22.6 Erythroid disorders 5354 22.6.1 Erythropoiesi

22.6 Erythroid disorders 5354 22.6.1 Erythropoiesis 5354 Vijay G. Sankaran

CONTENTS 22.6.1 Erythropoiesis  5354 Vijay G. Sankaran 22.6.2 Anaemia: pathophysiology, classification, and
clinical features  5359 David J. Weatherall and Chris Hatton 22.6.3 Anaemia as a challenge to world health  5366 David J. Roberts and David J. Weatherall 22.6.4 Iron metabolism and its disorders  5371 Timothy M. Cox and John B. Porter 22.6.5 Anaemia of inflammation  5402 Sant-​Rayn Pasricha and Hal Drakesmith 22.6.6 Megaloblastic anaemia and miscellaneous
deficiency anaemias  5407 A.V. Hoffbrand 22.6.7 Disorders of the synthesis or function of haemoglobin  5426 Deborah Hay and David J. Weatherall 22.6.8 Anaemias resulting from defective maturation
of red cells  5450 Stephen J. Fuller and James S. Wiley 22.6.9 Disorders of the red cell membrane  5456 Patrick G. Gallagher 22.6.10 Erythrocyte enzymopathies  5463 Alberto Zanella and Paola Bianchi 22.6.11 Glucose-​6-​phosphate dehydrogenase
deficiency  5472 Lucio Luzzatto 22.6.12 Acquired haemolytic anaemia  5479 Amy Powers and Leslie Silberstein 22.6.1  Erythropoiesis Vijay G. Sankaran ESSENTIALS Biological mechanisms of erythropoiesis Erythropoiesis is a highly regulated, multistep process in which stem cells, after a series of amplification divisions, generate multipotential progenitor cells, then oligo-​ and finally unilineage erythroid progen- itors, and then morphologically recognizable erythroid precursors and mature red cells. Ontogeny of erythropoiesis—​this involves a series of well-​ coordinated events during embryonic and early fetal life. In the fetus, the main site of erythropoiesis is the liver, which initially pro- duces mainly fetal haemoglobin (HbF, α2γ2) and a small component (10–​15%) of adult haemoglobin (HbA, α2β2), with the fraction of HbA rising to about 50% at birth. After birth, the site of erythroid cell production maintained throughout life is the bone marrow, with the final adult erythroid pattern (adult Hb with <1% fetal Hb) being reached a few months after birth. Regulation of erythropoiesis—​the main regulator is erythropoietin, a sialoglycoprotein that is produced by interstitial cells in the kidney in response to tissue hypoxia and exerts its effect by binding to a spe- cific receptor on erythroid burst-​forming units (BFU-​Es), erythroid colony-​forming units (CFU-​Es), and proerythroblasts. Abnormalities of erythroid production Abnormal erythropoietin production—​anaemia can be caused by acquired or congenital deficiency in erythropoietin production, most commonly in chronic kidney disease. Impaired tissue oxygen delivery is a common cause of erythropoietin-​driven secondary erythrocytosis, often caused by chronic lung disease, sometimes by 22.6 Erythroid disorders

22.6.1  Erythropoiesis 5355 congenital heart anomalies, and rarely by haemoglobin mutations. Some kidney cancers increase erythropoietin production and hence cause secondary erythrocytosis. Other causes of abnormal erythroid production—​these include (1)  acquired and congenital defects in erythropoietin signalling; (2) acquired and congenital defects in the transcription factors GATA1 or EKLF, which are required for activation of erythroid-​specific genes; (3) acquired or congenital abnormalities in ribosome synthesis or splicing factors (Diamond–​Blackfan anaemia and myelodysplastic syndromes); and (4) factors that lead to premature red cell destruc- tion, including inherited defects in protein structure (e.g. hereditary spherocytosis), enzyme defects in metabolic pathways (e.g. pyruvate kinase), and haemoglobin defects (e.g. sickle cell anaemia). Introduction Every second in humans, over two million red blood cells are pro- duced in the bone marrow. This process is carefully coordinated and is referred to as erythropoiesis. Alterations of erythropoiesis can result in a variety of pathological conditions due to a mismatch between the removal of red blood cells from the circulation and the production of red cells in the bone marrow. Anaemia is very common worldwide, with nearly 30% of the world population af- fected with some form of this condition. Anaemia may be due to nutritional, infectious, or systemic aetiologies, while other forms are a consequence of intrinsic defects affecting developing precursors or mature red blood cells