hem6: an ENU-induced recessive hypochromic microcytic anemia mutation in the mouse

Abstract

Mouse models have proven invaluable for understanding erythropoiesis. Here, we describe an autosomal recessive, inherited anemia in the mouse mutant hem6. Hematologic and transplantation analyses reveal a mild, congenital, hypochromic, microcytic anemia intrinsic to the hematopoietic system that is associated with a decreased red blood cell zinc protoporphyrin to heme ratio, indicative of porphyrin insufficiency. Intercross matings show that hem6 can suppress the porphyric phenotype of mice with erythropoietic protoporphyria (EPP). Furthermore, iron uptake studies in hem6 reticulocytes demonstrate defective incorporation of iron into heme that can be partially corrected by the addition of porphyrin precursors. Gene expression and enzymatic assays indicate that erythroid 5-aminolevulinic acid synthase (Alas2) is decreased in hem6 animals, suggesting a mechanism that could account for the anemia. Overall, these data lead to the hypothesis that hem6 encodes a protein that directly or indirectly regulates the expression of Alas2.

Figure 1

hem6 peripheral blood smear. Wright-Giemsa–stained peripheral blood smears demonstrating relatively prominent hypochromia with abundant target cells and minimal aniosopoikilocytosis in the mutant.

Figure 2

The hem6 anemia is transplantable. Irradiated C57BL/6J-Igh^a Thy1^a Gpi1 ^a +/+ or hem6/hem6 animals that had received C57BL/6J-Igh^b Thy1^b Gpi1^b hem6/hem6 (n = 7; black lines) or +/+ (n = 8; gray lines) fetal liver cells, respectively, were serially monitored for RBC chimerism (dotted lines) by Gpi1 isozyme analysis and for blood parameters. The decrease (in the case of hem6 → +) or increase (in the case of + → hem6) in MCV (solid lines) closely follows engraftment by the donor cells. Error bars indicate plus or minus 1 SD.

Figure 3

hem6 reticulocytes have a defect in iron incorporation into heme partially corrected by ALA. (A) Reticulocyte iron uptake and heme biosynthesis were measured in 129B6N₂F₁ hem6/hem6 (n = 4; ■) and +/+ (n = 4; ▩) control littermates following 30 minutes of incubation with ⁵⁵Fe₂-transferrin with or without 2 mM added ALA. The data are normalized to and presented as uptake per microgram of RNA per hour. (B) Average heme iron to whole-cell iron ratio from 3 independent experiments with or without ALA treatment is shown. Error bars represent plus or minus 1 SD.

Figure 4

hem6 modifies erythropoietic protoporphyria. (A) Flow cytometric analysis of erythroid PPIX levels in 129S6;C- fech^m1Pas/+ × 129S6;B6-hem6 intercross offspring, showing a representative set of 3 animals with key genotypes. Numbers in the upper left corner of each scattergram represent the percentage of cells in the fluorocyte gate. The horizontal lines separate the positive gate (above the line) from the negative gate (below the line). (B) Histogram of the average fluorocyte percentage. (C) Liver–body weight ratios of selected genotypes. (D) Hemoglobin levels of selected genotypes. All error bars represent plus or minus 1 SD.

Figure 5

hem6 reticulocytes are Alas2 deficient. Alas (Alas1 + Alas2) enzymatic activity normalized to reticulocyte RNA content, Alas2 mRNA levels normalized to β-actin mRNA levels, and the ratio of normalized Alas activity to normalized Alas2 mRNA. Each value is expressed in arbitrary units plus or minus 1 SD.