New Insights on ß-Thalassemia in the Palestinian Population of Gaza: High Frequency and Milder Phenotype Among Homozygous IVS-I-1 (HBB: c.92+1G>A) Patients with High Levels of Hb F.

ß-Thalassemia (ß-thal) is a very common disease in the Palestinian population of the Gaza Strip. We studied their mutation frequency and clinical features. Thirteen different mutations were identified. The most common mutation was IVS-I-1 (G>A) (HBB: c.92+1G>A), which was prevalent in 31.5% of the thalassemia alleles studied. The IVS-I-110 (G>A) (HBB: c.93-21G>A) mutation was found in 25.0% of the alleles. Homozygotes for the IVS-I-1 mutation had higher mean hemoglobin (Hb) levels, required less blood transfusions, and lower transferrin saturation than the homozygotes for the IVS-I-110 mutation. This milder phenotype was, most likely, the result of the persistent production of Hb F; it was 9-fold higher in absolute terms (g/dL) and 7.7-fold higher in relative terms (percentage of total Hb). About half of our IVS-I-1 patients carried the XmnI polymorphism, which is known to be associated with elevated Hb F levels.

The frequency of silent cerebral ischemia in patients with transfusion-dependent ß-thalassemia major.

The aim of this study is to determine the frequency of silent cerebral ischemia (SCI) in a group of patients with ß-thalassemia major and correlate them with demographic parameters, blood and spleen status, volume and frequency of transfusions. In this cross-sectional study, 40 ß-thalassemic patients over 10 years old who had no neurologic deficit were studied. Brain MRI was performed to detect SCI. Silent cerebral ischemia was classified according to number and size. Silent cerebral ischemia was found in 15 patients (37.5 %). Mean number of SCI was 6.73 ± 10.33 (1-40), and mean size of the brain lesions was 3.07 ± 2.81 mm (1-11 mm). The patients with SCI were significantly older (31.1 ± 6.5 vs. 25 ± 6.8 years, P = 0.009), and most of them were splenectomized (80% vs. 36 %, P = 0.01). Interestingly, 10 out of 15 patients with SCI had platelet count less than 500,000/mm(3). Eight of these patients (80 %) were splenectomized. Other variables had no statistically significant association with the presence of SCI. Older age and splenectomized multitransfused ß-thalassemic patients even with normal platelet count have a higher incidence of SCI. The effect of splenectomy is more significant in statistical analysis. In splenectomized patients with a high platelet count and even with normal platelet count, aspirin therapy is indicated. Based on the results, it seems that regular blood transfusions are not going to have a significant effect on the number and size of SCI.

How I treat thalassemia.

The purpose of this article is to set forth our approach to diagnosing and managing the thalassemias, including ß-thalassemia intermedia and ß-thalassemia major. The article begins by briefly describing recent advances in our understanding of the pathophysiology of thalassemia. In the discussion on diagnosing the condition, we cover the development of improved diagnostic tools, including the use of very small fetal DNA samples to detect single point mutations with great reliability for prenatal diagnosis of homozygous thalassemia. In our description of treatment strategies, we focus on how we deal with clinical manifestations and long-term complications using the most effective current treatment methods for ß-thalassemia. The discussion of disease management focuses on our use of transfusion therapy and the newly developed oral iron chelators, deferiprone and deferasirox. We also deal with splenectomy and how we manage endocrinopathies and cardiac complications. In addition, we describe our use of hematopoietic stem cell transplantation, which has produced cure rates as high as 97%, and the use of cord blood transplantation. Finally, we briefly touch on therapies that might be effective in the near future, including new fetal hemoglobin inducers and gene therapy.

Non-transfusion-dependent thalassemias.

Non-transfusion-dependent thalassemias include a variety of phenotypes that, unlike patients with beta (ß)-thalassemia major, do not require regular transfusion therapy for survival. The most commonly investigated forms are ß-thalassemia intermedia, hemoglobin E/ß-thalassemia, and α-thalassemia intermedia (hemoglobin H disease). However, transfusion-independence in such patients is not without side effects. Ineffective erythropoiesis and peripheral hemolysis, the hallmarks of disease process, lead to a variety of subsequent pathophysiologies including iron overload and hypercoagulability that ultimately lead to a number of serious clinical morbidities. Thus, prompt and accurate diagnosis of non-transfusion-dependent thalassemia is essential to ensure early intervention. Although several management options are currently available, the need to develop more novel therapeutics is justified by recent advances in our understanding of the mechanisms of disease. Such efforts require wide international collaboration, especially since non-transfusion-dependent thalassemias are no longer bound to low- and middle-income countries but have spread to large multiethnic cities in Europe and the Americas due to continued migration.

