**1. Introduction**

The thalassemias are a major cause of morbidity and mortality throughout much of the world [1–9]. Thalassemias are characterized by the disruption of the synthesis of normal adult hemoglobin (HbA; an α2β2 tetramer; **Figure 1**) consequent to a diverse array of genetic mutations/deletions to either the β or α-hemoglobin chain genes (Chromosomes 11 and 16, respectively). As a consequence of reduced/absent production of β-chains, β thalassemia is characterized by the presence of highly unstable monomeric α-chains as these chains cannot self-associate and indeed require a chaperone protein to prevent precipitation [10]. In contrast, α thalassemia is characterized by the presence of relatively stable tetrameric β chains. Interestingly, as schematically shown in **Figure 1**, unlike most genes, there are four copies of the α-globin genes;

#### **Figure 1.**

*Molecular basis and clinical diagnosis of the α and β thalassemias [1–9]. β\* denotes abnormal β hemoglobin gene such as HbS or HbE.*

this is in contrast to the expected two copies of the β-globin genes. The evolutionary duplication of the human α-chain genes may have been favored consequent to the inherent instability of monomeric α-chains. Indeed, the instability of the α-chains is the key factor underlying the pathophysiology of the β thalassemic red blood cell (RBC). Moreover, the pathophysiology of β thalassemia can be further complicated by the geographical prevalence, and high frequency, of a number of mutated β hemoglobin genes (e.g., sickle hemoglobin, hemoglobin E and hemoglobin C). If a mutated β-chain is the only functional β-chain present, the resultant disease will be more severe than that observed in β thalassemia Intermedia (a single normal β-globin gene). Loss of both β-chain genes gives rise to severe β Thalassemia Major which is fatal in the absence of transfusion therapy. The α thalassemias are characterized by a broader range of disease states due to the presence of 4 α genes. The loss of expression from a single gene (α Thalassemia1 Trait) is often asymptomatic and undiagnosed; though the individual is a carrier for α Thalassemia and, in high frequency geographic areas may be at elevated risk for symptomatic disease transmission to an offspring. Deletion of two or three α-genes results in severe disease as a single active α-gene cannot, due to the instability of the chain, produce sufficient mature α-chains to form sufficient HbA. Loss of all four α-genes is fatal (resulting in Hydrops fetalis) due to the crucial role that α-chains play in embryonic and fetal hemoglobin. In contrast to β thalassemia, stable mutated α-chains are rare so typically these do not pose a significant complication in the pathophysiology of α thalassemia.

In this chapter we will further explore the pathophysiology of the β thalassemic RBC. Surprisingly, while significant injury to the thalassemic erythrocyte arises from the excess α-chains, the underlying mechanisms by which these chains damage and subsequently destroy the thalassemic RBC in the bone marrow and peripheral blood have not been clearly delineated. Our lack of understanding of the mechanisms of α-chain mediated damage is due, in part, to three major factors: (1) studies of RBC from β thalassemic individuals are difficult to do since these cells, upon collection, already exhibit significant injury and represent a survivorship bias since up to 80% of erythroid precursors are destroyed within the bone; (2) β thalassemic patients are typically transfused to both correct the severe anemia accompanying the disease and to prevent endogenous erythropoiesis of defective RBC; and (3) the lack of a good experimental model by which the pathophysiology of excess globin chains on human RBC can be examined.

While little can be done to change the first two problems, researchers have attempted to tackle the third issue using murine models of thalassemia [11–17]. Original murine studies examining the knockout of the murine β-chains were not

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**Figure 2.**

vivo *survival.*

*Model Human β Thalassemic Erythrocytes: Effect of Unpaired Purified α-Hemoglobin Chains…*

productive as the murine α-chains behave significantly different from their human counterpart. To overcome this problem, human α-chain genes were inserted into the mouse genome in place of the murine genes. Again, these studies failed to give rise to as severe a phenotype as is seen in the human disease. Subsequent studies utilized additional mutations to produce symptomatic disease in the murine context—albeit with still substantial differences from the pathophysiology seen in the human β thalassemic RBC. Hence, an alternative approach for studying the pathophysiology

To this end, our laboratory developed an *in vitro* model of the HUMAN β thalassemic erythrocyte [18–27]. In this model, purified human α-chains are entrapped within normal human RBC (or, if desired, mouse RBC) by osmotic lysis and resealing (**Figure 2**) [18–34]. As previously shown, osmotic lysis and resealing results in RBC exhibiting normal hemoglobin concentration and volume (**Table 1**) as well as normal ATP concentration, oxidant sensitivity, morphology and

*Generation of model β thalassemic RBC from normal human donor cells via osmotic lysis and resealing [18–34]. Osmotically lysed and resealed RBC have normal morphology and metabolism and exhibit normal* in

*DOI: http://dx.doi.org/10.5772/intechopen.90288*

**2. Model human β thalassemic RBC**

of unpaired α-chains on the human RBC was needed.

*Model Human β Thalassemic Erythrocytes: Effect of Unpaired Purified α-Hemoglobin Chains… DOI: http://dx.doi.org/10.5772/intechopen.90288*

productive as the murine α-chains behave significantly different from their human counterpart. To overcome this problem, human α-chain genes were inserted into the mouse genome in place of the murine genes. Again, these studies failed to give rise to as severe a phenotype as is seen in the human disease. Subsequent studies utilized additional mutations to produce symptomatic disease in the murine context—albeit with still substantial differences from the pathophysiology seen in the human β thalassemic RBC. Hence, an alternative approach for studying the pathophysiology of unpaired α-chains on the human RBC was needed.
