A Transgenic Mouse Model of Homozygous HbE Disease.

Hemoglobin E (β26 Glu→Lys) is the most common abnormal hemoglobin (Hb) variant in the world. Homozygotes for HbE have a benign clinical picture (microcytic and mildly anemic) with rare clinical symptoms. However, when HbE coexists with β⁰-thalassemia, it is the most common severe hemoglobinopathy world-wide. National research priorities in the United States now include HbE related diseases because of the growing number of immigrants with these diseases, as well as the significant world impact of this life-threatening condition. In efforts to understand these diseases, a transgenic mouse model of homozygous HbE Disease was generated (Chen et al. Blood Cells Mol. Dis., 2004). Subsequent breeding has yielded knockout transgenic mice expressing exclusively human HbE: RBC smears show hypochromia, target cells, and stress reticulocytes. Measurement of PS exposure with Annexin V does not show a significantly increased population of PS exposing cells in the blood of these mice. Density gradients showing broader RBC density distributions correlate with increased RDW. RBC parameters determined by the ADVIA show low MCV (31.6±1.5fl vs. 46.9±2.1fl for C57) and MCH (9.8±0.1pg vs. 14.8±0.7pg for C57); and normal MCHC (32.0±2.0g/dl vs. 31.2±1.7 g/dl for C57) [n=5]. These RBC parameters mirror human HbE homozygotes. Increased levels of zinc protoporphyrin (ZPP), free protoporphyrin (PPIX), and fluorescent heme degradation products (FHDP) are found. To see if these porphyrins and FHDP arise from alterations in heme synthesis, real-time PCR technology methods were used. A comparison of the gene expression of enzymes involved in heme synthesis was made from RNA extracted from the spleens (the mouse hematopoietic organ). Upregulation in the rate-limiting enzyme erythroid delta-aminolevulinate synthase (ALAS) and other heme synthesis enzymes is suggested. These observations may be explained by increased erythropoiesis, compatible with the increase in % reticulocytes (7.2%±2.5, n=5); and/or elevated RBC porphyrins observed in these mice. This result is consistent with the effect of elevated porphyrins on ALAS in peripheral blood of erythropoietic protoporphyria-chemically induced mice (Inafuku et al., J Dermatol Sci, 1999). Considering the above, we conclude that the HbE full knockout mice are an excellent model for the benign human homozygous EE disease, and will serve as an important control for the proposed transgenic studies to generate HbE/β-thalassemia mice.