Frequency and distribution of asymptomatic brain lesions in patients with ß-thalassemia intermedia.

We aimed to determine the frequency of asymptomatic brain lesions in a group of patients with ß-thalassemia intermedia (ß-TI) and to evaluate correlation of asymptomatic brain lesions with splenectomy, thrombocytosis, blood transfusions, and clinical parameters. Ninety five neurologically intact patients with ß-TI were randomly enrolled in this cross-sectional study. Diffusion-weighted imaging brain MRI was performed in every patient to detect cerebral white matter lesions (WML). We found an overall frequency of 15 (15.8 %) for WMLs, 14 (23.7 %) in splenectomized, and 1 (2.8 %) in nonsplenectomized patients. The presence of WML was significantly associated with splenectomy (P = 0.008) and thrombocytosis (P = 0.009). However, after adjustment for splenectomy, thrombocytosis was not significantly associated with the presence of WML (P > 0.05). The number of patients with regular blood transfusions and normal MRI was not significantly higher compared to those with abnormal findings (52.5 % vs. 26.7 %; P = 0.092). In untransfused patients, hydroxyurea (HU) administration was associated with a lower incidence of WML (P < 0.001). Although in univariate analysis either splenectomy or thrombocytosis showed significant correlation with the presence of single or multiple WMLs, thrombocytosis by itself did not significantly contribute in developing asymptomatic brain lesions. The lack of significant correlation between lesions and regular blood transfusions could be related to the treatment with HU in untransfused patients, which increased fetal hemoglobin levels and improved the morphology and the pathological indices of the red blood cells. Larger prospective studies are suggested for the accurate evaluation of the correlation of these factors with developing asymptomatic brain lesions.

Evidence for tissue iron overload in long-term hemodialysis patients and the impact of withdrawing parenteral iron.

BACKGROUND/AIMS: Erythropoiesis in long-term hemodialyzed (LTH) patients is supported by erythropoietin (rHuEpo) and intravenous (IV) iron. This treatment may end up in iron overload (IO) in major organs. We studied such patients for the parameters of IO in the serum and in major organs. METHODS: Patients were treated with rHuEpo (6-8 x 10(3) units × 1-3/wk) and IV 100 mg ferric saccharate. RESULTS: Of 115 patients, 21 had serum ferritin (SF) > 1000 ng/mL. This group was further analyzed. Their SF and transferrin saturation (TSAT) were 2688 ± 1489 ng/mL and 54.2 ± 32.7%, respectively (vs. 125-360 ng/mL and 20-50% in normal controls). Serum hepcidin was 60.1 ± 29.5 nm (vs. 10.61 ± 6.44 nm in controls) (P < 0.001). Nineteen patients had increased malonyldialdehyde, a product of lipid peroxidation, indicating oxidative stress. T2* MRI disclosed in 19 of 21 patients moderate to severe IO in the liver and spleen, in three of eight patients in the pancreas, but in no patient in the heart. After stopping IV iron for a mean of 12 months, while continuing rHuEpo, the mean SF decreased in 11 patients to 1682 ng/mL and the mean TSAT decreased to 28%, whereas hemoglobin did not change indicating that tissue iron was utilized. CONCLUSION: High SF correlates with IO in the liver and spleen, but not in the heart.

Increased serum hepcidin levels during treatment with deferasirox in iron-overloaded patients with myelodysplastic syndrome.

Hepcidin is a major regulator of iron metabolism. We evaluated changes in serum hepcidin during 3 months of therapy with the iron-chelator deferasirox in patients with low-risk myelodysplastic syndrome and iron overload. Serum hepcidin was found to be high in these patients, correlated with their iron and oxidative status, and further increased by treatment with deferasirox. These findings support the concept that the hepcidin level represents a balance between the stimulating effect of iron overload and the inhibitory effects of erythropoietic activity and oxidative stress. These preliminary findings favour the rationale for iron chelation therapy in such patients.

