Two New Recessive Mouse Mutations Cause Severe Hemolytic Anemia and Reveal Unexpected Interactions in the C-Terminus of α-Spectrin.

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, sph^1J, sph^2J, sph^2BC, sph^Dem) and one in β-spectrin (ja) have been identified; all result in severe hemolytic anemia. We have identified two new mouse α-spectrin mutations, sph^3J and sph^4J, 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 sph^3J and sph^4J, novel mutations distinct from the previously described five sph alleles were subsequently identified. In sph^3J 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 sph^3J reticulocyte RNA. In sph^4J 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 sph^4J reticulocytes. Both mutations cause a phenotype of severe hemolytic anemia. In homozygous adult sph^3J 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 sph^4J mice show similar abnormally shaped RBCs and blood profile changes. SDS-PAGE analysis of sph^3J and sph^4J RBC membrane skeletons revealed unique changes in membrane skeleton proteins compared to each other and to the five known sph alleles. In sph^3J, 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 sph^3J RBCs. The presence of normal amounts of ankyrin, which binds band 3 tetramers, suggests that band 3 dimers are absent in sph^3J 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 sph^3J, all RBC membrane skeleton proteins appear normal by SDS-PAGE and western blot analyses of sph^4J 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 sph^3J and sph^4J mouse models provide powerful resources for identifying critical interactions within the membrane skeleton that are relevant to the pathogenesis of hereditary elliptocytosis and spherocytosis.