Impact of Haptoglobin Gene Polymorphism on Phenotypic Variability in β-thalassemia Patients: Relation to Iron Overload and Oxidative Stress

• This study assessed the effect of haptoglobin gene polymorphism on phenotypic variability in thalassemia patients and its relation to iron overload and oxidative stress.
• The study revealed higher incidence of haptoglobin 2-2genotype in thalassemic patients. Serum level of MDA & ferritin were higher and haptoglobin levels were lower in patients with Hp2-2 genotype.
• Haptoglobin polymorphism and phenotypic variability have a major influence on oxidative stress in patients with thalassemia and those with the Hp 2-2 phenotype are more vulnerable to iron-steered oxidative stress than those with different phenotypes.

Thalassemia has emerged as a significant problem in clinical practice.  Analysis of the molecular principles of the different types of thalassemia and the phenotype / genotype relationships have made great advances in understanding the processes behind the phenotypic variability in β-thalassemia [16]. This phenotypic variability is mainly due to genetic factors which still have been incompletely defined. In addition, little is known about the phenotypic heterogeneity complications of thalassemia and its pathophysiology. Repeated blood transfusions are necessary for the survival of thalassemia patients, but lead to excessive iron   overload, altered trace element levels and antioxidant enzymes, all of which may cause oxidative stress [17].

Nitric oxide (NO) can oxidize heme, the free hemoglobin molecule carrying the iron moiety, causing oxidative stress-mediated damage via hydrogen peroxides and lipid. Plasma Haptoglobin (HP) binds to free hemoglobin to scavenge it from oxidation, possibly halting additional heme-mediated nitric oxide damage, iron renal excretion, and additional oxidative damage [18].

In the current study, a significant decline in mean serum haptoglobin level in βTM patients compared to the control group was observed. Ragab et al [19] confirmed our results. They found that thalassemic children had intense Hp depletion when compared to the control group. They concluded that the presence of ineffective erythropoiesis as well as hemolysis results in a decrease in the level of plasma haptoglobin. Regardless of hemolysis site (extravascular or intravascular) or the existence of concordant inflammation, its depletion is an indication for the rapid diagnosis of enhanced destruction of red cells. Thus, Hp depletion in patients with thalassemia is assigned to hemolysis and ineffective erythropoiesis.

MDA is a lipid peroxidation and oxidative stress marker that is produced as the final product of oxygenation of polyunsaturated fatty acid [5]. Our results revealed a significant increase in MDA levels in thalassemia patients compared to controls which reflects the patient’s state of oxidative stress. This was in accordance with Atmakusuma et al [20] who demonstrated that thalassemia patients show increased MDA levels compared to healthy controls reflecting the state of oxidative stress of these patients. In addition, Basu et al [21] reported that Malonaldehyde (MDA) is increased significantly in both transfusion and non-transfusion-dependent β-thalassemia. Regardless of severity and blood transfusion dependency they signified MDA as a reliable surrogate indication of iron overload in β-thalassemia. As a result, it can be utilized instead of ferritin as a predictive biomarker of management, particularly in the consequence of chelation therapy.

In the present study, 56% of thalassemic patients had the Hp2-2 genotype, while in the control group Hp2-1 genotype predominated. Our results agree with Melamed- Frank et al, [22] who reported that Hp2-2 persons are more susceptible to oxidative stress than Hp1-1 and Hp2-1persons. Moreover, our results showed that Hp2-1 and Hp2-2 genotype are associated with lower serum haptoglobin levels while the genotype Hp1-1 is linked with the highest serum haptoglobin level. This result was confirmed by Van et al [23] who stated that persons with the Hp 2-2 phenotype have lower reference range for haptoglobin concentration than those with the Hp 1-1 and Hp 2-1 phenotypes.

Additionally, Kasvosve et al [24] described that the reference values of haptoglobin depend on the Hp phenotype. In the same context, Imrie et al [25] reported that Hp levels were related to Hb levels, Hp genotype, splenomegaly and age. Additionally, it appears that the rate of free hemoglobin clearance from circulation is phenotype-dependent as shown by Van et al [23] hence the clearance of Hp1-1-Hb complex is faster than that of Hp2-2Hb complex. Thus, the lower levels of serum haptoglobin in thalassemic patients could be attributed to presence of different polymorphisms of the haptoglobin gene. Kaiser et al [7] found that people with Hp2-2 have the lowest concentrations of Hp, while those with Hp1-1 have the highest, and the ones with Hp2-1 have concentrations fall somewhere in between.

Our study also revealed that thalassemic patients with Hp2-2 have the highest serum ferritin than other phenotypes which agrees with Ragab et al [19] who demonstrated that Hp2-2 genotype patients had higher serum ferritin compared to their analogues. When coupled to Hp 2-2 rather than Hp 1-1 or Hp 2-1, the Hb-Hp complex is more effectively internalized by CD163 receptors. Therefore, it was hypothesized that people with the Hp 2-2 phenotype experience more oxidative stress as a result of higher iron buildup in macrophages [26].

On the other hand, we demonstrated that patients with the Hp2-2 phenotype had the highest level of MDA when compared to the other phenotypes. Our results corroborate the findings of Blum et al [27] who stated that the Hp-1 protein is allied with enhanced production of the antioxidant cytokine IL 10, whereas Hp-2 protein is accompanied with excess of oxidative active iron production. Subsequently, the Hp1 phenotype has anti-inflammatory and antioxidant properties. Moreover, Awadallaha et al [28] reported that upon formation of the Hp-Hb complex, it is rapidly quenched from the circulation and hunted by the macrophages and hepatocytes CD163 receptors.

In the current study, lower level of serum haptoglobin could be interpreted by the presence of anti Hp antibody which needs to be further studied. The risk to produce anti Hp antibody by blood transfusion has been investigated by Shimada et al [29] who reported that haptoglobin deficiency was associated with Hp IgG antibodies and those subjects could experience fatal anaphylactic non hemolytic transfusion reactions. They also reported that Hp-2 allele has a superior ability to form antibodies than the Hp-1 allele. It is worth mentioning that our results revealed high Hp-2 allele frequency than Hp-1. Previous studies suggest that Hp- 1 allele may be beneficial from a biological standpoint due to its smaller size, faster Hb exportation, and increased antioxidative capabilities, so Hp 1-1 can be taken up by cells faster and pass the blood-brain barrier. On the other hand, Hp-2 genotype is associated with a lower Hp concentration and a diminished competency to bind to Hb [30].

CONCLUSION

We found functional variations in the antioxidant ability of different haptoglobin proteins towards hemoglobin, proposing that those with haptoglobin 1-1 protein may have better antioxidant protection than those with Hp 2-2 protein. Haptoglobin polymorphism and phenotypic variability have a major influence on oxidative stress in patients with thalassemia and those with the Hp 2-2 phenotype are more vulnerable to iron-steered oxidative stress than those with different phenotypes.

Funding: None

Conflict of interest:None.

Ethical consideration

This study was approved by Research Ethics Committee of Medical Research Institute, Alexandria University following principles of the declaration of Helsinki (IORG0008812). All studied patients signed for informed and written consent.