondrial iron importer essential for heme biosynthesis in vertebrate erythroblasts (Shaw GC, et al. 2006 Nature 440:96–100). To study the function of Mfrn in mammalian organisms, we identified an embryonic stem (ES) cell clone that harbors a gene trap b-geo cassette in intron 1 that inactivates the Mfrn locus. Homozygous disruption of the Mfrn locus results in embryonic lethality at E11.5 from profound anemia due to a failure of primitive erythropoiesis, confirming the requirement of Mfrn in mammalian development . Circumventing the embryonic lethality, we generated Mfrn−/− ES cells to study the role of Mfrn in definitive erythropoiesis by in vitro differentiation of embryoid bodies and mixed chimera assays. Mfrn−/− ES cells were defective in promoting the growth, differentiation, and hemoglobinization of both primitive and definitive erythroblasts by in vitro differentiation of embryoid bodies. In mixed chimera studies, Mfrn−/− ES cells failed to contribute to the erythroid compartment of adult mosaic mice, whereas measurable contribution of Mfrn−/− donor cells could be assayed in the non-erythroid, leukocyte compartment. Transcriptome microarray analysis, using the mouse Affymetrix GeneChip and the custom IronChip, revealed unexpected down-regulation of transcripts for heme-biosynthetic enzymes in Mfrn−/− erythroblasts. The block in protoprophyrin synthesis, as well as mitochondrial heme synthesis, could be partially rescued by the addition of aminolevulinic acid (ALA) to Mfrn−/− erythroblasts in vitro. Our data demonstrate that mitochondrial iron homeostasis, working through the Mfrn iron importer, coordinately regulates the synthetic pathways for porphyrin and heme in developing mammalian erythroblasts.

Description: The adducins are a family of three closely related proteins (α, β, γ) encoded by distinct genes. α- and γ-adducin are expressed ubiquitously, while β expression is restricted to hematopoietic cells and the brain. In red blood cells (RBCs) adducin localizes to spectrin-actin junctions in the membrane skeleton as αβ heterotetramers. Previously (Gilligan et. al., PNAS, 1999) we showed that deletion of β-adducin results in osmotically fragile, microcytic RBCs and an overall phenotype of hereditary spherocytosis (HS). Notably, α-adducin was significantly reduced in β-adducin null RBCs. We also demonstrated that γ-adducin is present in low amounts in normal mouse RBCs and is upregulated ∼5-fold in β-adducin null RBCs. The increase in γ-adducin suggests that αγ heterotetramers may be compensating for the absence of β-adducin. In an effort to analyze γ-adducin function in RBCs in greater detail, we generated a conditional γ-adducin knockout allele in mice using a Cre-loxP strategy to delete exon 2 containing the start codon. All mice were maintained on a segregating B6.129 genetic background. Western blotting confirmed the absence of γ-adducin in spleen homogenates and RBC ghost preparations from γ-adducin null mice. All other membrane skeleton proteins examined by a combination of SDS-PAGE and western blotting, including α- and β-adducin, are normal in γ-adducin null RBCs (spectrin, ankyrin, band 3, protein 4.1, protein 4.2, dematin). Phenotypically, γ-adducin null mice display normal growth curves and show no overt defects. γ-adducin null RBCs appear normal on Wright’s stained peripheral blood smears and by scanning electron microscopy (SEM). The RBC count, hemoglobin content, hematocrit, MCV, reticulocyte %, osmotic fragility, and all other hematopoietic parameters are normal in γ-adducin null mice vs. wildtype. The apparent compensation by γ-adducin in β-adducin null red cells previously observed was tested by intercrossing mice null for γ- and β-adducin to produce βγ null double homozygotes. The additional loss of γ-adducin did not exacerbate the β-adducin null RBC phenotype as judged by examination of peripheral blood smears and SEM. Moreover, RBC osmotic fragility and complete blood counts in βγ-adducin null mice did not differ from β-adducin null mice. Western blotting of RBC ghost proteins confirmed reduction of α-adducin to ∼20% of normal in β-adducin null mice, as previously described. Strikingly, α-adducin in βγ-null RBC ghosts is reduced to barely detectable levels (

Description: Five spontaneous, allelic mutations in the α-spectrin gene, Spna1, have been identified in mice (spherocytosis [sph], sph1J, sph2J, sph2BC, sphDem). All cause severe hemolytic anemia. Here, analysis of 3 new alleles reveals previously unknown consequences of red blood cell (RBC) spectrin deficiency. In sph3J, a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sphIhj, a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced RBC membrane spectrin content, decreased band 3, and absent β-adducin. Reevaluation of available, previously described sph alleles reveals band 3 and adducin deficiency as well. In sph4J, a missense mutation occurs in the C-terminal EF hand domain (C2384Y). Notably, an equally severe hemolytic anemia occurs despite minimally decreased membrane spectrin with normal band 3 levels and present, although reduced, β-adducin. The severity of anemia in sph4J indicates that the highly conserved cysteine residue at the C-terminus of α-spectrin participates in interactions critical to membrane stability. The data reinforce the notion that a membrane bridge in addition to the classic protein 4.1-p55-glycophorin C linkage exists at the RBC junctional complex that involves interactions between spectrin, adducin, and band 3.

