Haemoglobinopathies are the Commonest Single Gene Disorders in the Worl
Haemoglobinopathies are the commonest single gene disorders in the world, which result in anaemia due to premature destruction of red cells. Defective globin chain renders the cells vulnerable to oxidative damage. Such anaemic state can only be corrected by regular transfusion of blood. The only way to reduce the transfusional load, is through salvaging such defective red cells from free radical induced oxidative damage. In this research we will find the inducer which will shield the dyserythropoiesis and increase red cell survival, hence bring down the transfusional load of individuals suffering from thalassaemias and haemoglobinopathies.
Thalassemia syndromes, constitute the most common inherited single gene disorder, worldwide. It is due to autosomal mutations in the gene encoding Beta-globin or Alfa globin, which induce an absence or low-level synthesis of that protein in erythropoietic cells . The consequence of these mutations is an imbalance of Alfa / Beta-globin chain synthesis, mostly evident in the homozygous forms, leading to the accumulation of free Alfa-globin or non-Alfa chains forming highly toxic aggregates . Thalassemic patients suffer from anemia resulting from shortened red blood cell (RBC) survival, due to hemolysis, and erythroid precursors premature death in bone marrow (ineffective erythropoiesis), as a result of the toxic aggregates, destabilization of the Red Cell Membrane, by inducting free radicals.
So, the basic pathology of thalassaemia, leading to haemolysis, is accumulation of unpaired chain aggregates, Alfa chains in case of Beta Thalassaemias. It was never believed by clinicians that the ineffective erythropoiesis and dys-erythropoiesis, which lead to erythroptosis of late stage red cell precursors, hence anaemia could be controlled by any means other than post erythron ablation – stem cell transplantation or gene therapy. Luspaercept or Sotatercept, opened a new era of therapy for thalassaemias and haemoglobinopathies. It was identified that the breakdown of the defective red cells occurs through the transforming growth factor ?, which regulate the positive Smad2/3 signalling on growth differentiation factor 11 (GDF-11), this induces apoptosis of the late erythroid progenitors. RAP-011(Sotatercept) and RAP-536 (Luspatercept) are proteins, which include a extracellular part of ActRIIB linked to the Fc portion of IgG1, which makes it as decoy receptor for GDF-11, thus blocking ActRIIB, blocking the conversion R-SMAD to R-SMAD P to SMAD 4, thereby reducing the apoptosis of the apoptosis of the immature erythroblasts. The erythroptosis, which has been controlled, leads to less ineffective erythropoiesis, increasing the Red Cell life span and rendering less transfusion dependence to the transfusion dependent thalassaemics [1, 2, ].
These medicines are undergoing clinical trials, but are very expensive and requires parenteral route of administration. These compounds also are unable to control the explosive erythropoiesis in the early and intermediate phase of maturation of the erythrocytes, thus being unable to control the extramedullary haematopoiesis, one of the very debilitating complications of such types of haemolytic anaemias.
Depending on this background, we began searching for another pathway, which is equally affects the apoptosis of the immature red cells in the late stage of differentiation and maturation.
Review of literature:
Normal adult haemoglobin is assembled with a pair of Alfa and Beta (Non-Alfa) Globin, whose synthesis is very tightly controlled. Rate of synthesis of alfa chain is slightly higher than beta chains. Alfa chains have been synthesized in excess of Non-alfa chains from 1.0 to 2.0 till about reticulocyte stage, with an average of 0.65 . Under physiological conditions, this excess Alfa chain is stabilized by Alfa Haemoglobin Stabilizing Protein (AHSP), which forms a stable hemichrome, out of oxidized Alfa haemoglobin, playing a vital role in prevention of premature red cell destruction in the maturation stage . So, in normal erythropoiesis, not only there exists excess alfa chains, there also exists a mechanism of neutralising the oxidative damage and cellular destruction potential.