Iron overload in MDS-pathophysiology, diagnosis, and complications.

Many patients with myelodysplastic syndromes (MDS) become dependent on blood transfusions and develop transfusional iron overload, which is exacerbated by increased absorption of dietary iron in response to ineffective erythropoiesis. However, it is uncertain whether there is an association among iron accumulation, clinical complications, and decreased likelihood of survival in MDS patients. Here, we discuss our current understanding of the effects of transfusion dependency and iron overload in MDS, indicate our knowledge gaps, and suggest that particular emphasis should be placed on further characterizing the role of redox-active forms of labile iron, which may be as important as the total iron burden.

Decreased differentiation of erythroid cells exacerbates ineffective erythropoiesis in beta-thalassemia.

In beta-thalassemia, the mechanism driving ineffective erythropoiesis (IE) is insufficiently understood. We analyzed mice affected by beta-thalassemia and observed, unexpectedly, a relatively small increase in apoptosis of their erythroid cells compared with healthy mice. Therefore, we sought to determine whether IE could also be characterized by limited erythroid cell differentiation. In thalassemic mice, we observed that a greater than normal percentage of erythroid cells was in S-phase, exhibiting an erythroblast-like morphology. Thalassemic cells were associated with expression of cell cycle-promoting genes such as EpoR, Jak2, Cyclin-A, Cdk2, and Ki-67 and the antiapoptotic protein Bcl-X(L). The cells also differentiated less than normal erythroid ones in vitro. To investigate whether Jak2 could be responsible for the limited cell differentiation, we administered a Jak2 inhibitor, TG101209, to healthy and thalassemic mice. Exposure to TG101209 dramatically decreased the spleen size but also affected anemia. Although our data do not exclude a role for apoptosis in IE, we propose that expansion of the erythroid pool followed by limited cell differentiation exacerbates IE in thalassemia. In addition, these results suggest that use of Jak2 inhibitors has the potential to profoundly change the management of this disorder.

Amelioration of oxidative stress in red blood cells from patients with beta-thalassemia major and intermedia and E-beta-thalassemia following administration of a fermented papaya preparation.

In beta-hemoglobinopathies, such as beta-thalassemia (thal) and sickle cell anemia, the primary defects are mutations in the beta-globin gene. However, many aspects of the pathophysiology are mediated by oxidative stress. Fermented papaya preparation (FPP), a natural health food product obtained by biofermentation of carica papaya, has been shown to limit oxidative stress both in vitro and in vivo. We studied the effect of FPP on two groups of beta-thal patients: beta-thal, major and intermedia, (in Israel) and E-beta-thal (in Singapore). The results indicated that in both groups FPP treatment increased the content of reduced glutathione (GSH) in red blood cells (RBC), and decreased their reactive oxygen species (ROS) generation, membrane lipid peroxidation, and externalization of phosphatidylserine (PS), indicating amelioration of their oxidative status, without a significant change in the hematological parameters. Since the turnover of the erythron is relatively slow, it is possible that longer duration of treatment, probably with the addition of an iron chelator, is required in order to achieve the latter goals.

Fermented papaya preparation as redox regulator in blood cells of beta-thalassemic mice and patients.

Many aspects of the pathology in beta-hemoglobinopathies (beta-thalassemia and sickle cell anemia) are mediated by oxidative stress. Fermented papaya preparation (FPP) was tested for its antioxidant effects: the scavenging effect was determined spectrofluorometrically in a cell-free system using 2'-7'-dichlorofluorescin-diacetate (DCF). Both spontaneous and H(2)O(2)-induced DCF oxidations were decreased by FPP in a dose-dependent fashion. Using flow cytometry, it was shown that in vitro treatment of blood cells from beta-thalassemic patients with FPP increased the glutathione content of red blood cells (RBC), platelets and polymorphonuclear (PMN) leukocytes, and reduced their reactive oxygen species, membrane lipid peroxidation and externalization of phosphatidylserine. These effects result in (a) reduced thalassemic RBC sensitivity to hemolysis and phagocytosis by macrophages; (b) improved PMN ability to generate oxidative burst - an intracellular mechanism of bacteriolysis, and (c) reduced platelet tendency to undergo activation, as reflected by fewer platelets carrying external phosphatidylserine. Oral administration of FPP to beta-thalassemic mice (50 mg/mouse/day for 3 months) and to patients (3 g x 3 times/day for 3 months), reduced all the above mentioned parameters of oxidative stress (p < 0.001 in mice and p < 0.005 in patients). These results suggest that FPP, as a potent antioxidant, might alleviate symptoms associated with oxidative stress in severe forms of thalassemia.