directly. Erythropoiesis is frequently and commonly perturbed in these varied aetiologies that result in an- aemia. In this chapter, we discuss the process of erythropoiesis, how this process varies in the course of human development, and how erythropoiesis is regulated by both external factors and through in- trinsic regulation. This chapter will introduce erythropoiesis as a framework for subsequent chapters focused on the pathophysiology of the disorders that arise as a result of its disruption. The process of erythropoiesis To ensure that red blood cells can be rapidly produced to balance their continuous loss following a circulation time of approximately 120 days, an intricately regulated differentiation process has emerged for red blood cell production from haematopoietic stem cells in the bone marrow. Precise regulation of this process is necessary, since it must be able to compensate rapidly for haemolysis or blood loss. As described subsequently, this process is exquisitely sensitive to a variety of molecules, which can regulate the rate at which erythro- poiesis occurs. Feedback systems exist to ensure that the process of erythropoiesis maintains a red blood cell count at homeostatic levels in most individuals. All blood cells arise initially from a rare population of cells that are capable of lifelong maintenance of haematopoiesis, known as haematopoietic stem cells (see Chapter  22.2.1). Approximately 1 in 104 to 105 cells in the bone marrow are estimated to be haem- atopoietic stem cells. These haematopoietic stem cells can give rise to all the differentiated cells that compose the blood, including red blood cells, platelets, and the white blood cell lineages, including both myeloid (granulocytes, monocytes, eosinophils, and baso- phils) and lymphoid cells (T lymphocytes, B lymphocytes, and NK cells). Traditional models suggest that a series of differentiation di- visions can occur from the haematopoietic stem cells to give rise to multilineage and then more restricted progenitors that are in turn capable of producing the various terminally differentiated cells that compose the blood. Work over several years has elucidated surface markers that can allow enrichment of these progenitor populations within the bone marrow. While substantial support exists for this hierarchical model of haematopoiesis, more recent work suggests that lineage commitment may occur in earlier haematopoietic stem or progenitor cells that then give rise to each lineage through a sep- arate differentiation process. Earlier studies may have been con- founded by examining bulk populations of cells, rather than looking at cells individually. It is likely that some combination of these models exists in humans, and further work, particularly by exam- ining patients with blood disorders, will be needed to gain further insight into these models. Regardless of the uncertainty of the early steps in haemato- poietic differentiation, the subsequent stages of differentiation that define erythropoiesis are well understood and characterized (Fig. 22.6.1.1). The earliest committed erythroid progenitors are the burst-​forming unit erythroid cells (BFU-​Es). These progen- itors have traditionally been identified through the use of colony-​ forming assays in semisolid medium where a single BFU-​E is capable of giving rise to colonies containing hundreds to thousands ProE BasoE PolyE OrthoE Retic RBC Haemoglobin expression BFU-E CFU-E HSC Identified through colony assays Fig. 22.6.1.1  A schematic showing normal erythropoiesis. The haematopoietic stem cell (HSC) differentiates to a series of multipotential progenitors that can then give rise to the earliest erythroid-​restricted progenitor, the burst forming unit erythroid cell (BFU-​E). This cell can then give rise to the colony-​forming unit erythroid cell (CFU-​E). These early erythroid progenitors can only be identified through the use of colony assays, where the form colonies composed of numerous more differentiated erythroblasts. Subsequently, a series of morphologically identifiable precursors are produced that include the proerythroblast (ProE), basophilic erythroblast (BasoE), polychromatophilic erythroblast (PolyE), orthochromatophilic erythroblast (OrthoE), reticulocyte (Retic), and mature red blood cells (RBCs). The increase in haemoglobin during this process is depicted below this scheme.