Description: Erythrocyte dehydration is a distinguishing feature of hematological diseases including sickle cell anemia and hereditary spherocytosis (HS). Identification of genes influencing hydration status will increase our understanding of red cell pathologies and provide new targets for risk assessment and drug development. We mapped quantitative trait loci (QTLs) influencing red cell hydration using the cell hemoglobin concentration mean (CHCM) as a marker of hydration status. CHCM (analogous to the calculated mean cell hemoglobin content, MCHC) was obtained using an Advia 120 whole blood analyzer, which provides a direct measure of hemoglobin concentration on a cell-by-cell basis. We established seven F2 intercrosses and one backcross and phenotyped 200–500 offspring from each cross at 8–10 weeks of age. Genome-wide scans were performed to detect single loci associated with CHCM (main effect QTL) using the R/qtl software (www.rqtl.org). Significance thresholds were determined by permutation testing (1000 permutations). A highly significant locus was identified on chromosome 7 in 6 of the 8 crosses (maximum LOD score of 82) with a 95% confidence interval (CI) spanning from approximately 90–129 mega base pairs (Mb). Previously we identified two QTL for CHCM (Peters et al., Mamm. Genome17:2006); hence, we have designated the chromosome 7 locus Chcmq3 (CHCM quantitative locus 3). Chcmq3 has a significant impact on the baseline CHCM; it alone accounts for 48% of the variance of the trait. The 95% CI was subsequently narrowed using statistical and bioinformatic tools. First we performed combined cross analysis (Li et al., Genetics169:2005) where genotype data from multiple crosses in which the same QTL is detected is re-coded to a phenotype-based designation based on allele effects and re-analyzed as a single dataset to increase statistical power. This analysis reduced the CI to 7 Mb containing 289 genes. Using a dense single nucleotide polymorphism (SNP) map, including imputed SNPs (Center for Genome Dynamics, http://cgd.jax.org) and phenotypic data from 32 inbred strains, we performed interval-specific haplotype analysis and haplotype association mapping to eliminate regions identical by descent and obtain a final interval of 0.38 Mb containing 30 candidate genes. Twenty one were olfactory receptor (Olfr) genes, poor biological candidates, leaving 9 candidates including the beta-globin cluster. Two major ancestral haplotype blocks span the beta globin cluster, and the parental strains contributing the high allele in each cross (129S1/SvImJ, DBA/2J, NZW/LacJ, WSB/EiJ, MRL/MpJ, CBA/J) are of the hemoglobin-diffuse electrophoretic variant (Hbbd), while the low allele strains (C57BL/6J, SM/J) are hemoglobin-single (Hbbs) variants. Notably, examination of the Mouse Phenome Database (www.jax.org/phenome, project Peters1) reveals that Hbbd strains all have relatively high CHCM values, while Hbbs strains have lower CHCM values. Moreover, in the two crosses in which Chcmq3 was not detected, the parental strains shared the same haplotype at the beta globin locus. As a QTL can not be detected in the absence of genetic variation in the underlying gene, this observation supports beta globin as a strong candidate gene for Chcmq3. Furthermore, in preliminary quantitative PCR studies, strain 129S1/SvImJ, a diffuse variant, showed higher expression of adult beta globin, Hbb-b1, in the spleen than C57BL/6J (single variant). The data suggest that polymorphisms in beta globin itself modify hemoglobin concentration in mice and reinforce the notion that hemoglobin charge and/or structure may directly or indirectly modulate membrane ion transport and erythrocyte hydration in both mice and humans.

