A transgenic mouse model expressing exclusively human hemoglobin E: Indications of a mild oxidative stress

Abstract

Hemoglobin (Hb) E (β26 Glu→ Lys) is the most common abnormal hemoglobin (Hb) variant in the world. Homozygotes for HbE are mildly thalassemic as a result of the alternate splice mutation and present with a benign clinical picture (microcytic and mildly anemic) with rare clinical symptoms. Given that the human red blood cell (RBC) contains both HbE and excess α-chains along with minor hemoglobins, the consequence of HbE alone on RBC pathophysiology has not been elucidated. This becomes critical for the highly morbid βE-thalassemia disease. We have generated transgenic mice exclusively expressing human HbE (HbEKO) that exhibit the known aberrant splicing of βE globin mRNA, but are essentially non-thalassemic as demonstrated by RBC α/β (human) globin chain synthesis. These mice exhibit hematological characteristics similar to presentations in human EE individuals: microcytic RBC with low MCV and MCH but normal MCHC; target RBC; mild anemia with low Hb, HCT and mildly elevated reticulocyte levels and decreased osmotic fragility, indicating altered RBC surface area to volume ratio. These alterations are correlated with a mild RBC oxidative stress indicated by enhanced membrane lipid peroxidation, elevated zinc protoporphyrin levels, and by small but significant changes in cardiac function. The C57 (background) mouse and full KO mouse models expressing HbE with the presence of HbS or HbA are used as controls. In select cases, the HbA full KO mouse model is compared but found to be limited due to its RBC thalassemic characteristics. Since the HbEKO mouse RBC lacks an abundance of excess α-chains that would approximate a mouse thalassemia (or a human thalassemia), the results indicate that the observed in vivo RBC mild oxidative stress arises, at least in part, from the molecular consequences of the HbE mutation.

The G6PD flow-cytometric assay is a reliable tool for diagnosis of G6PD deficiency in women and anaemic subjects

“…Distribution of abnormal haemoglobin variants is super-imposable on that of G6PD deficiency and malaria 18 ; in certain populations of Southeast Asia, Hb E prevalence reaches peaks of 60–70% 19 . Both Hb E and beta thalassaemia have been found to be associated with increased intracellular oxidative stress 12 , 20 , 21 . In contrast in early observations of the Sardinian population where beta-thalassaemia and G6PD deficiency co-exist, it was noted that subjects with G6PD deficiency and concomitant beta-thalassaemic trait were less likely to have favism as compared to G6PD deficient subjects with a normal haemoglobin type 22 , 23 .…”

Generating S-Nitrosothiols from Hemoglobin

“…Human HbE was purified from red blood cells (RBC) obtained from transgenic mice expressing human HbE as described previously (57,64,65). HbE was shown previously to have similar allosteric and oxygen-binding properties as HbA, but relative to HbA, it appears to have a higher redox potential as manifested in a lower rate for nitrite reductase activity in both the T and R state and a faster rate of reduction by L-cysteine (57).…”

HbE/β-Thalassemia and Oxidative Stress: The Key to Pathophysiological Mechanisms and Novel Therapeutics

“…In contrast, these parameters of oxidative stress were unremarkable in HbE trait controls. It should be noted that a full knockout HbE transgenic mouse (HbEKO) was generated that expressed solely human HbE in a mouse RBC (38). The HbEKO mouse exhibited a mild oxidative stress similar to that seen in human The role of RBC oxidative stress, as a key player initiating the pathophysiological mechanisms of this disease, becomes convincing in a study of HbE/b-thal RBCs and a human plasma proteome analysis (76).…”