Current Molecular Medicine - Volume 8, Issue 7, 2008

The hemoglobinopathies are the most common genetic disorders in the world. It is estimated that about 5% of the world population, located mainly in South East Asia and Africa, are carriers of variants in either α or β globin genes and consequently result in two major red blood cell disorders, the thalassemia syndrome and sickle cell anemia. It is no wonder that studies on the genetics, pathophysiology and treatment of these diseases have been carried out extensively for more than half a century. With the rapid progress in research technology and methodology, which enables a better understanding of the basic and clinical pathophysiology, as well as new treatment modalities, there is room to update the scientific and medical community every few years on all the recent developments in this area by leading experts in the field, which will be presented in the following volume of the journal. The up-to-date information on the geographical distribution of the two major hemoglobinopathies, sickle cell disease and thalassemia, with an emphasis on ways and means of prevention are summarized by Professor D. Weatherall. In the following chapter, Drs. D. Rund and S. Fuchareon shed light on the effect of genetic modifiers, which influence the distribution and expression of the various hemoglobinopathies worldwide. Several papers present recent information on the pathophysiology of both diseases. Drs. E. Fibach and E. Rachmilewitz discuss the role of oxidative hemolysis in hemoglobinopathies, with new data that have not been emphasized before. Another aspect discussed by Drs. C. Morris, M. Gladwin and G. Kato is the role of disregulation of nitrous oxide and arginine on the development of pulmonary hypertension in hemoglobinopathies. One of the consequences of the oxidative insult is severe changes in the structure and function of the cell membrane, described by Dr. F. Kuypers, both in sickle cell anemia and thalassemia. Another outcome of the pathological changes in the composition of the red blood cell lipid membrane bilayer is the activation of the coagulation system inducing a hypercoagulable state. This chapter has been written by Drs. S. Singer and K. Ataga. Two papers are dedicated to the issue of iron overload in hemoglobinopathies. Drs. S. Rivella and G. Rechavi present the current status on the regulation of iron absorption and particularly on the role of hepcidin, a protein which regulates iron absorption from the intestines, while Professor M. Cappellini and Dr. A. Piga present the current status of iron chelation in hemoglobinopathies, mainly in thalassemia, using the 3 available iron chelators, deferoxamine, deferipone and deferasirox. Last but not the least, available options for curing thalassemia by bone marrow transplantation are reported with very positive and impressive results by Drs. J. Michlitsch and M. Walters, and what is the present status and future of gene therapy in hemoglobinopathies has been written by Dr. M. Sadelain. Taken together, we hope and believe that going through the different chapters, the reader will be able to obtain an objective up-to-date progress report on all the new epidemiological, pathophysiological, clinical and therapeautical innovations in patients with hemoglobinopathies.

The genetic disorders of hemoglobin, the commonest monogenic diseases, occur at some of their highest frequencies in the developing countries, particularly those of Sub-Saharan Africa and Asia. Although progress towards their control and management continues to be made, the prospects for curing them, apart from marrow transplantation, remain uncertain. In many countries expertise and facilities for their control are extremely limited. Although a great deal can be done to help the situation by developing further North/South and South/South partnerships for disseminating better practice, the major problem for the future lies in the unwillingness of governments and international health agencies to accept that the hemoglobinopathies represent a health burden comparative to that of communicable and other major diseases. However, preliminary analyses suggest that, at least in the case of Asia, this may not be true. Further work of this type, together with more detailed frequency and economic data, is required to provide solid evidence for the health burden posed by the hemoglobin disorders, particularly in the developing world. Unless this is done, the increasingly large populations of patients with these diseases will continue to be neglected.

Hereditary anemias show considerable variation in their clinical presentation. In some cases, the causes of these variations are easily apparent. In thalassemia (or in HbE/thalassemia), genetic variation is primarily caused by the severity of the thalassemia mutation. However, not uncommonly, there is variation unexplained by the globin gene mutations themselves, which may be caused by genetic modifiers. In sickle cell disease, the primary mutation is the same in all patients. Therefore, variations in disease severity generally are due to genetic modifiers. In most genetic diseases involving beta globin, the most clearcut influence on phenotype results from elevated fetal hemoglobin levels. In addition, alpha globin gene number can influence disease phenotype. In thalassemia major or intermedia, reduction in the number of alpha globin genes can ameliorate the disease phenotype; conversely, excess alpha globin genes can convert beta thalassemia trait to a clinical picture of thalassemia intermedia. In sickle cell disease, the number of alpha globin genes has both ameliorating and exacerbating effects, depending on which disease manifestation is being examined. Unlinked genetic factors have substantial effects on the phenotype of hereditary anemias, both on the anemia and other disease manifestations. Recently, studies using genome-wide techniques, particularly studying QTLs causing elevated HbF, or affecting HbE/thalassemia, have revealed other genetic elements whose mechanisms are under study. The elucidation of genetic modifiers will hopefully lead to more rational and effective management of these diseases.

