Haemoglobinopathies Are The Commonest Single Gene Disorders In The World

Abstract:

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.

Background:

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 [3]. 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 [4]. 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.

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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 [3]. 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 [4]. 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 [5]. 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 [6]. 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 [10].

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 [13]. 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 [19].

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.

Research problem:

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.

Hypothesis:

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 [22]. 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 [24]. 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 [25]. 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.

Objectives:

  • 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.

Research methodology:

General:

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.

Objective 1:

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.

Objective 2:

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.

Objective 3:

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.

References:

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2. Dussiot M, Maciel TT, Fricot A, et al.. An activin receptor IIA ligand trap corrects ineffective erythropoiesis in ?-thalassemia.Nat Med. 2014 Apr;20(4):398-407.

3. Wood WG1, Stamatoyannopoulos G, Globin synthesis in fractionated Normoblasts of beta-thalassemia heterozygotes.J Clin Invest. 1975 Mar;55(3):567-78.

4. Weiss MJ1, Zhou S, Feng L, Gell DA, Mackay JP, Shi Y, Gow AJ, Role of alpha-hemoglobin-stabilizing protein in normal erythropoiesis and beta-thalassemia. Ann N Y Acad Sci. 2005;1054:103-17.

5. D. J. Weatherall, “Phenotype-genotype relationships in monogenic disease: lessons from the thalassaemias,” Nature Reviews Genetics, vol.2, no.4, pp.245–255,2001.

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8. F. Centis, L. Tabellini, G. Lucarelli et al., “The importance of erythroid expansion in determining the extent of apoptosis in erythroid precursors in patients with ?-thalassemia major,” Blood, vol.96, no.10, pp.3624–3629,2000.

9. J. Yuan, E. Angelucci, G. Lucarelli et al., “Accelerated programmed cell death (apoptosis) in erythroid precursors of patients with severe ?-thalassemia (Cooley’s anemia),” Blood, vol.82, no.2, pp.374–377,1993.

10. S. L. Schrier. Pathophysiology of the thalassaemia the Albion Walter Hewlett Award presentation,” Western Journal of Medicine, vol.167,no.2,pp.82–89,1997.

11. L. A. Mathias, T. C. Fisher, L. Zeng et al., “Ineffective erythropoiesis in ?-thalassemia major is due to apoptosis at the polychromatophilic normoblast stage,” Experimental Hematology, vol.28, no.12, pp.1343–1353,2000.

12. S. N. Wickramasinghe and V. Bush, “Observations on the ultrastructure of erythropoietic cells and reticulum cells in the bone marrow of patients with homozygous ? thalassaemia, ”The British Journal of Haematology, vol. 30, no.4,pp.395–399,1975.

13. Leecharoenkiat, T. Wannatung, P. Lithanatudom et al., “Increased oxidative metabolism is associated with erythroid precursor expansion in ?0-thalassaemia/Hb E disease,” Blood Cells, Molecules and Diseases, vol.47, no.3, pp.143–157,2011.

14. L.de Franceschi, M. Bertoldi, L.de Falcoetal.,“Oxidative stress modulates heme synthesis and induces peroxiredoxin-2 as a novel cytoprotective response in ?-thalassemic erythropoiesis,” Haematologica,vol.96,no.11,pp.1595–1604,2011.

15. Y. Liu, R. Pop, C. Sadegh, C. Brugnara, V. H. Haase, and M. Socolovsky, “Suppression of Fas-FasL co-expression by erythropoietin mediates erythroblast expansion during the erythropoietic stress response in vivo,” Blood, vol. 108, no. 1, pp. 123–133, 2006.

16. S. L. Schrier, F. Centis, M. Verneris, L. Ma, and E. Angelucci, “The role of oxidant injury in the pathophysiology of human thalassemias, ” Redox Report, vol.8, no.5, pp.241–245,2003.

17. D.G. Nathan and R.B. Gunn, “Thalassemia: the consequences of unbalanced haemoglobin synthesis, ”The American Journal of Medicine,vol.41,no.5,pp.815–830,1966.

18. N.F. Olivieri, “The ?-thalassemias,” The New England Journal of Medicine, vol.341,no.2,pp.99–109,1999.

19. M. Aljurf, L. Ma, E. Angelucci et al., “Abnormal assembly of membrane proteins in erythroid progenitors of patients with ? thalassemia major,” Blood, vol.87, no.5, pp.2049–2056,1996.

20. J. Yuan, R. Kannan, E. Shinar, E. A. Rachmilewitz , and P. S. Low, “Isolation, characterization, and immunoprecipitation studies of immune complexes from membranes of ?-thalassemic erythrocytes,”Blood,vol.79,no.11,pp.3007–3013,1992.

