A Mouse Model for Human Unstable Hemoglobin Santa Ana

Abstract

In the present study, we described the phenotype, histologic morphology, and molecular etiology of a mouse model of unstable hemoglobin Santa Ana. Hematologic evaluation of anemic mice (Anem/+) discovered after N-ethyl-N-nitrosourea mutagenesis revealed moderate anemia with intense reticulocytosis and polychromasia, followed by anisocytosis, macrocytosis, hypochromia, and intraerythrocytic inclusion and Heinz bodies. The mice also demonstrated hemoglobinuria, bilirubinemia, and erythrocytic populations with differing resistance to osmotic lysis. Splenomegaly (particularly in older mutant mice) and jaundice were apparent at necropsy. Histopathologic examination revealed dramatically increased hematopoiesis and hemosiderosis in hematopoietic organs and intracellular iron deposition in tubular renal cells. These data are characteristic of a congenital hemolytic regenerative anemia, similar to human anemias due to unstable hemoglobin. Genetic mapping assigned the affected gene to mouse chromosome 7, approximately 50 cM from the Hbb locus. The sequence of the mutant Hbb gene exhibited a T→C transversion at nucleotide 179 in Hbb-b1, leading to the substitution of proline for leucine at amino acid residue 88 and thus homologous to the genetic defect underlying Santa Ana anemia in humans.

Figure 1.

Positional cloning of the Hbb–b1 Santa Ana allele. (A) The chromosome 7 haplotypes from 35 backcross mice are indicated. (B) The haplotype of 86 mice from the same backcross are indicated. Panels A and B respectively show 2 recombinant mice and 1 recombinant mouse that were critical for determining the minimal region for the Hbb-b1 gene. (C) Point mutation in the Hbb-b1 gene from anemic mice. A T→C transition causes an amino acid change from leucine to proline at residue 88. This point mutation is located in the 2nd exon of the Hbb-b1gene of anemic mice.

Figure 2.

Blood smears from control and anemic mice. (A) Control mice. Note the uniformity of RBC size, shape, and color. (B–D) Anemic mice. Note the variation in RBC size, shape, and color. ABI, amorphous basophilic inclusion; BS, basophilic stippling; HJB, Howell–Jolly body; HYPOC, hypochromia; POIK, poikilocyte; POL, polychromasia. May–Grümwald–Giemsa stain; magnification, 1000×.

Figure 3.

Cumulative and derivative curves for RBC osmotic fragility. The continuous lines represent the percentage of hemolysis; the dotted lines indicated the increment of hemolysis according to NaCl concentration.

Figure 4.

(A) Hemoglobin cellulose acetate and (B) hemoglobin polypeptide chain electrophoresis in alkaline pH (Ponceau S stain) showing electrophoretic curves of blood from anemic and control mice.

Figure 5.

Methemoglobin curve of control and anemic blood. Data from 2 different of 10-wk-old anemic and control female mice.

Figure 6.

Histologic sections obtained from spleen, liver, and kidney of control and anemic mice and stained with Perl iron stain. (A) Spleen, control (magnification, 100×): discrete distribution pattern of iron deposits revealed in blue. (B) Spleen, anemic (magnification, 100×): moderate quantity of iron (blue). (C) Liver, control (magnification, 400×): no iron deposits present. (D) Liver, anemic (magnification, 400×): moderate quantity of iron in Kupfer cells (arrows). (E) Kidney, control (magnification, 400×): no iron deposits. (F) Kidney, anemic (magnification, 400×): note the blue staining (indicating iron deposits) in tubular cells.