Description: Abstract 776 The spontaneous recessive mutation scat (severe combined anemia and thrombocytopenia) arose on the inbred BALB/cBy (BALB) mouse strain. The phenotype of scat is cyclic. All homozygotes are severely anemic and thrombocytopenic at birth. Leukocytes are also significantly depleted (Table). Approximately 13% die during this first “crisis” episode that lasts, on average, until the 9th postnatal day. Remarkably, a spontaneous remission ensues in those surviving the neonatal crisis period wherein all peripheral blood values revert to normal. A second crisis follows, and 94% of the mice die by 30 days of age. The recessive ENU (N-ethyl-N-nitrosourea)-induced mutation, hlb381, on the C57BL/6J (B6) background, is characterized by severe thrombocytopenia and leukopenia with mild anemia. Unlike scat, hlb381 is not cyclical. The phenotype is present at birth and persists throughout life. Despite the phenotypic differences, scat and hlb381 interact genetically; double heterozygotes show non-cyclical severe thrombocytopenia and leukopenia, and mild anemia (Table). This interaction implies that the scat and hlb381 genetic defects affect the same gene or distinct genes within the same pathway. Both scat and hlb381 mapped to overlapping intervals on mouse chromosome 8. Sequence analysis of genes within the interval identified Rasa3 (GAPIII, GAP1IP4BP) as a strong candidate gene for both scat and hlb381. In scat, Rasa3 carries a missense mutation near the N-terminus (G125V, exon 5) and, in hlb381, a missense mutation near the C-terminus (H794L, exon 23). RASA3 is a GTPase activating protein (GAP) that negatively regulates p21 Ras function by accelerating GTP hydrolysis and converting Ras to the inactive GDP bound form. Analysis in Panther and SIFT predicts that both residues are highly conserved and substitutions are likely to be deleterious. Rasa3 is widely expressed throughout embryonic and fetal development in mice, and is ubiquitously expressed in zebrafish 24 hours post fertilization (hpf). RASA3 protein is detected in erythroid tissues and platelets in the adult mouse. Analysis of scat spleen and bone marrow erythrocyte populations by FACS (dual staining for Ter119 and CD71 followed by forward scatter of the Ter119 high population) reveals a severe block in erythropoiesis during crisis periods. The proerythroblast, EryA (basophilic erythroblasts), and EryB (late basophilic and polychromatophilic erythroblasts) populations are significantly increased in frequency vs. wild type, and the EryC (orthochromatophilic erythroblasts and reticulocytes) population is markedly decreased. Annexin V staining revealed no significant differences in any of these populations. Notably, a similar delay in erythroid maturation, albeit much milder, is also seen in hlb381. In pull-down assays using the Ras-binding domain of Raf1 to affinity purify active GTP-bound Ras followed by detection by western blotting with pan-Ras antibody, active GTP-bound Ras is deficient in scat crisis red cells but recovers during remission. Finally, injection of two independent splice-blocking morpholinos designed to disrupt exon 5 and induce disruption of rasa3 mRNA processing resulted in a major decrease in the number of hemoglobinized cells when stained with o-dianisidine at 48 and 72 hpf in zebrafish. Over 90% of morphants showed no hemoglobinized cells at all, or vastly reduced numbers (

Description: Hermansky-Pudlak syndrome (HPS), a disorder of organelle biogenesis, affects lysosomes, melanosomes, and platelet dense bodies. Seven genes cause HPS in humans (HPS1-HPS7) and at least 15 nonallelic mutations cause HPS in mice. Where their function is known, the HPS proteins participate in protein trafficking and vesicle docking/fusion events during organelle biogenesis. HPS-associated genes participate in at least 4 distinct protein complexes: the adaptor complex AP-3; biogenesis of lysosome-related organelles complex 1 (BLOC-1), consisting of 4 HPS proteins (pallidin, muted, cappuccino, HPS7/sandy); BLOC-2, consisting of HPS6/ruby-eye, HPS5/ruby-eye-2, and HPS3/cocoa; and BLOC-3, consisting of HPS1/pale ear and HPS4/light ear. Here, we report the cloning of the mouse HPS mutation reduced pigmentation (rp). We show that the wild-type rp gene encodes a novel, widely expressed 195-amino acid protein that shares 87% amino acid identity with its human orthologue and localizes to punctate cytoplasmic structures. Further, we show that phosphorylated RP is part of the BLOC-1 complex. In mutant rp/rp mice, a premature stop codon truncates the protein after 79 amino acids. Defects in all the 5 known components of BLOC-1, including RP, cause severe HPS in mice, suggesting that the subunits are nonredundant and that BLOC-1 plays a key role in organelle biogenesis.