section 22  Haematological disorders 5356 of erythroblasts. The BFU-​E is thought to divide and give rise to an- other more-​differentiated committed erythroid progenitor, known as the colony-​forming unit erythroid cell (CFU-​E). CFU-​Es form colonies earlier than BFU-​Es in semisolid culture medium, which are composed of tens to hundreds of erythroblasts. Recent studies have suggested methods that allow for the prospective enrichment of both BFU-​Es and CFU-​Es in humans and mice using cell surface molecules, although further validation of these methods is needed before they can regularly supplant the traditional colony forming assays used to assess such progenitor populations. It is important to note that BFU-​E and CFU-​E cells in the bone marrow cannot be dis- tinguished using morphology, since they appear identical to other blast cells in the bone marrow. Subsequent to the CFU-​E, a series of morphologically dis- tinct and further differentiated erythroblast populations emerge (Fig. 22.6.1.1). The CFU-​E initially differentiates into a proery­ throblast. The proerythroblast differentiates into the basophilic erythroblast, which then differentiates into a polychromatophilic erythroblast. The final stages of differentiation involve dramatically increased haemoglobinization and nuclear condensation, which characterizes the orthochromatic erythroblast (which is notable for its haemoglobin-​rich cytoplasm and condensed nucleus). The orthochromatic erythroblast then undergoes enucleation, thereby giving rise to a reticulocyte. The reticulocyte can then enter the circulation and terminally matures by losing the remaining ribo- nucleic acids (RNAs) to become a mature red blood cell with the characteristic biconcave shape. Throughout this process, with every cell division, there is amplification in the number of cells pre- sent, thus allowing for effective production of the numerous red blood cells necessary to replace those continuously lost from the circulation. Developmental erythropoiesis The previous framework describes the general process of erythro- poiesis, as it occurs to maintain red blood cell numbers in the circulation of the adult. However, erythropoiesis is critical for en- suring sufficient oxygen transport capability throughout the body at all stages of development. In the early stages of development, red blood cells are produced via transient populations of progen- itors, which are eventually supplanted by the haematopoietic stem cells which maintain blood production throughout the life of the organism. In humans, the first erythroid cells are detected beginning at week 3 of gestation and continue to supply the key oxygen transport cap- ability until week 8 of gestation. This early and transient population of cells, which are produced in the yolk sac and enter the circulation as nucleated cells, are known as the primitive erythroid cells. These cells have been noted to eventually enucleate in both humans and mice. This population has a key role in ensuring that development can proceed effectively once passive oxygen diffusion can no longer keep pace with the rapid growth of the embryo. The function and development of the primitive erythroid population, which largely occurs in the circulation of the embryo, has been studied in some detail. While the differentiation process resembles that in the adult, as discussed earlier, there are key differences. For example, there ap- pears to be less expansion of this population compared to that seen at later stages of erythropoiesis in the fetal liver or bone marrow. The molecular regulators of this process are largely conserved, but some differences exist here too, and in humans a unique set of embry- onic haemoglobin genes are expressed, composing the majority of haemoglobin at this stage. Starting at week 7 to 8 of human gestation, the fetal liver begins to be colonized by erythroblasts and the majority of red blood cell production for the remainder of gestation shifts to this organ. The enucleated red blood cells produced from these erythroid pre- cursors enter the circulation by week 8 of gestation and fetal liver erythropoiesis maintains the circulating population of red blood cells for the remainder of gestation. The initial erythroid progen- itors that seed the fetal liver are derived from a transient wave of haematopoietic progenitors that are produced in the yolk sac. As gestation proceeds, these progenitors are supplanted by those pro- duced from haematopoietic stem cells, which arise in the major ar- teries of the embryo proper (including the aorta, which is a major site of haematopoietic stem cell production). The exact stage at which the haematopoietic stem cell-​derived progenitors supplant the yolk sac-​derived populations in the fetal