Red cells, in its life cycle passes through two distinct phases – maturation and release into circulation. Complete haemoglobinization of the red cells comes after sacrificing the nucleus and important organelles. This is made possible through controlled apoptosis, in such a way that a mature Red cell is considered to be a post apoptotic creature, presumably by transient mitochondria triggered caspase activation, in the maturation cum haemoglobinization phase. In the senescent phase, calcium influx occurs, triggered by the Ca++ exposure, ?-calpain is cleaved into its active fragments, which in-turn degrades the spectrin, this being a mitochondria independent phase . None of these two mechanisms seems to be entirely responsible for the excess chain aggregation and precipitation induced red cell destruction.
It has been observed that the bone marrow of patients suffering from beta Thalassaemia, contain about 5 to 6 times more erythroid precursors, that compared to healthy normal . There is corresponding increase in early and intermediate normoblasts and decrease in late normoblasts [6, 7, 8, 9] and electron dense alfa-globin inclusions have been detected, in the early stage intermediate normoblasts, which becomes more as maturation proceeds .
These results suggest that dys-erythropoiesis in Beta Thalassaemia is characterized by expansion of proerythroblast, early and intermediate normoblasts and reduction in population of late normoblasts, so this is characterized by accelerated erythroid differentiation, blockage of maturation at intermediate stage and lastly death of the late erythroid precursors [11, 12]
Study of apoptotic death receptor reveal that Fas and FasL are co-expressed at all stages of terminal differentiation . Increased death at late-intermediate Normoblastic and Late normoblastic stage also coincides with stages of intense haemoglobinization. Accumulation and precipitation of un-paired Alfa chains as aggregates also appear at this stage. This highly unstable free Alfa globin, generate reactive oxygen species, which damage cellular proteins, lipids and nucleic acids. This hypothesis was proved in both early and late normoblasts acquired from HbE / Beta thalassaemia patients when compared with normal red cells [14,15,16].
Alfa globin accumulation occur also from the intermediate erythroblastic stage and such deposits were seen to be colocalizing with areas of defective assembly of membrane skeletal proteins Spectrin and Protein 4.1 and in very early normoblast stage, there is defective assembly of the transmembrane band 3. Oxidant injury led to clustering of Band 3, producing a neoantigen that binds to IgG and Complement – a complex providing signal for macrophages to remove such affected red cell precursors [17,18].
Red blood cell membrane of thalassaemic patients carry abnormal deposits of iron. Several pathobiological consequences in thalassaemic RBS membrane have been linked to the deposition of generic iron on the cytosolic leaflet of the plasma membrane, contributing to apoptosis .
So, it can be concluded that the basic pathology behind ineffective erythropoiesis and red cell destruction of late normoblasts is damage to red cell membrane, due to defective, protein assembly by reactive oxygen species. If the vicious cycle of robust early erythropoiesis and destructive late erythropoiesis could be interrupted, not only would the expansion of early stage normoblasts be controlled but the late stage erythroptosis would be reduced, transfusion dependency and iron overload would be largely reduced.
JAK-STAT pathway controls the erythropoietin induced, positive feedback mechanism for erythroid maturation and differentiation from Haematopoietic stem and progenitor cells, if JAK inhibitor – Ruxolitinib is used, it should be able to control the massive erythroid expansion and extramedullary haematopoiesis and the stress erythropoiesis would be reduced – controlling one of the key complications of Transfusion dependent and non-transfusion dependent thalassaemia patients.
Haem-Oxigenase – I (HO-1), is the common pathway by which apoptosis of membrane disrupted erythroblasts occur, principally due to accumulation and precipitation of unpaired Alfa globin chains in case of Beta Thalassaemia. Nrf-2 is the inhibitor of HO-I and is stimulated by Alkaloids of the Catechin family, extracted from Green Tea, of which Epigallocatechin and Epicatechin are the most effective members.
If Catechins are used, in therapeutic doses, it should be able to reduce the apoptosis of the late erythroid precursors, due to precipitation of the excess Alfa chains. This is because AHSP, which is present in normal individuals, will produce stable compound with unpaired Alfa Globin chains, known as Alfa Haemoglobin, in Oxygenated form, an
d contribute to extension of the life of the affected Red Cells. This enhanced RBC survival will reduce the transfusion burden in transfusion dependent thalassaemics.