Description: The red blood cell (RBC) lipid bilayer is supported by an underlying membrane skeleton. Erythroid spectrin, which is composed of flexible alpha and beta subunits, is encoded by the α (Spna1) and β (Spnb1) genes and is the major protein in the membrane skeleton. In mice, five independent autosomal recessive mutations in α-spectrin (sph, sph1J, sph2J, sph2BC, sphDem) and one in β-spectrin (ja) have been identified; all result in severe hemolytic anemia. We have identified two new mouse α-spectrin mutations, sph3J and sph4J, on the NOD.B10 and C57BL/6J background strains, respectively. Linkage analysis in F2 intercrosses localized both mutations to the distal portion of mouse chromosome 1 near Spna1, an obvious candidate gene. In both sph3J and sph4J, novel mutations distinct from the previously described five sph alleles were subsequently identified. In sph3J a cytosine to thymine transition in exon 43 causes a histidine to tyrosine substitution within the αβ nucleation site of α-spectrin (H2012Y). Spna1 message levels are significantly reduced in sph3J reticulocyte RNA. In sph4J a guanine to adenine transition in exon 52 results in a cysteine to tyrosine substitution near the C-terminus (C2384Y). Spna1 message levels are normal in sph4J reticulocytes. Both mutations cause a phenotype of severe hemolytic anemia. In homozygous adult sph3J mice, dramatic decreases in the RBC count (−67%), hemoglobin (−68%), and hematocrit (−65%) are seen. On Wright’s stained peripheral blood smears and by scanning electron microscopy, large numbers of elliptocytes and spherocytes are evident. Significantly increased spleen-to-body weight ratio (+1,200%), bilirubin (+98%), iron (+74%) and circulating reticulocytes are also present. Homozygous adult sph4J mice show similar abnormally shaped RBCs and blood profile changes. SDS-PAGE analysis of sph3J and sph4J RBC membrane skeletons revealed unique changes in membrane skeleton proteins compared to each other and to the five known sph alleles. In sph3J, α- and β-spectrin are significantly decreased but ankyrin, protein 4.1 and protein 4.2 levels are normal. Surprisingly, band 3 is reduced to ~30% of normal, and both α- and β-adducin are nearly undetectable in sph3J RBCs. The presence of normal amounts of ankyrin, which binds band 3 tetramers, suggests that band 3 dimers are absent in sph3J RBCs. These observations indicate that previously unsuspected interactions, direct or indirect, exist between spectrin and band 3 (probably dimers) and between spectrin and adducin within the RBC membrane skeleton. In contrast to sph3J, all RBC membrane skeleton proteins appear normal by SDS-PAGE and western blot analyses of sph4J RBC membranes. Coupled with the severe hemolytic anemia present in these mice, these data suggest that interactions involving the C-terminus of α-spectrin, specifically cysteine 2384, are critical to RBC membrane integrity. Together, the sph3J and sph4J mouse models provide powerful resources for identifying critical interactions within the membrane skeleton that are relevant to the pathogenesis of hereditary elliptocytosis and spherocytosis.