21. G.L. Forni, M. Podesta’, M. Mussoetal., “Differential effects of the type of iron chelator on the absolute number of hematopoietic peripheral progenitors in patients with ?-thalassemia major,” Haematologica,2012

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Brown Vs. Board Of Education Summary

This landmark case began in Topeka, Kansas during the 1950s. This case was based around segregation in schools. Linda Brown’s, Family and many other families sued for their children because public schools were not letting them enroll in these schools because they only allowed whites. These kids didn’t have have many options. From Linda Brown´s house there was a white only school closer, but she had to go to the other schools for African Americans. There was a school close to their house but it was for whites only, so they couldn’t go.

There were only four schools for African Americans. L?inda Brown and her family believed that the segregated school system violated the 14th Amendment and took their case to court. This case started to become huge because of how big of an issue it was people decided to fight harder and came out to support this issue. History The Thirteenth, fourteenth and fifteenth amendments are known as the civil rights amendments. The 13th amendment abolished slavery. The 14th amendment allowed people born in the United States to be American citizens including African Americans. The 14th amendment also states that you could not deny African Americans anything that the whites could do they. Everyone had an equal protection of the laws. Lastly, the 15th amendment, prohibited people for denying anyone race or color from voting. In this case the 14th amendment was being broken by not allowing these kids to even enroll into these white schools. Since this was not allowing the same treatment as a white person they believed it was unconstitutional. In this period of time the practice of ¨Separate but equal¨ was still happening. Because of the plessy vs ferguson case. ¨At the time of the Brown case, a Kansas statute permitted, but did not require, cities of more than 15,000 people to maintain separate school facilities for black and white students.¨ Since Topeka had 15,000 or more people they allowed these schools to be seperate.

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They law stated the schools could be separate but also they had to be equal. In which the african american schools were not equal to the whites at all. Results The courts had a lot to take into consideration. One thing being are the schools being separate but equal against the 14th amendment. Another is seperating people by race in busses, schools, and other public places actually unconstitutional. The courts believed that they were breaking the 14th amendment and it was unconstitutional. This case was the case that ended separate but equal. A year later, they decided to desegregate the schools and everything because of the unanimous ruling. “in addition to the obvious disapproving segregationists were some constitutional scholars who felt that the decision went against legal tradition by relying heavily on data supplied by social scientists rather than precedent or established law.” The courts decided to take away separate but equal as soon as possible. Desegregating everything at a very fast pace. Amendment violation The amendment that was said to be broken was the 14th amendment. Which states in section one, ¨All persons born or naturalized in the United States, and subject to the jurisdiction thereof, are citizens of the United States and of the State wherein they reside. No State shall make or enforce any law which shall abridge the privileges or immunities of citizens of the United States; nor shall any State deprive any person of life, liberty, or property, without due process of law; nor deny to any person within its jurisdiction the equal protection of the laws.¨

This amendment granted equality to all people know matter the race. It also granted the people born in the united states are American citizens and they should get the same treatment as whites born in America. Not allowing this children into specific schools was depriving them for property. They were not allowing citizens of america into the schools which is violating their rights. Impact on future generations I picked this landmark case because it was the most interesting to me. Seeing especially our school and how diverse it is now. I could not imagine living in those days and the different faces being in different schools would be so different. I think that this case was very important to society. There still is some issues with racism but everyone is more accepting. I feel like the school segregation taught the kids that you can be better than someone because of your skin tone. Our generation and many other generations will mostly never think like that based on the diversity in the United states. The education system is also so different. Back then the white schools were getting way better education. The white schools would get the educated teachers and the better books. The schools for African american people were old and they would only get the hand-me-down books when they were all used up, and their teachers were not as good. Now, everyone gets the same education and everyone is treated as equally as possible. The teachers would get fired if they treated anyone like they did when this case started. This case showed the people that everyone deserves the same opportunities no matter the color of the skin color.

Work Cited

  1. “Brown v. Board of Education (1954).” Our Documents – Brown v. Board of Education (1954), www.ourdocuments.gov/doc.php?flash=false&doc=87.
  2. Editors, History.com. “Brown v. Board of Education.” History.com, A&E Television Networks, 27 Oct. 2009, www.history.com/topics/black-history/brown-v-board-of-education-of-topeka.
  3. “The 14th Amendment of the U.S. Constitution.” National Constitution Center – The 14th Amendment of the U.S. Constitution, constitutioncenter.org/interactive-constitution/amendments/amendment-xiv.
  4. “The Supreme Court . Expanding Civil Rights . Landmark Cases . Brown v. Board of Education (1954) | PBS.” THIRTEEN, www.thirteen.org/wnet/supremecourt/rights/landmark_brown.html.
  5. Tunes, History. “Learn American History through 50 Pop Songs.” History Tunes: 13th 14th and 15th Amendments, www.historytunes.com/13th14thand15thAmendments.php.

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