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.