Iron overload in hematological disorders.

While most common symptom of impairment of iron homeostasis is iron deficiency anemia, some hematological disorders are associated with iron overload (IO). These disorders are related mainly to chronic severe hemolytic anemia, where red blood cells (RBC) or their precursors are destroyed prematurely (hemolyzed), leading to anemia that cannot be compensated by increased production of new RBC. In such cases, IO is mainly due to repeated RBC transfusions and/or increased uptake of iron in the gastrointestinal tract. Normally, iron is present in the plasma and in the cells bound to compounds that render it redox inactive. Iron overload leaves a fraction of the iron free (labile iron pool) and redox active, leading to the generation of excess free radicals such as the reactive oxygen species. This condition upsets the cellular redox balance between oxidants and antioxidants, leading to oxidative stress. The free radicals bind to various cellular components, thereby becoming toxic to vital organs. Oxidative stress may also affect blood cells, such as RBC, platelets and neutrophils, exacerbating the anemia, and causing recurrent infections and thrombotic events, respectively. The toxic effect of IO can be decreased by treating the patients with iron chelators that enter cells, bind free iron and remove it from the body through the urine and feces. Iron toxicity may be also ameliorated by treatment with anti-oxidants that scavenge free radicals and/or correct their damage. The use of iron chelators is widely accepted when started in young patients with severe chronic anemia, but is still debatable as a therapeutic modality for older patients suffering from IO due to myelodysplastic syndromes. It should be noted that in addition to preventing iron toxicity, some compounds with iron chelator activity may also benefit other aspects of hematological disorders. These aspects include stimulation of platelet production, inhibition of leukemic cell proliferation and induction of their differentiation. Compounds with such multiple activities may prove beneficial for at least some patients with leukemia and myelodysplastic syndromes.

Pathophysiology and treatment of patients with beta-thalassemia - an update.

Thalassemia (thal) is an autosomal recessive, hereditary, chronic hemolytic anemia due to a partial or complete deficiency in the synthesis of α-globin chains (α-thal) or ß-globin chains (ß-thal) that compose the major adult hemoglobin (α 2ß 2). It is caused by one or more mutations in the corresponding genes. The unpaired globin chains are unstable; they precipitate intracellularly, resulting in hemolysis, premature destruction of red blood cell [RBC] precursors in the bone marrow, and a short life-span of mature RBCs in the circulation. The state of anemia is treated by frequent RBC transfusions. This therapy results in the accumulation of iron (iron overload), a condition that is exacerbated by the breakdown products of hemoglobin (heme and iron) and the increased iron uptake for the chronic accelerated, but ineffective, RBC production. Iron catalyzes the generation of reactive oxygen species, which in excess are toxic, causing damage to vital organs such as the heart and liver and the endocrine system. Herein, we review recent findings regarding the pathophysiology underlying the major symptoms of ß-thal and potential therapeutic modalities for the amelioration of its complications, as well as new modalities that may provide a cure for the disease.

Prevalence of thromboembolic events among 8,860 patients with thalassaemia major and intermedia in the Mediterranean area and Iran.