liver is not well under- stood, and in mouse models the yolk sac-​derived blood progenitors can maintain sufficient blood production to allow the mouse em- bryos to survive for the entirety of gestation. The fetal liver-​derived erythroid progenitors display similar characteristics to their adult counterparts and are both commonly referred to as definitive erythroid cells. The red blood cells produced from the fetal liver ex- press different haemoglobin subtypes to their adult counterparts, as discussed later, and are larger in volume. In addition, the precursors to these red cells appear to have higher cell cycle rates and may have increased sensitivity to the key hormone erythropoietin, which is discussed later. While the bone marrow is seeded with haematopoietic progen- itors in the first few weeks of gestation as rudimentary cartilage and endothelial cells appear in this region, only a small amount of haem- atopoiesis occurs here before birth, with the fetal liver being respon- sible for the bulk of erythropoiesis. That which does occur in the fetal marrow has a myeloid bias. However, after birth, haematopoi- esis shifts primarily to the bone marrow. Developmental haemoglobin switching While the sites of haematopoiesis shift as noted previously, the erythroid lineages are also noted to have unique switches in the production of haemoglobin in these different populations. As al- ready discussed, the yolk sac-​derived primitive erythroid cells ex- press a variety of embryonic haemoglobins. The embryonic form of α-​globin is known as ζ-​globin and is expressed only in the primitive erythroid cells along with the adult α-​globin molecule. ε-​Globin is an embryonic β-​like globin, the expression of which is restricted to primitive erythroid cells. Subsequently, once the de- finitive erythroid lineage is produced from the fetal liver, there is expression of adult α-​globin along with production of the fetal β-​ like globins, known as γ-​globins, as well as a small amount of adult β-​globin. Shortly before the time of birth, the predominance of γ-​ globin production within the definitive erythroid lineage switches to high-​level expression of adult β-​globin—​a process termed the fetal-​to-​adult haemoglobin switch. It should be noted that all α-​like

22.6.1  Erythropoiesis 5357 globins are encoded by genes at the same locus on chromosome 16 and similarly all β-​like globin genes are found at a single locus on chromosome 11, where the genes are controlled by a common set of regulatory elements. The developmental regulation of the genes encoding these different globins has been studied extensively and there is recent insight into specific transcriptional regulators of this process, which is discussed later in the section on intrinsic regu- lators of erythropoiesis. The process of haemoglobin switching can be altered with specific mutations that occur in rare individuals and there can be persistence of certain haemoglobins at later develop- mental stages. For example, a variety of mutations, which primarily occur within the β-​globin gene locus itself, can cause persistence of fetal haemoglobin (composed of α-​ and γ-​globin) into adulthood in a condition known as hereditary persistence of fetal haemoglobin. The mutations that cause this condition are primarily deletions in the β-​globin gene locus, but point mutations and alterations of regu- latory factors have also been identified in rare cases. The regulation of fetal haemoglobin is of clinical importance, since increased pro- duction of γ-​globin can compensate for the defective production of β-​globin in β-​thalassaemia and can prevent the clinical symp- toms resulting from the mutant version of β-​globin found in sickle cell disease (see also Chapter 22.6.7). Therefore, there has been a long-​standing interest in attempting to stimulate fetal haemoglobin production for therapeutic purposes in both sickle cell disease and β-​thalassaemia. Extrinsic regulation of erythropoiesis As discussed earlier, the process of erythropoiesis must be carefully coordinated to ensure that the production of red blood cells can be appropriately tailored to the need for oxygen delivery throughout the body. The regulation of erythropoiesis is primarily controlled by the hormone erythropoietin (EPO), which is a single chain glyco- protein. Camot and Deflandre initially hypothesized that a hormone with the function of EPO existed in 1906. Work from Reissman, Stohlman, and their colleagues in the 1950s demonstrated its ex- istence. EPO was purified using biochemical approaches in the late 1970s, and in the early 1980s the gene encoding EPO was cloned. This resulted in the production of recombinant forms of EPO, which are in widespread clinical use today. EPO has a short