A novel ENU-generated truncation mutation lacking the spectrin-binding and C-terminal regulatory domains of Ank1 models severe hemolytic hereditary spherocytosis

Abstract

Objective: Hereditary spherocytosis (HS) is a heterogeneous group of spontaneously arising and inherited red blood cell disorders ranging from very mild subclinical cases to severe and life-threatening cases, with symptoms linked directly to the severity of the mutation at the molecular level. We investigated a novel mouse model in which the heterozygotes present with the diagnostic hallmarks of mild HS and surviving homozygotes phenocopy severe hemolytic HS.

Materials and methods: We used N-ethyl-N-nitrosourea mutagenesis to generate random point mutations in the mouse genome and a dominant screen to identify mouse models of human hematopoietic disease. Gene mapping of the HS strain revealed a unique in-frame nonsense mutation arising from a single base transversion in exon 27 of Ank1 (strain designation: Ank1(E924X)). Employing conventional hematopoietic, pathological, biochemical, and cell biology assays, we characterized heterozygous and homozygous Ank1(E924X) mice at the biochemical, cellular, and pathophysiological levels.

Results: Although Ank1(E924X/E924X) red blood cell ghosts lack abundant full-length ankyrin-1 isoforms, N-terminal epitope ankyrin-1 antibodies reveal a band consistent with the theoretical size of a truncated mutant ankyrin-1. Using domain-specific antibodies, we further show that this protein lacks both a spectrin-binding domain and a C-terminal regulatory domain. Finally, using antisera that detect C-terminal residues of the products of alternative Ank1 transcripts, we find unique immunoreactive bands not observed in red blood cell ghosts from wild-type or Ank1(E924X) heterozygous mice, including a band similar in size to full-length ankyrin-1.

Conclusions: The Ank1(E924X) strain provides a novel tool to study Ank1 and model HS.

Figure 1

The RBC phenotype of an ENU-generated mutant mouse strain ENU7192 demonstrates diagnostic features of human hereditary spherocytosis. (A) Enumeration and MCV of RBC parameters of individual mice from a B6.C3H control hybrid strain (gray) and generation 2 and 3 (G2/3) progeny of mutagenized strain ENU7192 (black). Individual affected mice with RBC count and MCV parameters deviating by more than 2 SDs (box) from nonaffected littermates and nonmutagenized hybrid controls were selected for heritability testing and mapping. (B) Wright-Geimsa stain of PB smear of unaffected (upper panel) and affected (lower panel) ENU7192 at 400× magnification. (C) Histogram of mean fluorescence intensity (MFI) of RBCs labeled with eosin-5′-maleimide (EMA) where affected (red) and nonaffected (open peak) mice are defined by the low MCV parameter and confirmed by prospective PCR genotyping for the E924X or WT Ank1 genotypes (see Supplementary Figure E1; online only, available at www.exphem.org). The mean MFI values and SDs are shown. (D) Osmotic hemolysis plot of RBCs from affected (● red) and nonaffected (■ blue) ENU7192 (C3H N8–9) mice and C3H/HeJ parental strain (▲ black, dashed lined) (n = 4–7 mice per strain). (E) Affected mice (red) have increased percent of circulating reticulocytes compared to nonaffected littermates (blue). *Significantly different than unaffected mice with p < 0.05.

Figure 2

A single ENU-generated point mutation (G → T transversion) in Ank1 exon 27 yields a new mouse model of hereditary spherocytosis. (A) Table indicating the number of affected and unaffected ENU7192 mice heterozygous (red) or null (white) for Chr. 8 marker loci derived from the mutagenized strain (B6). The indicated candidate region of Chr. 8 indicated is linked to the mutant phenotype of affected mice. (B) Schematic mouse Ank1 locus highlight transcript variant 1 and the mapped position of the ENU7192 point mutation (in Ank1). A portion of Ank1 exon 27 is expanded to show the genomic sequence with the position of the ENU-generated point mutation (G → T transversion) encoding a stop codon (*) in place of glutamate (E) at residue 924 (ankyrin-1, isoform 1 [NP001104253.1]) of the theoretical translated product. The locations of the E924X point mutation within the Ank1 genomic locus relative to other reported Ank1 mutant mouse strains (Ank1^nb (nb) [17] and Ank1^RBC2 (RBC2) [31]) is shown. ^‡Note that the nb mutation is in exon 38 using the exon numbering for Ank1, transcript variant 1(NM001110783.1) but is reported as exon 36 in the original mapping publication [17]). sAnk1 indicates the position of the alternative start site for the short ankyrin-1 isoform using the exon numbering scheme of Ank1, transcript variant 1 [12]. (C) The theoretical truncated ankyrin-1 protein products generated by the known Ank1 mouse mutant alleles. Note that ankyrin-1 protein attributable to the RBC2 allele has not been detected in reticulocytes or erythroid progenitors [31].