To identify specific agent/s which will induce reduce early stage erythroid expansion and effective late stage erythroid maturation by interrupting the common pathway for reactive oxygen species, and rescue of the functional red cells.
Late erythroid precursors are protected from apoptosis due to haem auto-oxidation by a transcription factor Nrf2, other than being identified as a transcription factor, which is located in the beta globin gene regulator region. The system that protect against oxidative stress is the Keap1-Nrf2 pathway. So it has been concluded that NF-E2p45 regulates the basal expression of antioxidant enzymes and Nrf2 stimulates the response and Nrf2 is activated in the erythroid lineage cells which are susceptible to oxidative stress . It has been shown that Nrf2 also controls the expression of gamma globin gene by directly binding to its regulatory region [23, 24]. Nrf2 also binds to regulatory loci of the various heme biosynthesis genes, like, ferrochelatase gene. CNC transcription factors may regulate heme and globin synthesis as well as antioxidant gene expression in the erythroblast stages . It has been observed that Catechins extracted from Green Tea and Theaflavins from Black Tea, can directly act as free radical scavengers and can indirectly exert its effect through activation of transcription factors and antioxidant enzymes. Tea polyphenols, which have been dried and purified from black and green tea have been shown to have a profound protective effect on red cells challenged with exogenous oxidants . Resveratrol also is another potent anti oxidant agent.
It is hypothesized that the immature red cells, specially those of late erythroblastic stage, undergo apoptosis in thalassaemia haemoglobinopathy model, upon introduction of such antioxidant agents from plant sources, should survive the free radical induced damage. This protection should decrease the degree of eryroptosis and induce red cell survival, thereby reducing transfusion load in thalassaemics.
- To identify the active ingredients, present in liquid drinks as inducer of late erythroid oxidation protector.
- To study the expression of different applicable transcription factors in erythroid cells, in the presence or absence of inducer obtained from natural products.
- To study the transcriptome profile of the defective erythroid progenitor cells after challenging with the haematopoiesis inducer.
1.1. Peripheral blood would be collected from healthy individuals and thalassaemic subjects of confirmed genotype.
1.2. The collected peripheral blood would undergo buffy coat enrichment and cultured in vitro.
1.3. The cultured cells would be sampled at intervals and the staging of maturation would be done using flowcytometry ?4-integrin vs Band 3. The cell population of healthy and thalassaemic individuals would be compared and the hypothesis of expansion of early and intermediate normoblasts and erythroptosis at late normoblasts would be confirmed.
1.1. 4 sets of culture will be established, one pair each from normal and thalassaemic individuals.
1.2. Inducing agents would be introduced in the initiation stage of culture, in one from each normal and abnormal culture and will not be introduced in another pair set.
1.3. Inducing agents would be introduced, at different concentrations, both individually and in combination, and the cultured cells would be analysed at fixed time intervals, i.e., at different stages of maturation and the results would be compared
1.4. The expression of Spectrin, Band 3 and Nrf-2 would be studied from cells obtained from both the test and control population cell samples.
1.5. The specific concentration at which there is maximum survival of late stage red cell precursors will be noted and confirmed in similar cell cultures obtained from buffy coat enriched peripheral blood of thalassaemic individuals. Significant and reproducible results will be accepted.
1.1. Transcriptome profiling of HbA and HbF would be done by isolation of mRNA will be done in the corresponding stages of erythroid differentiation.
1.2. Increase of HbA transcriptome profile in between Inducer treated and untreated cultures would be compared to prove increase HbA.
1.1. If the hypothesis is proved to be corrected, alteration in expression of Nrf2 , would be studied and the difference between the control and test population established.
1.2. In addition expression of GATA 1 and JAK-1 would be studied in the cell population by quantitative estimation of mRNA at different stages of erythroid maturation in both control and test population.
Impact of their research studies:
If the intervention strategy is able to prove that introduction of such agents either singly or in combination is able to reduce the erythroid expansion in the early and intermediate normoblastic stage and reduce erythroptosis in the late normoblastic stage, these agents can be developed as medications for increasing transfusion interval in transfusion dependent thalassaemics.
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