half-​life of less than 5 h in the circulation, as a re- sult of degradation in the liver and urinary secretion. It is primarily produced by a group of oxygen-​sensitive cells in the cortex and outer medulla of the kidney and therefore its secretion can be increased by over 100-​fold in cases of hypoxia when oxygen delivery to tissues needs to be increased. EPO acts by binding to the erythropoietin receptor (EPOR) that is primarily expressed on erythroid progenitors and some early precursors. This binding event promotes expansion and differen- tiation of erythroid progenitors and precursors to produce mature red blood cells. EPOR is a cytokine receptor that can be dimer- ized by binding to a single molecule of EPO in an asymmetric manner. This dimerization following binding triggers a conform- ational change in the EPOR-​binding Janus kinase, JAK2, which allows its phosphorylation and subsequent activation of a variety of downstream signalling factors. This includes the STAT5 tran- scription factor, which can trigger the transcriptional activation of a variety of genes in the nucleus. In addition, JAK2 activates several downstream cellular signalling pathways, including the mitogen ­activated protein kinase and phosphatidylinositol-​3 kinase path- ways to mediate its activities. In addition to the use of recombinant EPO, other therapeutic ap- proaches have been taken to modulate this pathway. Peptides that bind and activate the EPOR have been tested in clinical trials, but are not currently in routine clinical use. In addition, increasing EPO ex- pression in the kidney can be accomplished by activation of hypoxia-​ inducible factors, a group of transcription factors that stimulate EPO gene transcription in the setting of hypoxia. A group of drugs known as prolyl hydroxylase inhibitors are able to prevent the degradation of the hypoxia-​inducible factors and thereby activate EPO transcrip- tion. These drugs are in clinical trials as therapeutic agents to be used in place of recombinant EPO. The importance of this pathway for erythropoiesis in humans has been elegantly demonstrated by rare human mutations that affect this process. For example, in Chuvash polycythemia and some re- lated conditions, the von Hippel–​Lindau (VHL) gene can be mu- tated, resulting in excessive activation of the hypoxia-​inducible factors that in turn cause EPO levels to increase in an unregulated fashion. Rare mutations that cause an increased oxygen affinity in red cells due to mutations in haemoglobin or in metabolic enzymes, such as 2,3-​diphosphoglycerate mutase, can result in decreased oxygen sensing by the kidney (as a result of impaired oxygen re- lease from the red cell) and thus can cause increased EPO levels and erythrocytosis (excessive red blood cell production). Rare mutations in the EPOR can eliminate negative regulatory pathways and cause excessive activation downstream from the receptor, again causing erythrocytosis. In addition, in an acquired condition known as poly- cythemia vera, mutations in JAK2 can result in excessive activation downstream of EPOR and cause excessive red blood cell production (see also Chapter 22.3.5). Another well-​studied growth factor, stem call factor (SCF), binds to the KIT receptor that is expressed on erythroid progenitors and to some extent on proerythroblasts. SCF can synergize with EPO to promote erythropoiesis. A variety of naturally occurring and in- duced mouse mutations in either the SCF or KIT receptor cause im- paired erythropoiesis with anaemia, macrocytosis, and a reduction in the number of erythroid progenitors, including CFU-​Es, that are present in the mice. However, to date, no human mutations in SCF or KIT have been identified that affect erythropoiesis. Recent work has also uncovered other regulatory pathways that affect red blood cell production. During clinical testing of activin ligand traps for use in bone disease, the effectiveness of these agents for red blood cell production was serendipitously discovered. Follow-​up studies showed that these ligand traps appear to block the activity of the ligand GDF11, which may normally be a negative regulator of erythropoiesis. Further studies are necessary to under- stand whether this is the only target of these drugs and also how GDF11 may normally affect erythropoiesis. In addition, in the setting of inflammation or infections, a variety of proinflammatory cytokines can be produced that block erythro- poiesis. This can occur indirectly due to effects on EPO production from the kidney or on iron availability, but this can also be due to direct effects on the process of erythropoiesis. Proinflammatory cytokines, including tumour necrosis factor (TNF)-​α and interferon (IFN)-​γ, have been shown to block normal erythropoiesis.