Figure 2

A single ENU-generated point mutation (G → T transversion) in Ank1 exon 27 yields a new mouse model of hereditary spherocytosis. (A) Table indicating the number of affected and unaffected ENU7192 mice heterozygous (red) or null (white) for Chr. 8 marker loci derived from the mutagenized strain (B6). The indicated candidate region of Chr. 8 indicated is linked to the mutant phenotype of affected mice. (B) Schematic mouse Ank1 locus highlight transcript variant 1 and the mapped position of the ENU7192 point mutation (in Ank1). A portion of Ank1 exon 27 is expanded to show the genomic sequence with the position of the ENU-generated point mutation (G → T transversion) encoding a stop codon (*) in place of glutamate (E) at residue 924 (ankyrin-1, isoform 1 [NP001104253.1]) of the theoretical translated product. The locations of the E924X point mutation within the Ank1 genomic locus relative to other reported Ank1 mutant mouse strains (Ank1^nb (nb) [17] and Ank1^RBC2 (RBC2) [31]) is shown. ^‡Note that the nb mutation is in exon 38 using the exon numbering for Ank1, transcript variant 1(NM001110783.1) but is reported as exon 36 in the original mapping publication [17]). sAnk1 indicates the position of the alternative start site for the short ankyrin-1 isoform using the exon numbering scheme of Ank1, transcript variant 1 [12]. (C) The theoretical truncated ankyrin-1 protein products generated by the known Ank1 mouse mutant alleles. Note that ankyrin-1 protein attributable to the RBC2 allele has not been detected in reticulocytes or erythroid progenitors [31].

Figure 3

Homozygous Ank1^E924X mice display severe, hemolytic anemia and RBC structural defects. (A) E17.5 embryos and (B) day 1 neonates obtained from Ank1^E924X/+ intercross matings of a hybrid strain (C3J;129 [N2]). The Ank1^E924X genotype abbreviations are as indicated for wild-type (+/+), heterozygous (E924X/+), or homozygous mutants (E924X/E924X). The arrow indicates a jaundiced pup. (C) Flow cytometric analysis histograms of thiazole orange–stained PB collected from WT (+/+) and E924X/E924X mice. Cells were first gated for forward scatter (FSC) and side scatter (SSC) properties on a log-scale to exclude platelets and white blood cells from the analysis. The peak in the third decade includes nucleated erythroblasts present in the PB of E924X/E924X mice and these were excluded from the retic % calculation. There is no significant contribution of nucleated erythroblasts in the analysis of WT mice. (D) Wright-Geimsa stains of PB smears of WT (+/+) (upper panel) and Ank1^E924X homozygous mutant mice (lower panel) at 400× magnification. (E) Scanning electron micrographs (SEM) (HitachiS4700 FESEM at 2 kV) of fixed red cells sampled from +/+ (upper panels) and E924X/E924X (lower panels). Scale bars for SEM: 10 μm (left panels) and 3 μm (right panels). Micrographs and photographs were processed in Adobe Illustrator (11.0) with equal scaling and color correction (where appropriate).

Figure 3

Homozygous Ank1^E924X mice display severe, hemolytic anemia and RBC structural defects. (A) E17.5 embryos and (B) day 1 neonates obtained from Ank1^E924X/+ intercross matings of a hybrid strain (C3J;129 [N2]). The Ank1^E924X genotype abbreviations are as indicated for wild-type (+/+), heterozygous (E924X/+), or homozygous mutants (E924X/E924X). The arrow indicates a jaundiced pup. (C) Flow cytometric analysis histograms of thiazole orange–stained PB collected from WT (+/+) and E924X/E924X mice. Cells were first gated for forward scatter (FSC) and side scatter (SSC) properties on a log-scale to exclude platelets and white blood cells from the analysis. The peak in the third decade includes nucleated erythroblasts present in the PB of E924X/E924X mice and these were excluded from the retic % calculation. There is no significant contribution of nucleated erythroblasts in the analysis of WT mice. (D) Wright-Geimsa stains of PB smears of WT (+/+) (upper panel) and Ank1^E924X homozygous mutant mice (lower panel) at 400× magnification. (E) Scanning electron micrographs (SEM) (HitachiS4700 FESEM at 2 kV) of fixed red cells sampled from +/+ (upper panels) and E924X/E924X (lower panels). Scale bars for SEM: 10 μm (left panels) and 3 μm (right panels). Micrographs and photographs were processed in Adobe Illustrator (11.0) with equal scaling and color correction (where appropriate).