The oxidative status of cells is determined by the balance between pro-oxidants and antioxidants. Pro-oxidants, referred to as reactive oxygen species (ROS), are classified into radicals and nonradicals. The radicals are highly reactive due to their tendency to accept or donate an electron and attain stability. When cells experience oxidative stress, ROS, which are generated in excess, may oxidize proteins, lipids and DNA - leading to cell death and organ damage. Oxidative stress is believed to aggravate the symptoms of many diseases, including hemolytic anemias. Oxidative stress was found in the β-hemoglobinopathies (sickle cell anemia and thalassemia), glucose-6-phosphate dehydrogenase deficiency, hereditary spherocytosis, congenital dyserythropoietic anaemias and Paroxysmal Nocturnal Hemoglobinuria. Although oxidative stress is not the primary etiology of these diseases, oxidative damage to their erythroid cells plays a crucial role in hemolysis due to ineffective erythropoiesis in the bone marrow and short survival of red blood cells (RBC) in the circulation. Moreover, platelets and polymorphonuclear (PMN) white cells are also exposed to oxidative stress. As a result some patients develop thromboembolic phenomena and recurrent bacterial infections in addition to the chronic anemia. In this review we describe the role of oxidative stress and the potential therapeutic potential of anti-oxidants in various hemolytic anemias.

Secondary pulmonary hypertension (PH) is emerging as one of the leading causes of mortality and morbidity in patients with hemolytic anemias such as sickle cell disease (SCD) and thalassemia. Impaired nitric oxide (NO) bioavailability represents the central feature of endothelial dysfunction, and is a major factor in the pathophysiology of PH. Inactivation of NO correlates with hemolytic rate and is associated with the erythrocyte release of cell-free hemoglobin, which consumes NO directly, and the simultaneous release of the argininemetabolizing enzyme arginase, which limits bioavailability of the NO synthase substrate arginine during the process of intravascular hemolysis. Rapid consumption of NO is accelerated by oxygen radicals that exists in both SCD and thalassemia. A dysregulation of arginine metabolism contributes to endothelial dysfunction and PH in SCD, and is strongly associated with prospective patient mortality. The central mechanism responsible for this metabolic disorder is enhanced arginine turnover, occurring secondary to enhanced plasma arginase activity. This is consistent with a growing appreciation of the role of excessive arginase activity in human diseases, including asthma and pulmonary arterial hypertension. New treatments aimed at improving arginine and NO bioavailability through arginase inhibition, suppression of hemolytic rate, oral arginine supplementation, or use of NO donors represent potential therapeutic strategies for this common pulmonary complication of hemolytic disorders.

The complex mixture of lipids and proteins of the red blood cell membrane is well maintained during the life of the cell. Lipid analysis of the red cell reveals hundreds of phospholipid molecular species and cholesterol that differ with respect to their (polar) head group, and (apolar) side chains. These molecules move rapidly in the plane, as well as across the lipid bilayer. This dynamic movement is highly organized. In the plane of the bilayer, areas enriched in certain lipids accommodate protein structure and modulate function. While lipids move across the bilayer, the organization is highly asymmetric. Amino phospholipids are mainly found on the inside and choline containing phospholipids on the outside. Both the composition and organization of the red cell membrane is maintained throughout the life of the red cell by an intricate mechanism that involves enzymes, transporters and cytosolic factors. Key proteins that maintain red blood cell lipid organization have recently been identified. Alterations in these mechanisms, as the result of the globin mutations in sickle cell disease or thalassemia will lead to loss of membrane viability, apoptosis during erythropoiesis, early demise of the cell in the circulation, and when these cells are not removed appropriately their presence has pathologic consequences.

Sickle cell disease (SCD) and beta-thalassemia (also referred to as β-thalassemia) are common hereditary hemoglobinopathies with differing pathophysiologies and clinical courses. However, patients with both diseases exhibit increased platelet and coagulation activation, as well as decreased levels of natural anticoagulant proteins. In addition, they are characterized by thrombotic complications that may share a similar pathogenesis. The pathogenesis of hypercoagulability is likely multifactorial, with contributions from the abnormal red blood cell (RBC) phospholipid membrane asymmetry, ischemia-reperfusion injury, and chronic hemolysis with resultant nitric oxide depletion. More studies are needed to better define the contribution of hemostatic activation to the pathophysiology of SCD and beta-thalassemia. Furthermore, adequately controlled studies using anticoagulants and antiplatelet agents are warranted to define the role of hypercoagulability in specific complications of these diseases.

Beta-thalassemia and sickle cell anemia (SCD) represent the most common hemoglobinopathies caused, respectively, by deficient production or alteration of the beta chain of hemoglobin (Hb). Patients affected by the most severe form of thalassemia suffer from profound anemia that requires chronic blood transfusions and chelation therapies to prevent iron overload. However, patients affected by beta-thalassemia intermedia, a milder form of the disease that does not require chronic blood transfusions, eventually also show elevated body iron content due to increased gastrointestinal iron absorption. Even SCD patients might require blood transfusions and iron chelation to prevent deleterious and painful vaso-occlusive crises and complications due to iron overload. Although definitive cures are presently available, such as bone marrow transplantation (BMT), or are in development, such as correction of the disease through hematopoietic stem cell beta-globin gene transfer, they are potentially hazardous procedures or too experimental to provide consistently safe and predictive clinical outcomes. Therefore, studies that aim to better understand the pathophysiology of the hemoglobinopathies might provide further insight and new drugs to dramatically improve the understanding and current treatment of these diseases. This review will describe how recent discoveries on iron metabolism and erythropoiesis could lead to new therapeutic strategies and better clinical care of these diseases, thereby yielding a much better quality of life for the patients.