section 22  Haematological disorders 5358 While the regulation of iron homeostasis is beyond the scope of this chapter, it should be noted that a variety of extrinsic regu- lators of iron homeostasis can significantly impact the iron avail- able for erythropoiesis (and iron is absolutely essential for the production of the haem prosthetic group found in every subunit of haemoglobin). Most notable among these regulators is the liver-​produced peptide hormone, hepcidin, which prevents iron absorption by the intestine and limits iron release from macro- phages. The regulation of iron is complex, but has a significant impact on the availability of iron for erythropoiesis and can there- fore impact normal red blood cell production profoundly (see also Chapters 22.6.4 and 22.6.5). Intrinsic regulation of erythropoiesis While factors outside of the cell, including EPO and potentially other factors such as GDF11, play an important role in regulating erythropoiesis, much of the ultimate control of this process occurs at the level of gene expression. A group of factors that regulate the pro- cess of messenger RNA transcription plays a key role in regulating erythropoiesis. Studies in model organisms and analysis of rare mu- tations in humans have highlighted the importance of a few specific factors that are necessary for erythropoiesis. The transcription factor GATA1 has been shown to have a key role in erythropoiesis and has therefore been termed a master regu- lator of erythropoiesis. Most genes expressed during erythropoi- esis require GATA1 for their expression. Mutations in GATA1 can result in severe forms of anaemia, such as rare cases of Diamond–​ Blackfan anaemia or other forms of anaemia due to impaired erythroid cell maturation. GATA1 plays a key role in both pro- moting transcription of genes necessary for erythropoiesis and in repressing other genes that would normally be expressed in other lineages. The transcription factor KLF1 has been shown to have key roles in erythropoiesis, particularly in the late stages of this process. Mutations in KLF1 in humans cause a variety of phenotypes, all of which are characterized by late-​stage defects in erythropoiesis. This includes a condition known as congenital dyserythropoietic anaemia, which is characterized by impaired maturation of late-​ stage erythroblasts. In addition, some mutations in KLF1 have been shown to result in increased fetal haemoglobin production. The ex- tent to which this function can be separated from its role in normal erythropoiesis needs to be studied further. A variety of other transcription factors, including TAL1, GFI1B, ZFPM1, LMO2, LDB1, and others, have also been implicated in erythropoiesis, although they may also have pleiotropic roles in other cell types. Many of these transcription factors appear to act to- gether in complexes with GATA1 and KLF1 and this combinatorial activity may explain how some genes are activated at different times or in varying contexts during normal erythropoiesis. Recent genomic studies have also highlighted the role of other transcription factors that appear to have key roles in the produc- tion of fetal haemoglobin. By following up on genome-​wide asso- ciation studies, which were focused on examining common genetic variation underlying the normal distribution of fetal haemoglobin levels, the transcription factors BCL11A and MYB were found to be important silencing factors for fetal haemoglobin. Humans with reduced expression of BCL11A can have substantial persistence of fetal haemoglobin, while erythropoiesis appears to be otherwise essentially unperturbed. However, BCL11A and MYB have other roles and approaches to target these and other factors are under ac- tive investigation as potential therapies for sickle cell disease and β-​thalassaemia. Much more work needs to be done to gain an im- proved understanding of how factors including BCL11A, MYB, and others act to regulate the haemoglobin genes during erythropoiesis; this could also suggest further therapeutic avenues for stimulating fetal haemoglobin production. Recent studies have suggested that by simply altering the looping of regions of chromatin within a gene locus, specific genes, such as the γ-​globin genes, can be activated in adult erythroid cells. This suggests that factors such as BCL11A may carry out their activity in γ-​globin silencing, at least in part, by al- tering such looping interactions. While a great deal is known about the transcriptional regulators of erythropoiesis, far less is known about other processes that also contribute to gene expression. Protein translation plays an increas- ingly appreciated role in the control of gene expression in a variety of settings. In erythropoiesis, the key role of translation has been highlighted as a result of a rare disorder that results in a paucity of erythroid progenitors and precursors in the bone marrow without alterations in other lineages—​a condition known as Diamond–​ Blackfan or hypoplastic anaemia. The majority of mutations causing Diamond–​Blackfan anaemia are in ribosomal protein genes that compose the ribosome in every cell. Why mutations in these genes could cause an anaemia is poorly understood. However, recent work has identified rare gene mutations in GATA1 that can cause Diamond–​Blackfan anaemia and this has led to the finding that ribosomal protein mutations appear to impair the protein trans- lation of GATA1. Much more work is needed to understand how protein translation contributes to erythropoiesis both in healthy in- dividuals and in pathological states, but this recent work suggests that promising new insight into both normal erythropoiesis and some forms of anaemia can be gained through such studies. Concluding remarks In this chapter, we have provided a brief overview of the process by which red blood cells are produced. Erythropoiesis is critical for en- suring that red blood cells are available at all stages of development, and, since this process varies during gestation, we have discussed the current understanding of its developmental regulation. In add- ition, we have attempted to provide an overview of the key external and intrinsic regulators of erythropoiesis. We have provided a few examples to highlight how these processes are perturbed in disease and subsequent chapters will build upon this framework to discuss specific disorders that affect erythropoiesis in more detail. FURTHER READING An X, et  al. (2014). Global transcriptome analyses of human and murine terminal erythroid differentiation. Blood, 123, 3466–​77. Arlet JB, et  al. (2014). HSP70 sequestration by free alpha-​globin promotes ineffective erythropoiesis in beta-​thalassaemia. Nature, 514, 242–​6.