Beta-thalassaemia is a congenital haemolytic anaemia characterized by partial (intermedia, TI) or complete (major, TM) deficiency in the production of beta-globin chains. The primary aim of this study was to determine the prevalence of thromboembolic events in patients with beta-thalassaemia. To achieve this, a multiple choice questionnaire was sent to 56 tertiary referral centres in eight countries (Lebanon, Italy, Israel, Greece, Egypt, Jordan, Saudi Arabia and Iran), requesting specific information on patients who had experienced a thromboembolic event. The study demonstrated that thromboembolic events occurred in a clinically relevant proportion (1.65%) of 8,860 thalassaemia patients (TI - 24.7% or TM - 75.3%) from the Mediterranean and Iran. Thromboembolism occurred 4.38 times more frequently in TI than TM (p < 0.001), with more venous events occurring in TI and more arterial events occurring in TM. Thrombosis in thalassaemia was also more common in females, splenectomized patients and those with profound anaemia (haemoglobin <9 g/dl). Due to the increased risk of thromboembolic events, the rationale for splenectomy should perhaps be re-assessed and the role of transfusion therapy for the prophylaxis of thrombosis, among other complications, be evaluated prospectively.

Role of iron in inducing oxidative stress in thalassemia: Can it be prevented by inhibition of absorption and by antioxidants?

The pathophysiology of thalassemia is, to a certain extent, associated with the generation of labile iron in the pathological red blood cell (RBC). The appearance of such forms of iron at the inner and outer cell surfaces exposes the cell to conditions whereby the labile metal promotes the formation of reactive oxygen species (ROS) leading to cumulative cell damage. Another source of iron accumulation results from increased absorption due to decreased expression of hepcidin. The presence of labile plasma iron (LPI) was carried out using fluorescent probes in the FACS. RNA expression of hepcidin was measured in two models of thalassemic mice. Hepcidin expression was also measured in human hepatoma HepG2 cells following incubation with thalassemic sera. LPI was identified and could be quantitatively measured and correlated with other parameters of iron overload. Hepcidin expression was downregulated in the livers of thalassemic mice, in major more than in intermedia. Thalassemic sera down regulated hepcidin expression in HepG2 liver cells. A possible way to decrease iron absorption could be by modulating hepcidin expression pharmacologically, by gene therapy or by its administration. Treatment with combination of antioxidants such as N-acetylcysteine for proteins and vitamin E for lipids in addition to iron chelators could neutralize the deleterious effects of ROS and monitored by quantitation of LPI.

Exploring the role of hepcidin, an antimicrobial and iron regulatory peptide, in increased iron absorption in beta-thalassemia.

To develop new treatments for beta-thalassemia, it is essential to identify the genes involved in the relevant pathophysiological processes. Iron metabolism in thalassemia mice being investigated, focusing on the expression of a gene called hepcidin (Hamp), which is expressed in the liver and whose product (Hamp) is secreted into the bloodstream. In mice, iron overload leads to overexpression of Hamp, while Hamp-knockout mice suffer from hemochromatosis. The aim of this study is to investigate Hamp in the mouse model of beta-thalassemia and to address the potential gene transfer of Hamp to prevent abnormal iron absorption.

The Effect of Fermented Papaya Preparation on Radioactive Exposure.

Exposure to ionizing radiation causes cellular damage, which can lead to premature cell death or accumulation of somatic mutations, resulting in malignancy. The damage is mediated in part by free radicals, particularly reactive oxygen species. Fermented papaya preparation (FPP), a product of yeast fermentation of Carica papaya Linn, has been shown to act as an antioxidant. In this study, we investigated the potential of FPP to prevent radiation-induced damage. FPP (0-100 µg/ml) was added to cultured human foreskin fibroblasts and myeloid leukemia (HL-60) cells either before or after irradiation (0-18 Gy). After 1-3 days, the cells were assayed for: intracellular labile iron, measured by staining with calcein; reactive oxygen species generation, measured with dichlorofluorescein diacetate; apoptosis, determined by phosphatidylserine exposure; membrane damage, determined by propidium iodide uptake; and cell survival, determined by a cell proliferation assay. DNA damage was estimated by measuring 8-oxoguanine, a parameter of DNA oxidation, using a fluorescent-specific probe and by the comet assay. These parameters were also assayed in bone marrow cells of mice treated with FPP (by adding it to the drinking water) either before or after irradiation. Somatic mutation accumulation was determined in their peripheral red blood cells, and their survival was monitored. FPP significantly reduced the measured radiation-induced cytotoxic parameters. These findings suggest that FPP might serve as a radioprotector, and its effect on DNA damage and mutagenicity might reduce the long-term effects of radiation, such as primary and secondary malignancy.