Figure 4

Ank1^E924X homozygous mice display several phenotypes analogous to the pathological features of severe hereditary spherocytosis in humans. (A) Photographs of spleen (left panel), liver (center panel), gall bladder with gallstones (i, ii, inset) and heart (right panel) dissected from 8- to 12-week-old WT (+/+), heterozygous mutant (E924X/+) (only spleen shown) and homozygous mutant (E924X/E924X) mice (C3H:129 [N2] hybrid). (B) Micrographs of hematoxylin and eosin–stained sections of formalin-fixed BM (400×), spleen (200×, inset 400×). Arrows in the liver sections indicate sites of extramedullary erythropoiesis in the liver (images acquired with Olympus C-5060 digital camera). Photographs and micrographs were processed in Adobe Illustrator (11.0) with equal scaling and color correction where appropriate for presentation.

Figure 5

Ank1^E924X homozygous mutant mice demonstrate profound compensatory erythropoiesis in the spleen, liver, and peripheral blood. (A) Representative CD71/Ter119 flow cytometry profiles of BM, spleen, and PB. RBCs were lysed to exclude mature erythrocytes and reticulocytes. The percentage of CD71⁺Ter119⁺ cells (erythroblasts) (upper right quadrant) as a percentage of total cells for the indicated tissues is shown. (B) CFU-C present (per 1 × 10⁵ nucleated cells plated) in the indicated tissues of WT (+/+) (open bars), E924X/+ (shaded bars) and E924X/E924X (solid bars) mutant mice. *Significantly different than both WT and E924X/+ with p < 0.05. **Significantly different than E985X/+ with p < 0.05.

Figure 6

Several components of the band 3 macrocomplexes are destabilized in Ank1^E924X homozygous mice. (A) Coomasie Blue stain of fractionated RBC ghosts (10% sodium dodecyl sulfate polyacrylamide gel electrophoresis) prepared from PB of WT (+/+) and homozygous Ank1^E924X (E924X/E924X) mutant mice. Positions of the major RBC membrane proteins are indicated with dashed lines. (B) Immunoblot (α– ankyrin-1 monoclonal antibody [clone 8C3]) of fractionated RBC ghosts prepared from (1) WT (+/+), (2) E924X/+, and (3) E924X/E924X blood. Arrows indicate major immunoreactive bands. (C) Immuno-blot of fractionated RBC ghosts prepared from WT (+/+) and E924X/E924X blood probed with antibodies raised against major RBC membrane and cytoskeletal proteins. For all experiments, gel lanes were loaded with equal concentrations of total protein as measured by BCA assay. The size of molecular weight markers (in kD) and the identification of protein bands are indicated. Digital scans and LI-COR images were processed in Adobe Illustrator (11.0) with equal scaling and contrast enhancement, where required.

Figure 7

Ank1^E924X homozygous mutant mice express several unique truncated forms of ankyrin. (A) Schematic of the structural domains of erythroid ankyrin protein and the site of the theoretical E924X truncation. Peptides (p89, p65, αC-term [A], [B], and [C]) identical to the indicated residues of the products of canonical (isoforms 1 and 2) and alternatively spliced variants of mouse Ank1 were used to raise domain-specific antibodies (rabbit primary antibodies). The location of residues (ankyrin 1, isoform 1) of canonical peptides used as immunogens are indicated in the schematic and the alternative C-terminal peptides are shown (inset box). (B) Immunoblots of RBC ghosts prepared from (1) WT (+/+) and (2) E924X/E924X mice, fractioned by sodium dodecyl sulfate polyacrylamide gel electrophoresis and probed with ankyrin domain-specific anti-sera as indicated. LI-COR images were processed in Adobe Illustrator (11.0) with equal scaling and contrast enhancement where required.