Although blood transfusions are important for patients with hemoglobinopathies, chronic transfusions inevitably lead to iron overload as humans cannot actively remove excess iron. The cumulative effects of iron overload lead to significant morbidity and mortality, if untreated. Desferrioxamine (DFO) is the referencestandard iron chelator whose safety and efficacy profile has been established through many years of clinical use. DFO side effects are acceptable and manageable however the prolonged subcutaneous infusion regimen of 5-7 days per week is very demanding and results in poor adherence to therapy. Deferiprone (Ferriprox, L1) is a bidentate molecule, orally administrable three-times/day, licensed in Europe and in other regions but in the USA and Canada, for the treatment of iron overload in patients for whom DFO therapy is contraindicated or inadequate. Preliminary evidences suggest that Deferiprone may be more effective than DFO in chelating cardiac iron. The side effects include gastrointestinal symptoms, liver dysfunction, joint pain, neutropenia and agranulocytosis. A weekly assessment of white blood cell counts is recommended because of the risk of agranulocytosis. Deferasirox is a new, convenient, once-daily oral iron chelator that has demonstrated in various clinical trials good efficacy and acceptable safety profile in adult and pediatric patients affected by transfusion-dependent thalassemia major and by different chronic anemias (SCD, BDA, MDS). The long half-life of Deferasirox (16-18 hours) provides sustained 24 hr iron chelation coverage. The efficacy and safety profile have been evaluated in more than 1000 patients in clinical trials allowing FDA registration. Patient satisfaction with Deferasirox was superior than with DFO therapy.

Allogeneic hematopoietic cell transplantation (HCT) is currently the only treatment with curative potential for sickle cell disease (SCD) and β-thalassemia. HCT was first used to treat SCD and thalassemia more than two decades ago, and with increasing experience this treatment modality has shifted from being an experimental intervention to one in which selected patient populations are targeted for treatment. Recent multicenter clinical studies show an event-free survival (EFS) of 85% after human leukocyte antigen (HLA)- identical sibling transplantation for SCD, using conventional myeloablative conditioning with a backbone of busulfan (BU) and cyclophosphamide (CY) [1-3]. Results of HCT for thalassemia show very similar outcomes, with EFS probabilities that range from 81%-87% [4,5]. However, the risk of graft failure, recurrent disease, graft-versus-host-disease (GVHD), infections, and long-term sequelae of chronic GVHD and endocrinopathies related to Fe overload and myeloablative BU limit broader application of this therapy. Non-myeloablative conditioning regimens may offer a lower risk of toxicity, and investigations to identify a regimen that is sufficiently immunosuppressive to ensure stable engraftment of donor cells are ongoing. Alternative sources of donor hematopoietic cells that include HLA-matched unrelated donor (URD) and umbilical cord blood (UCB), are being pursued for hemoglobinopathies, with promising initial results. This review discusses the successes, challenges and future direction of HCT for SCD and thalassemia.

The ß-thalassemias and sickle cell anemia are severe congenital anemias for which there is presently no curative therapy other than allogeneic bone marrow transplantation. This therapeutic option, however, is not available to most patients due to the lack of an HLA-matched bone marrow donor. Emerging modalities based on cell engineering offer new prospects for potentially curative approaches that are applicable to more patients. The first is based on the transfer of a regulated globin gene in autologous hematopoietic stem cells (HSCs). This strategy, simple in principle, raises major challenges in terms of controlling transgene expression, which ideally should be erythroid-specific, differentiation and stage-restricted, elevated, position-independent, and sustained over time. Following the original report by May et al., several groups have reported that lentiviral vectors encoding slightly different combinations of proximal and distal transcriptional control elements of the normal human ß-globin gene permit lineage-specific and elevated ß-globin expression in vivo, resulting in therapeutic hemoglobin production and correction of anemia in ß-thalassemic mice. Clinical studies utilizing the TNS.3 vector are likely to be initiated in the US in 2009. While the addition of the wild-type ß-globin gene is naturally suited for treating ß-thalassemia, several alternatives have been proposed for the treatment of sickle cell disease, using either γ- or mutant ß-globin gene addition, trans-splicing or RNA interference. The recent discovery that adult somatic cells can be reprogrammed to become pluripotent stem cells from which HSCs can be derived, provides yet another venue for developing stem cell engineering using either lentiviral vectors or homologous recombination techniques. Altogether, these recent advances bode well for the advent of curative stem cell-based therapies.